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Rapid reorganization of resistance gene homologues in cereal genomes. Proc Natl Acad Sci USA 95: 370-375
Abstract
We used conserved domains in the major class (nucleotide binding site plus leucine-rich repeat) of dicot resistance (R) genes to isolate related gene fragments via PCR from the monocot species rice and barley. Peptide sequence comparison of dicot R genes and monocot R-like genes revealed shared motifs but provided no evidence for a monocot-specific signature. Mapping of these genes in rice and barley showed linkage to genetically characterized R genes and revealed the existence of mixed clusters, each harboring at least two highly dissimilar R-like genes. Diversity was detected intraspecifically with wide variation in copy number between varieties of a particular species. Interspecific analyses of R-like genes frequently revealed nonsyntenic map locations between the cereal species rice, barley, and foxtail millet although tight collinear gene order is a hallmark of monocot genomes. Our data suggest a dramatic rearrangement of R gene loci between related species and implies a different mechanism for nucleotide binding site plus leucine-rich repeat gene evolution compared with the rest of the monocot genome.
Supplementary resources (25)
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... A nucleotide binding site (NBS) and a stretch of LRRs that characterize most of the functionally described R genes make up the fourth class, examples of which are N of tobacco, L6 of fl ax, and RPP5 and RPS2 of Arabidopsis spp. (Bent et al., 1994;Lander et al., 1987;Leister et al., 1998;Michelmore & Meyer, 1998;Pan et al., 2000b;Thomphson et al., 1994). Lopez et al. (2003) reported that they also have been an NBS upstream domain, which is either a region contained coiled coils (CC) or a Toll/Interleukin-1 receptor-like region (TIR; so named because of its homology with the cytoplasmic domain of the corresponding proteins in Drosophilla spp. ...
... Based on sequence similarity between R genes, a method using degenerate primers to target the conserved motifs has been successfully employed to isolate resistance gene analogs (RGAs) from potato (Lawrence et al., 1995), soybean (Joyeuk et al., 1999;Witham et al., 1994), rice (Leister et al., 1996), Arabidopsis spp. (Aatrs et al., 1998), lettuce (Saitou & Nei, 1987), common bean (Geffroy et al., 1999;Leister et al., 1998;Pitrat & Lecoq, 1980), melon (Brotman et al., 1998), and other monocotyledonous and dicotyledonous. ...
... The primers used in this study were designed according to Leister et al. (1996) and Brotman et al. (1998). Another primer combination (Leister et al., 1998) that had not been used for this purpose in melon and which matches the TIR domain was used to isolate another type of putative resistance genes in melon. As the results, two families include 11 MRGAs have been isolated. ...
Source of resistance to an Indonesia isolate of Cucumber mosaic virus (CMV-B2) in melon cultivar
Yamatouri has been reported. Moreover, Creb-2, a locus that confers resistance to CMV-B2 in Yamatouri has
been determined as a single dominant gene. To elucidate the resistance mechanism conferred by Creb-2 in
more detail, it is necessary to clone the Creb-2 gene and determine its molecular structure. One approach is
by amplification and cloning of melon resistance gene analogs (MRGAs) based on degenerated PCR primers
designed from conserved amino acids in the NBS-LRR motifs (P-loop, Kinase-2, and the GLPL) and Toll/
Interleukin-1 receptor-like region (TIR). This study was aimed to identify and characterize the resistance gene
analogs from Cucumis melo L. cv. Yamatouri by employing polymerase chain reactions (PCR) as a molecular
biology tools with degenerate primers based on conserved motifs of cloned R genes. The application of molecular
biology such as DNA isolation, degenerate primers and PCR condition, cloning, sequencing, linkage analysis
and mapping of resistance gene analogs to Creb-2 gene in melon will be widely discussed in this paper
... Knox et al. (2010) have observed amplification of C-Repeat Binding Factor genes at the Frost Resistance 2 locus in winter relative to spring barleys, hinting at a potential role in winter hardiness. And several studies (discussed below) provided evidence for the importance of large-scale rearrangements and duplications in the evolution of resistance genes (Leister et al. 1998;Wei et al. 2002;Himmelbach et al. 2010). These studies support the notion that a thorough understanding of genic structural variation can contribute mechanistic insights into the basis of plant performance. ...
... We believe this approach to be most fruitful for loci involved in the response to pathogens. Resistance genes in cereals (and other plant) genomes have been reported to occur in clusters composed of arrays of nucleotide-binding site leucine-rich repeat (NBS-LRR) genes, and these loci may have experienced large-scale rearrangement and CNV (Leister et al. 1998;Wei et al. 2002). In addition to NBS-LRR genes, also the family of germin-like proteins (GER4) involved in resistance to powdery mildew occurs in clusters that evolved by tandem duplication (Himmelbach et al. 2010). ...
The concept of a pan-genome refers to intraspecific diversity in genome content and structure, encompassing both genes and intergenic space. Pan-genomic studies employ a combination of de novo sequence assembly and reference-based alignment to discover and genotype structural variants. The large size and complex structure of Triticeae genomes were for a long time an obstacle for genomic research in barley and its relatives. Now that a reference genome is available, computational pipelines for high-quality sequence assembly are in place, and sequence costs continue to drop, investigations into the structural diversity of the barley genome seem within reach. Here, we review the recent progress on pan-genomics in the model grass Brachypodium distachyon, and the cereal crops rice and maize, and devise a multi-tiered strategy for a pan-genome project in barley. Our design involves: (1) the construction of high-quality de novo sequence assemblies for a small core set of representative genotypes, (2) short-read sequencing of a large diversity panel of genebank accessions to medium coverage and (3) the use of complementary methods such as chromosome-conformation capture sequencing and k-mer-based association genetics. The in silico representation of the barley pan-genome may inform about the mechanisms of structural genome evolution in the Triticeae and supplement quantitative genetics models of crop performance for better accuracy and predictive ability.
... Our whole-genome analysis showed that RGA were clustered, confirming the previous results from maize (Xiao et al. 2007) and other grasses such as rice, sorghum, and brachypodium (Li et al. 2010;Monosi et al. 2004). The clustered distribution of R genes may provide a reservoir of genetic variation from which new pathogen specificity can evolve via gene duplication, unequal crossing-over, interlocus recombination, or diversifying selection (Leister et al. 1998). Gene clusters may also be important for activation and expression of the R genes. ...
... Many authors mapped RGA within disease resistance QTL intervals (Graham et al. 2000, Wang et al. 2001Xiao et al. 2007), and used them to develop markers associated with resistance genes in wheat (Chen et al. 2006), barley (Leister et al. 1998), and maize Xiao et al. 2006). RGA were also used to isolate disease-resistance genes, such as the maize Rp1-D conferring resistance to rust (Collins et al. 1999). ...
Maize white spot (MWS), caused by the bacterium Pantoea ananatis, is one of the most important maize foliar diseases in tropical and subtropical regions, causing significant yield losses. Despite its economic importance, genetic studies of MWS are scarce. The aim of this study was to map quantitative trait loci (QTL) associated with MWS resistance and to identify resistance gene analogs (RGA) underlying these QTL. QTL mapping was performed in a tropical maize F2:3 population, which was genotyped with simple-sequence repeat and RGA-tagged markers and phenotyped for the response to MWS in two Brazilian southeastern locations. Nine QTL explained approximately 70% of the phenotypic variance for MWS resistance at each location, with two of them consistently detected in both environments. Data mining using 112 resistance genes cloned from different plant species revealed 1,697 RGA distributed in clusters within the maize genome. The RGA Pto19, Pto20, Pto99, and Xa26.151.4 were genetically mapped within MWS resistance QTL on chromosomes 4 and 8 and were preferentially expressed in the resistant parental line at locations where their respective QTL occurred. The consistency of QTL mapping, in silico prediction, and gene expression analyses revealed RGA and genomic regions suitable for marker-assisted selection to improve MWS resistance.
... RGAs mapping revealed linkage to the identified and characterized resistance (R) genes which ensured the presence of mixed clusters. This investigation of identified R-genes showed nonsystemic mapped locations between cereals like foxtail millet (Setaria italica), rice (Oryza sativa), and barley species (Leister et al. 1998). The study implies the rearrangement of R gene loci, suggesting various NBS and LRR mechanisms for R gene evolution compared with other monocot genomes. ...
Barley is regarded as the globe’s fourth major cereal crop. A variety of airborne, seedborne, and soilborne infective agents attack barley, causing a variety of barley diseases and substantial losses in agricultural output. Brown and yellow rusts, smut, net blotches, spot blotches, barley yellow dwarf, and molya disease are among the most serious diseases. In general, employing integrated disease management approaches is the best way to handle barley diseases. Growing resistant or tolerant varieties with the fewest foliar fungicides is the most effective approach for barley disease treatments. However, managing soilborne pathogens in barley plants is problematic due to a deficiency in distinguishing symptoms for diagnosis and the absence of fungicides or nematicides that are effective for these pathogens. Recently, nanotechnology has driven the advancement of creative concepts and agricultural productivity with a broad scope for managing plant infections and pests. The antimicrobial properties of metallic and metal oxide nanoparticulates such as silver, selenium, titanium dioxide, zinc oxide, and iron oxide have been extensively researched. In this chapter, we go over barley disease and the role of nanomaterials in reducing the incidence of disease and diagnosis, as well as barley seed germination, physiology, and nutritional quality of barley grain.KeywordsLeaf rust diseaseNet Blotch diseasePowdery mildewBarley yellow dwarfBarley smutSpot blotchFungicidesNanoparticulate
... Here, QTL is determined in interval generated between two markers at various points. It gives more accurate results compare to single marker approach but less than CIM and MIM technique (Leister, 1998). In this technique, likelihood ratio test is used to determine every QTL position in interval created by both markers. ...
QTL mapping is process of locating genes with effects on quantitative traits using molecular markers. It is basic research activity requiring careful planning of crosses and high precision phenotyping. It is used to offer direct mean to investigate the number of genes influencing the trait, to find out the location of the gene and to know the effect of dosage of these genes on variation of the trait. Genetic mapping is the first step to map based cloning. It is used for DNA based Marker Assisted Selection (MAS) and carrying out study on linkage between genes of interest. Genetic properties Of QTL, environmental factors, experimental errors in phenotyping and size of population are main factors affecting the QTL detection. The environment directly affects the expression of quantitative traits and when some experiments are conducted on the same sites for various seasons, it helps to detect the effects of environments on the QTL having influence on the traits of interest.
... The paralog hypothesis seems plausible for the underlying R-genes; however, our data were insufficient to rule out the single locus, multiple allele hypothesis. Although additional studies are needed to resolve this question, the classes of R-genes hypothesized to underlie these loci (see below) are commonly found in tandemly duplicated clusters in plants (Leister et al. 1998;Cook et al. 2012; The genotypic means, effects, and PVE estimates for SNP markers tightly linked with FW2 and FW5 were nearly identical to estimates for SNP markers associated with FW1 in the Fronteras and Portola S 1 populations (Table 1). FW2 was nearly completely dominant ( d∕a = 0.85 ). ...
Key Message
Several Fusarium wilt resistance genes were discovered, genetically and physically mapped, and rapidly deployed via marker-assisted selection to develop cultivars resistant to Fusarium oxysporum f. sp. fragariae , a devastating soil-borne pathogen of strawberry .
Abstract
Fusarium wilt, a soilborne disease caused by Fusarium oxysporum f. sp. fragariae , poses a significant threat to strawberry ( Fragaria $$\times$$ × ananassa ) production in many parts of the world. This pathogen causes wilting, collapse, and death in susceptible genotypes. We previously identified a dominant gene ( FW1 ) on chromosome 2B that confers resistance to race 1 of the pathogen, and hypothesized that gene-for-gene resistance to Fusarium wilt was widespread in strawberry. To explore this, a genetically diverse collection of heirloom and modern cultivars and octoploid ecotypes were screened for resistance to Fusarium wilt races 1 and 2. Here, we show that resistance to both races is widespread in natural and domesticated populations and that resistance to race 1 is conferred by partially to completely dominant alleles among loci ( FW1 , FW2 , FW3 , FW4 , and FW5 ) found on three non-homoeologous chromosomes (1A, 2B, and 6B). The underlying genes have not yet been cloned and functionally characterized; however, plausible candidates were identified that encode pattern recognition receptors or other proteins known to confer gene-for-gene resistance in plants. High-throughput genotyping assays for SNPs in linkage disequilibrium with FW1 - FW5 were developed to facilitate marker-assisted selection and accelerate the development of race 1 resistant cultivars. This study laid the foundation for identifying the genes encoded by FW1-FW5 , in addition to exploring the genetics of resistance to race 2 and other races of the pathogen, as a precaution to averting a Fusarium wilt pandemic.
... There appear to be a higher fraction of mutant genes in polyploid species compared to diploid species; however, there are presently not many polyploid pan-genomes accessible to corroborate this tendency. Dominant sub-genomes can affect allopolyploid gene content, as seen in Fragaria × ananassa [55,56] and B. napus [57,58], with the dominant sub-genomes containing a higher number of significant genes. In the case of WGDs (whole-genome duplications), the entire gene complement is doubled and is often followed by gene loss, renowned as fractionation. ...
The comparison of several associated species and plant genome sequencing efforts has increased in recent years. The inflated level of the genomic variety leads to the discovery that the single reference genomes may not reflect the variability in a species, resulting in the evolution of a pan-genome idea. Pan-genomes exhibit a species' genetic variability and contain mutant genes lacking in some individuals and essential genes present in all individuals. Mutant gene classifications often reveal cross-species parallels, including genes for abiotic and biotic stresses generally concentrated within mutant gene groupings. Here we discuss the history of pan-genomics in plants, investigate the causes of gene variation, deletion, and existence and demonstrate why pan-genomes might assist crop genetics and breeding research.
... It is speculated that either individual gene or its allelic variations, or the effects of the gene cluster may contribute to sorghum resistance against their respective diseases. Additionally, comparative mapping experiments may not benefit deep dissection of these QTLs underlying diseases resistance manipulated by R genes in cereals since their relative positions may be less conserved on account of various evolution events than other types of genes (Leister et al. 1998). This disadvantage slows the progress of discovery of functional R genes and their applications. ...
Plants are constantly exposed to numerous biotic stresses; thus, they have evolved to defend themselves from their enemies such as pathogen or insect pests. Plant resistance (R) genes encoded by nucleotide binding site-leucine rich repeat (NBS-LRR) or LRR-containing transmembrane-receptor proteins play important roles in plant defense against various biotic stresses. In this paper, we report the identification of 308 R genes, including NBS-LRR, 175 genes encoding receptor-like kinases (RLKs) and 65 genes coding for receptor-like proteins (RLPs) in sorghum [Sorghum bicolor (L.) Moench] through genome-wide sequence analysis. Those genes were dispersed across all ten chromosomes of sorghum. Phylogenetic analysis of the newly identified NBS-LRR, RLP and RLK genes classified them into five, eleven, and six clades, respectively, of which region-specific subclades were established. Investigation of exon/intron organization demonstrates the variations in number and location of introns within the family, and the genes that evolved close to their originals share identical gene structures. Also, gene duplication was noted, including 142 pairs of duplicated genes in the NBS family, 12 RLK genes and one RLP gene, respectively. Chromosomal locations analysis of these newly-identified R genes and 17 published QTLs indicated two hot-spot regions referring resistance to at least two biotic stresses. Among those R genes, some of them showed significantly differential expression during the infestation by greenbug biotype I based on the expression profile. Particularly, three R genes were validated by quantitative real time PCR for their up-regulation in response to aphids. In summary, the results of this study contribute not only a valuable genetic resource for further functional characterization of those newly-identified R genes, but also provide insight into genetic improvement of sorghum for resistance to greenbug aphids.
... The degenerate primers were developed from the conserved motifs in these NBS domains. PCR amplification was made to isolate RGAs (Kanazin et al., 1996 andLeister et al., 1998). Sequencing of brinjal R-gene-related genomic region could provide valuable knowledge for the development of DNA markers linked to a specific disease resistant phenotypes. ...
The experiment was conducted at C-Block Farm of Bidhan Chandra Krishi Viswavidyalaya, Kalyani, West Bengal, India during 2017–18 to screen eight brinjal germplasm lines against BW disease using tollinterleukin-1 receptors (TIR)-NBS-LRR type R-gene specific degenerate primer. The study showed that wild genotype S. torvum was highly resistant to bacterial wilt incidence with no wilting symptom whereas two cultivated genotypes (Utkal Anushree and Utkal Madhuri) and one wild genotype S. sisymbriifolium were found to be resistant to BW disease. Out of the 7 germplasm sequences, 2 had no match with R-genes whereas the remaining 5 sequences have 70-93% homology with R-genes of other plant species submitted in Gene Bank sequence database. Nearly 90% sequence identity of brinjal NBS-LRR RGA was found by analyzing through BLASTn with NBS-LRR RGAs of other solanaceous crops. Two cultivated resistant genotypes (Utkal Madhuri and Utkal Tarini) were similar to the wild resistant type S. sisymbriifloium, while cultivable resistant genotype Utkal Anushree was highly different at sequence level. Two cultivable susceptible genotypes (BCB-30 and Garia) showed high level of similarity among them and they were strongly associated with the wild susceptible genotype S. macrocarpum. Two cultivable genotypes Utkal Anushree and Utkal Madhuri could be utilized in future breeding programme and two wild genotypes S. torvum and S. sisymbriifolium could be used as resistant rootstocks in brinjal grafting.
... For all genes, except for the LRR genes, orthologs were present in syntenic positions in all three species analyzed (rice, sorghum, and foxtail millet). Disease resistance genes tend to not be evolutionary conserved (Leister et al., 1998), so the lack of orthology for LRR genes across species was not unexpected. More surprising was the fact that for only three of the eight NBS-LRR genes analyzed, both homoeologous gene copies were structurally intact. ...
Finger millet [Eleusine coracana (L.) Gaertn.] is a critical subsistence crop in eastern Africa and southern Asia but has few genomic resources and modern breeding programs. To aid in the understanding of finger millet genomic organization and genes underlying disease resistance and agronomically important traits, we generated a F2:3 population from a cross between E. coracana (L.) Gaertn. subsp. coracana accession ACC 100007 and E. coracana (L.) Gaertn. subsp. africana , accession GBK 030647. Phenotypic data on morphology, yield, and blast (Magnaporthe oryzae) resistance traits were taken on a subset of the F2:3 population in a Kenyan field trial. The F2:3 population was genotyped via genotyping‐by‐sequencing (GBS) and the UGbS‐Flex pipeline was used for sequence alignment, nucleotide polymorphism calling, and genetic map construction. An 18‐linkage‐group genetic map consisting of 5,422 markers was generated that enabled comparative genomic analyses with rice (Oryza sativa L.), foxtail millet [Setaria italica (L.) P. Beauv.], and sorghum [Sorghum bicolor (L.) Moench]. Notably, we identified conserved acrocentric homoeologous chromosomes (4A and 4B in finger millet) across all species. Significant quantitative trait loci (QTL) were discovered for flowering date, plant height, panicle number, and blast incidence and severity. Sixteen putative candidate genes that may underlie trait variation were identified. Seven LEUCINE‐RICH REPEAT‐CONTAINING PROTEIN genes, with homology to nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) disease resistance proteins, were found on three chromosomes under blast resistance QTL. This high‐marker‐density genetic map provides an important tool for plant breeding programs and identifies genomic regions and genes of critical interest for agronomic traits and blast resistance.
... For example, clustering occurs in the maize Rp1 (Bennetzen et al. 1994), the rice Xa21 (Song et al. 1997), the lettuce Dm3 (Farrara et al. 1987;Maisonneuve et al. 1994), the tomato Cf (Jones et al. 1994;Dixon et al. 1996;Thomas et al. 1997), I2C (Ori et al. 1997), and Pto (Martin et al. 1993), the flax M (Anderson et al. 1997), and the Arabidopsis RPP5 (Parker et al. 1997) loci. RGA sequences have also been shown to be clustered, often in close genetic linkage to known resistance genes (Kanazin et al. 1996;Yu et al. 1996;Leister et al. 1998). In tomato, Parniske et al. (1997) showed that R-gene analogues clustered at the Cf4/9 locus (Hcr9 genes) could discriminate between different Avr-genes of C. fulvum. ...
... In the Triticeae, it shows more rapid movement across the genome as well as rapid local rearrangements. Although the exact mechanisms of NLR-ID formation remain to be uncovered, we predict that it involves double-stranded DNA breaks and could be driven either Mla locus in barley(Leister et al., 1998), or alternatively by local activity of transposable elements and endogenous DNA repair machinery as has been previously documented for other types of gene duplications in cereals(Wicker, Buchmann and Keller, 2010). ...
Background
The plant immune system is innate, encoded in the germline. Using it efficiently, plants are capable of recognizing a diverse range of rapidly evolving pathogens. A recently described phenomenon shows that plant immune receptors are able to recognize pathogen effectors through the acquisition of exogenous protein domains from other plant genes.
Results
We showed that plant immune receptors with integrated domains are distributed unevenly across their phylogeny in grasses. Using phylogenetic analysis, we uncovered a major integration clade, whose members underwent repeated independent integration events producing diverse fusions. This clade is ancestral in grasses with members often found on syntenic chromosomes. Analyses of these fusion events revealed that homologous receptors can be fused to diverse domains. Furthermore, we discovered a 43 amino acids long motif that was associated with this dominant integration clade and was located immediately upstream of the fusion site. Sequence analysis revealed that DNA transposition and/or ectopic recombination are the most likely mechanisms of NLR-ID formation.
Conclusions
The identification of this subclass of plant immune receptors that is naturally adapted to new domain integration will inform biotechnological approaches for generating synthetic receptors with novel pathogen ‘baits’.
... Des RGA ont été cartographiés chez de nombreuses espèces. Dans la plupart des études, des RGA se cartographient dans des régions génomiques où des gènes ou des QTL de résistance ont déjà été localisés (Chen et al. 1998, Geffroy et al. 1998, Hamalainen et al. 1998, Kanazin et al. 1996, Leister et al. 1996, Leister et al. 1998, Pan et al. 2000, Pflieger et al. 1999 Le chromosome XII de la pomme de terre est représenté dans la même orientation que sur les cartes génétiques des autres Solanacées. Cette orientation est inversée par rapport à celle observée en cytologie (Dong et al. 2000). ...
* INRA Centre d'Avignon, Documentation, Domaine St Paul, Site Agroparc, 84914 Avignon cedex 9 Diffusion du document : INRA Centre d'Avignon, Documentation, Domaine St Paul, Site Agroparc, 84914 Avignon cedex 9 Diplôme : Dr. d'Université
... These authors detected closely linked multicopy RGA families in Cicer representing a cluster of tightly linked NBS-LRR genes. A similar pattern of genomic organization was described in species such as soybean, pea, rice or barley, with some loci tightly linked to the trait of interest (Leister et al. 1998). These results suggest that RGA05 may belong to the same NBS-LRR cluster, and opens the possibility to detect new members of the same family by tagging the genomic area. ...
... In contrast to these methods, a huge set of different sequencing methods has been established to identify DNA methylation ( Kurdyukov and Bullock, 2016) Furthermore, large chromosomal modifications might be CNVs including duplication and deletions, which were especially identified in gene clusters ( Boycheva et al., 2014). Exemplary, tandem and segmental duplications have been reported to be important for the distribution of genes involved in plant disease resistance ( Leister et al., 1998;Leister, 2004;Himmelbach et al., 2010) and are of increased interest for breeding. Lu et al. (2015) developed a first approach to map the presence/absence of GBS tags genetically and incorporated these into genome-wide association scans in maize. ...
Markers linked to agronomic traits are of the prerequisite for molecular breeding. Genotyping-by-sequencing (GBS) data enables to detect small polymorphisms including single nucleotide polymorphisms (SNPs) and short insertions or deletions (InDels) that can be used, for instance, for marker-assisted selection, population genetics, and genome-wide association studies (GWAS). Here, we aim at detecting large chromosomal modifications in barley and wheat based on GBS data. These modifications could be duplications, deletions, substitutions including introgressions as well as alterations of DNA methylation. We demonstrate that GBS coverage analysis is capable to detect Hordeum vulgare/Hordeum bulbosum introgression lines. Furthermore, we identify large chromosomal modifications in barley and wheat collections. Hence, large chromosomal modifications, including introgressions and copy number variations (CNV), can be detected easily and can be used as markers in research and breeding without additional wet-lab experiments.
... A possible explanation could be found in the organisation of resistance genes. It has been observed in different species that genes for resistance to pathogens are clustered in complex loci on the host genome [40,50,52,55]. The probe RPLJl and the resistance to anthracnose strains were mapped at such a cluster in a cross between two cultivars [31]. ...
... Colinearity, i.e. conserved gene order, is a fundamental feature of grass genomes (see 1.2). However, R genes are frequently not present at orthologous loci in different grass species (Leister et al., 1998). For example, although the Lr10 leaf rust R gene from wheat has significant similarity with the powdery mildew R genes Mla1, Mla6, and Mla13 from barley, the positions on the genetic maps of the homeologous chromosomes 1 does not suggest orthology between Lr10 and the barley genes . ...
... Comparative genomic studies have shown that although segmental duplication drives the formation of clusters of closely related genes, duplicated sequences can translocate to different chromosomal locations, resulting in dispersal of paralogs throughout the genome (Freeling et al., 2008;Lai et al., 2004;Mendivil Ramos and Ferrier, 2012). For example, the plant disease resistance NBS-LRR genes are particularly prone to being transposed (Ameline-Torregrosa et al., 2008;Baumgarten et al., 2003;Leister et al., 1998;Richly et al., 2002), vividly attesting to segmental duplication and translocation occurring in plants. This natural rearrangement of DNA fragments occurs spontaneously at a low frequency in maize and has been exploited by breeders over the decades for Table 1 Grain yield of ARGOS8 genome-edited variants and wild type under flowering stress, grain-filling stress and optimal (wellwatered) conditions. ...
Summary
Maize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought-tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR-Cas-enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 50
-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought-tolerant crops.
... Cortez on 1BS using microsatellites and in both cases the alleles at the linked marker loci occur in both resistant and susceptible varieties. R-gene clusters have been shown to rapidly evolve in cereals (Leister et al., 1998) and are frequently found in regions of high recombination (e.g. near the telomeres), which accounts for the low level of LD. ...
Papers from BSPP Presidential Meeting,
Queen Mary, University of London
16-17 December 2008
http://www.bspp.org.uk/archives/bspp2008/docs/bspppres2008papers.pdf
... We considered three possible mechanisms of NLR-ID formation: (1) retrotransposition of complementary DNA derived from the parental gene; (2) transposition of the parental gene; and (3) ectopic recombination during which double-stranded DNA breaks are repaired using a non-homologous exogenous parental gene as a template. All three mechanisms have been observed previously in cereal genomes [30] and both retrotransposition and ectopic recombination have been suggested as diversification mechanisms of NLRs [31]. We extracted the coding DNA sequences of IDs for 40 T. aestivum NLR-ID genes from MIC1 and aligned them back to the genome (BLASTN, e-value 1e-3). ...
Background
The plant immune system is innate and encoded in the germline. Using it efficiently, plants are capable of recognizing a diverse range of rapidly evolving pathogens. A recently described phenomenon shows that plant immune receptors are able to recognize pathogen effectors through the acquisition of exogenous protein domains from other plant genes. ResultsWe show that plant immune receptors with integrated domains are distributed unevenly across their phylogeny in grasses. Using phylogenetic analysis, we uncover a major integration clade, whose members underwent repeated independent integration events producing diverse fusions. This clade is ancestral in grasses with members often found on syntenic chromosomes. Analyses of these fusion events reveals that homologous receptors can be fused to diverse domains. Furthermore, we discover a 43 amino acid long motif associated with this dominant integration clade which is located immediately upstream of the fusion site. Sequence analysis reveals that DNA transposition and/or ectopic recombination are the most likely mechanisms of formation for nucleotide binding leucine rich repeat proteins with integrated domains. Conclusions
The identification of this subclass of plant immune receptors that is naturally adapted to new domain integration will inform biotechnological approaches for generating synthetic receptors with novel pathogen “baits.”
... Sequence and structural variation such as deletion, insertion, duplication, inversion and translocation underlies the phenotypic characteristics of every species (Sorrells et al. 2003;Bruggmann et al. 2006). Previous studies have also shown that genes with economic benefits are usually located in non-collinear regions (Gallego et al. 1998;Leister et al. 1998;Brown et al. 2003;Klein et al. 2005;Hu et al. 2012;Matsuhira et al. 2012). Therefore, we attempted to use a map-based cloning strategy to obtain the target gene after genetic mapping of N23601 by constructing a 13.8-fold bacterial artificial chromosome (BAC) library of the entire genome. ...
This study pioneered the use of multiple technologies to combine the bacterial artificial chromosome (BAC) pooling strategy with high-throughput next- and third-generation sequencing technologies to analyse genomic difference. To understand the genetic background of the Chinese soybean cultivar N23601, we built a BAC library and sequenced 10 randomly selected clones followed by de novo assembly. Comparative analysis was conducted against the reference genome of Glycine max var. Williams 82 (2.0). Therefore, our result is an assessment of the reference genome. Our results revealed that 3517 single nucleotide polymorphisms (SNPs) and 662 insertion-deletions (InDels) occurred in ∼1.2 Mb of the genomic region and that four of the 10 BAC clones contained 15 large structural variations (72887bp) compared with the reference genome. Gene annotation of the reference genome showed that Glyma.18g181000 was missing from the corresponding position of the 10 BAC clones. Additionally, there may be a problem with the assembly of some positions of the reference genome. Several gap regions in the reference genome could be supplemented by using the complete sequence of the 10 BAC clones. We believe that accurate and complete BAC sequence is a valuable resource that contributes to the completeness of the reference genome.
... One solution is to use degenerate primers to target and amplify the sequences encoding conserved NBS domains of NBS-containing maize R genes. This method has been used to clone the NBS-containing R genes from soybean (Glycine max) (Kanazin et al. 1996;Yu et al. 1996;Penuela et al. 2002), potato (Solanum tuberosum) (Leister et al. 1996), lettuce (Lactuca sp.) (Shen et al. 1998), bean (Vicia sp.) (Creusot et al. 1999;Rivkin et al. 1999), the Arabidopsis (Aarts et al. 1998), maize (Collins et al. 1998), rice (Oryza sativa) (Yuan et al. 2011), barley (Hordeum vulgare) (Leister et al. 1998), and other angiosperms. The present study builds on earlier work in which we identified a putative NBS-LRR gene that was expressed differentially in different maize lines under artificial inoculation of the fungus Sporisorium reilianum (Kühn) Clint. ...
The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family comprises the largest number of known disease resistance (R) genes and is one of the largest gene families in plants. In the present study, the full-length cDNA of ZmNL (GenBank Accession Number KF765443) was isolated using Rapid Amplification of cDNA Ends. The nucleotide sequence of ZmNL contains an open reading frame of 3156 bp that encodes the ZmNL protein, which is comprised of 1051 amino acid residues. This putative protein has high homology to other known resistance proteins (84% to Triticum aestivum LR10) and belongs to the CC–NBS–LRR type R gene family. The ZmNL gene was introduced into the maize inbred line of Huangzao4 which was highly susceptible to head smut under the control of the maize ubiquitin promoter by Agrobacterium-mediated transformation. The head smut disease incidence of 3 T2 transgenic lines was significantly reduced (by 18.38–29.40%) compared with the wild type, which indicated that the overexpression of ZmNL gene in maize enhanced the resistance to the fungus Sporisorium reilianum (Kühn) Clint of these plants.
... Gene ontology term enrichment analysis of the 672 genes located within CNV regions in soybean revealed that genes related to disease resistance response were significantly over-represented (McHale et al. 2012). In addition, it has been reported that resistance gene function is adapted to frequent re-arrangements and copy number variations (Leister et al. 1998). Copy number variation of a 31-kb repeat segment observed in different haplotypes of the Rhg1 locus encodes multiple gene products in soybean cyst nematode (SCN)-resistant varieties . ...
Plant genome diversity varies from single nucleotide polymorphisms to large-scale deletions, insertions, duplications, or re-arrangements. These re-arrangements of sequences resulting from duplication, gains or losses of DNA segments are termed copy number variations (CNVs). During the last decade, numerous studies have emphasized the importance of CNVs as a factor affecting human phenotype; in particular, CNVs have been associated with risks for several severe diseases. In plants, the exploration of the extent and role of CNVs in resistance against pathogens and pests is just beginning. Since CNVs are likely to be associated with disease resistance in plants, an understanding of the distribution of CNVs could assist in the identification of novel plant disease-resistance genes. In this paper, we review existing information about CNVs; their importance, role and function, as well as their association with disease resistance in plants.
... The conserved motifs within these NBS domains make it possible to use degenerate primers and PCR to isolate RGAs (Kanazin et al. 1996;Leister et al. 1998). These RGAs have the potential application in development of closely linked markers or functional resistant gene markers for marker assisted breeding (Hammond-Kosack and Jones 1997). ...
Viruses are serious threat to chilli crop production worldwide. Resistance screening against several viruses resulted in identifying a multiple virus resistant genotype ‘IHR 2451’. Degenerate primers based on the conserved regions between P-Loop and GLPL of Resistance genes (R-genes) were used to amplify nucleotide binding sites (NBS)—encoding regions from genotype ‘IHR 2451’. Alignment of deduced amino acid sequences and phylogenetic analyses of isolated sequences distinguished into two groups representing toll interleukin-1 receptor (TIR) and non-TIR, and different families within the group confirming the hypotheses that dicots have both the types of NBS-LRR genes. The alignment of deduced amino acid sequences revealed conservation of subdomains P-loop, RNBS-A, kinase2, RNBS-B, and GLPL. The distinctive five RGAs showing specific conserved motifs were subjected to BLASTp and indicated high homology at deduced amino acid level with R genes identified such as Pvr9 gene for potyvirus resistance, putative late blight resistance protein homolog R1B-23 and other disease resistance genes suggesting high correlation with resistance to different pathogens. These pepper RGAs could be regarded as candidate sequences of resistant genes for marker development.
... Such enrichment in the accessory part of the genome has been reported in other crops, such as Brassica oleracea (Golicz et al., 2016) or soybean (Mchale et al., 2012). This can be related to the pattern of evolution of this protein family, presenting specific mechanisms to generate diversity and elevated rates of non-synonymous substitutions (Leister et al., 1998;Michelmore and Meyers, 1998), as NBS-LRR genes are an important part of the immune system of plants (Jones and Dangl, 2006). In wheat, the domestication process caused loss of NBS-LRR ancestor genes, ensued by gene gain events through gene duplication and diversification, to keep up with pathogen evolution (Gu et al., 2015), resulting in large differences in NBS-LRR content between accessions. ...
The pan-genome of a species is defined as the union of all the genes and non-coding sequences found in all its individuals. However, constructing a pan-genome for plants with large genomes is daunting both in sequencing cost and the scale of the required computational analysis. A more affordable alternative is to focus on the genic repertoire by using transcriptomic data. Here, the software GET_HOMOLOGUES-EST was benchmarked with genomic and RNA-seq data of 19 Arabidopsis thaliana ecotypes and then applied to the analysis of transcripts from 16 Hordeum vulgare genotypes. The goal was to sample their pan-genomes and classify sequences as core, if detected in all accessions, or accessory, when absent in some of them. The resulting sequence clusters were used to simulate pan-genome growth, and to compile Average Nucleotide Identity matrices that summarize intra-species variation. Although transcripts were found to under-estimate pan-genome size by at least 10%, we concluded that clusters of expressed sequences can recapitulate phylogeny and reproduce two properties observed in A. thaliana gene models: accessory loci show lower expression and higher non-synonymous substitution rates than core genes. Finally, accessory sequences were observed to preferentially encode transposon components in both species, plus disease resistance genes in cultivated barleys, and a variety of protein domains from other families that appear frequently associated with presence/absence variation in the literature. These results demonstrate that pan-genome analyses are useful to explore germplasm diversity.
... The largest class of R genes encode proteins that contain a nucleotide-binding site plus leucine-rich repeat domains (NBS-LRR proteins) (Dangl and Jones 2001;Meyers et al. 2003;Howles et al. 2005). By making use of the conserved domains, it has been possible to design degenerate oligonucleotide primers that permit the amplification of similar regions from the genomes of diverse plant species by polymerase chain reaction (PCR) (Kanazin et al. 1996;Aarts et al. 1998;Leister et al. 1998). This strategy provides a powerful tool which facilitates isolation of candidate R genes. ...
... and Ferrier, 2012). For example, the plant disease resistance NBS-LRR genes are particularly prone to being transposed (Ameline-Torregrosa et al., 2008;Baumgarten et al., 2003;Leister et al., 1998;Richly et al., 2002), vividly attesting to segmental duplication and translocation occurring in plants. This natural rearrangement of DNA fragments occurs spontaneously at a low frequency in maize and has been exploited by breeders over the decades for maize improvement. ...
Maize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought tolerance breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR-Cas enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5'-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by 5 bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought tolerant crops. This article is protected by copyright. All rights reserved.
... The structures of R-genes are highly diverse according to comparative genomic analyses in vertebrates and plants. Evolutionary studies suggested R-gene families as some of the most plastic families in plants, which were associated with intense structural shuffling leading to synteny erosion [23]. What is more, tandem and segmental duplications are thought to contribute to the structural plasticity of NBS-LRR domains in different plant genomes [24,25]. ...
Background
Legumes are the second-most important crop family in agriculture for its economic and nutritional values. Disease resistance (R-) genes play an important role in responding to pathogen infections in plants. To further increase the yield of legume crops, we need a comprehensive understanding of the evolution of R-genes in the legume family.
Results
In this study, we developed a robust pipeline and identified a total of 4,217 R-genes in the genomes of seven sequenced legume species. A dramatic diversity of R-genes with structural variances indicated a rapid birth-and-death rate during the R-gene evolution in legumes. The number of R-genes transiently expanded and then quickly contracted after whole-genome duplications, which meant that R-genes were sensitive to subsequent diploidization. R proteins with the Coiled-coil (CC) domain are more conserved than others in legumes. Meanwhile, other types of legume R proteins with only one or two typical domains were subjected to higher rates of loss during evolution. Although R-genes evolved quickly in legumes, they tended to undergo purifying selection instead of positive selection during evolution. In addition, domestication events in some legume species preferentially selected for the genes directly involved in the plant-pathogen interaction pathway while suppressing those R-genes with low occurrence rates.
Conclusions
Our results provide insights into the dynamic evolution of R-genes in the legume family, which will be valuable for facilitating genetic improvements in the disease resistance of legume cultivars.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2736-9) contains supplementary material, which is available to authorized users.
... In that way, resistance gene analogs (RGA) have been isolated from dicot (Kanazin et al. 1996, Leister et al. 1996, Yu et al. 1996, Gentzbittel et al. 1998, Shen et al. 1998, Pflieger et al. 2000) and monocot species (Chen et al. 1998, Collins et al. 1998, Leister et al. 1998). Specific PCR primers derived from conserved regions of an NBS-LRR sequence at the Cre3 cereal cyst nematode resistance locus in Triticum tauschii L. and other known R genes (Grant et al. 1995) have been applied to isolate resistance gene-like sequences in wheat and barley ...
A higher plant contains a minimum of 20,000 genes (Kaul et al. 2000). A successful new variety — the end product of the function of genetic material — is never the result of the addition of just one gene but rather a better combination of several genes. Thus, the challenge in plant breeding is the optimal combination of many genes. Good luck and the “green thumb” of the breeder, are still important prerequisites for successful plant breeding. The question is whether increased knowledge of the function of genetic material will help in offering reliable tools for optimal combinations of the [(n+1) · n]k alleles (n=number of alleles per k loci) in a better genome (Rommens and Kishore 2000).
The presence of various micro-organisms causing diseases poses a significant threat to crop cultivation, leading to reduced economic yields. Plant ailments can lead to yield losses of around 40% or even more, implying that only about 60% of the crop's economic potential is realized, with the rest compromised by diseases. Researchers have studied the molecular cloning of R genes, which confer resistance to a wide array of pathogens, and have discovered common characteristics among the proteins encoded by these genes. Breeding crops for resistance is seen as the most efficient and practical method of control, as it avoids additional costs and is environmentally friendly. In the pursuit of disease-resistant genes, molecular techniques have proven to be highly effective, requiring less time and effort for identification, isolation, cloning, and transfer of these genes between different species. Molecular geneticists are now emphasizing the mapping and tagging of disease resistance genes, intending to share this information within the global scientific community.
Albugo candida, the causal organism of white rust, is an oomycete obligate pathogen infecting crops of Brassicaceae family occurred on aerial part, including vegetable and oilseed crops at all growth stages. The disease expression is characterized by local infection appearing on the abaxial region developing white or creamy yellow blister (sori) on leaves and systemic infections cause hypertrophy and hyperplasia leading to stag-head of reproductive organ. To overcome this problem, several disease management strategies like fungicide treatments were used in the field and disease-resistant varieties have also been developed using conventional and molecular breeding. Due to high variability among A. candida isolates, there is no single approach available to understand the diverse spectrum of disease symptoms. In absence of resistance sources against pathogen, repetitive cultivation of genetically-similar varieties locally tends to attract oomycete pathogen causing heavy yield losses. In the present review, a deep insight into the underlying role of the non-host resistance (NHR) defence mechanism available in plants, and the strategies to exploit available gene pools from plant species that are non-host to A. candida could serve as novel sources of resistance. This work summaries the current knowledge pertaining to the resistance sources available in non-host germ plasm, the understanding of defence mechanisms and the advance strategies covers molecular, biochemical and nature-based solutions in protecting Brassica crops from white rust disease.
In plant-pathogen interactions, signal activation and transduction confer resistance in plants against various pathogens. Communication between host and pathogen is the prime step for a pathogen to cause infection. The molecular basis of pathogen response in plants depends on the pathogen types. Hypersensitive reactions usually result from Avr-R interactions that restrict pathogens’ development through cell death. These avr genes can be recognized directly and indirectly by the resistance (R) gene. The NBS-LRR family is an important resistance gene (R gene) family in plants, which is divided into subclasses. Resistant gene analogues (RGAs) are candidates for R genes that have a significant role in defense response against disease-causing pathogens and are classified into two classes. The first class is based on the immediate recognition of a pathogen called resistance genes (R genes), while the second class is based on the defense response generated by recognition events. Hence, this chapter attempts to delineate a comprehensive overview of resistance genes, their classes, identification, and characterization in plants.KeywordsDisease resistanceR genesResistance genes analogsPlant-pathogen interaction
Wheat (Triticum aestivum L.) is a staple crop, contributing about a fifth of total calories intake by humans. The objective of worldwide wheat breeding program is to meet the global food demand for projected 9.8 billion populations by 2050. Therefore breeders need to accelerate the genetic gain, increase production, enhance protection, and improve quality traits. The completion of wheat-genome sequencing project and availability of the well-annotated wheat reference genome with advanced comparative and structural bioinformatics tools widens the scope of understanding of wheat biology and the molecular basis of important agronomic traits. Wheat has A, B, and D homologous subgenome, each having seven chromosomes encoding 107,891 high-confidence genes. The comparative analysis of wheat genome with rice, maize, sorghum, barley, Brachypodium, and two diploid wheat progenitor species genome revealed blocks of orthologous and paralogous genes consisting of approximately 22,000–28,000 orthologous genes and about 6000–18,000 paralogous genes. The agronomically important genes of rice, barley, or maize breeding program could be prioritized based on synteny and homologous block study on the specific wheat chromosomes. The functional characterization of these blocks is attained through marker–trait association studies. Advancements in high-throughput sequence-based genotyping, field phenotyping, and genome-wide association studies provide information about the regions of interest on wheat chromosomes, referred to as genomic hotspots. The physical position of the gene sequences and the position of markers flanking such regions are also important in pyramiding of genes. In this chapter, we discussed the wheat hotspots for recent agronomic, stress tolerance, and wheat quality traits. We also discussed the pipelines, tools, and web resources used for identification and analysis of homologous blocks and hotspot regions in wheat that might be useful for students and early career wheat researchers.
The sections in this article are
Introduction
Features of Cloned Resistance Genes
Control of Resistance Gene Specificity
Do Resistance Proteins Interact Directly with Avirulence Determinants?
Organisation of Resistance Gene Loci
Evolution of Resistance Genes by Divergent Selection
Evolution of Resistance Genes by Recombination
Concluding Remarks
Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is a causal pathogen of sugarcane mosaic disease, and it is widespread in regions where sugarcane (Saccharum spp. hybrids) is grown. It is difficult to investigate the molecular mechanism of pathogen infection in sugarcane because of limited genomic information. Here, we demonstrated that SCMV strain FZ1 can systemically infect Brachypodium distachyon inbred line Bd21 and Nicotiana benthamiana through inoculation, double antibody sandwich enzyme-linked immunosorbent, transmission electron microscopy, and reverse transcription PCR assays. The leaves of Bd21 developed mosaic symptoms, while the leaves of N. benthamiana showed no obvious symptoms under the challenge of SCMV-FZ1. We concluded that B. distachyon inbred line Bd21 is a promising experimental model plant compared with N. benthamiana for study on the infectivity of SCMV. This is the first report on the SCMV infection of model plants B. distachyon inbred line Bd21 and N. benthamiana, which will shed light on the mechanism of SCMV infection of sugarcane and benefit sugarcane breeding against sugarcane mosaic disease.
Recent years have seen a surge in plant genome sequencing projects and the comparison of multiple related individuals. The high degree of genomic variation observed led to the realization that single reference genomes do not represent the diversity within a species, and led to the expansion of the pan-genome concept. Pan-genomes represent the genomic diversity of a species and includes core genes, found in all individuals, as well as variable genes, which are absent in some individuals. Variable gene annotations often show similarities across plant species, with genes for biotic and abiotic stress commonly enriched within variable gene groups. Here we review the growth of pan-genomics in plants, explore the origins of gene presence and absence variation, and show how pan-genomes can support plant breeding and evolution studies. This Review summarizes the growth of plant pan-genome studies, explore the origins of gene presence and absence variation, and introduces the impacts of pan-genomes on plant biology, breeding and evolutionary studies.
In order to meet global food security demands in the next decades, considerable changes are required for sustainable agriculture in the context of plant disease, with sufficient food production depending on the development of durable genetically disease resistant crops. For this, further advances are required in our understanding of the plant innate immune system and how plants respond to invading pathogenic micro-organisms. Over the past 20 years, considerable research has been conducted into the characterization and cloning of plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors. These intracellular receptors can recognize directly or indirectly pathogen effector proteins, resulting in effector-triggered immunity (ETI). Elucidation, however, of the diversity of NLR resistance gene families and the molecular basis of NLR-driven effector recognition and defense signaling is incomplete. Here, we present a summary of the understanding of NLR structure, function, genomic organization and diversity in plants. Recent advances in target enrichment approaches for NLR characterization and function validation are highlighted in the context of NLR engineering possibilities for accelerated durable genetic resistance to biotic stresses.
Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.
Beet cyst nematode (Heterodera schachtii Schm.) is one of the most damaging pests in sugar beet growing areas around the world. The Hs1pro-1 and cZR3 genes confer resistance to the beet cyst nematode, and both were cloned from sugar beet translocation line (A906001). The translocation line carried the locus from B. procumbens chromosome 1 including Hs1pro-1 gene and resistance gene analogs (RGA), which confer resistance to Heterodera schachtii. In this research, BvHs1pro-1 and BvcZR3 genes were transferred into oilseed rape to obtain different transgenic lines by A. tumefaciens mediated transformation method. The cZR3Hs1pro-1 gene was pyramided into the same plants by crossing homozygous cZR3 and Hs1pro-1 plants to identify the function and interaction of cZR3 and Hs1pro-1 genes. In vitro and in vivo cyst nematode resistance tests showed that cZR3 and Hs1pro-1 plants could be infested by beet cyst nematode (BCN) juveniles, however a large fraction of penetrated nematode juveniles was not able to develop normally and stagnated in roots of transgenic plants, consequently resulting in a significant reduction in the number of developed nematode females. A higher efficiency in inhibition of nematode females was observed in plants expressing pyramiding genes than in those only expressing a single gene. Molecular analysis demonstrated that BvHs1pro-1 and BvcZR3 gene expressions in oilseed rape constitutively activated transcription of plant-defense related genes such as NPR1 (non-expresser of PR1), SGT1b (enhanced disease resistance 1) and RAR1 (suppressor of the G2 allele of skp1). Transcript of NPR1 gene in transgenic cZR3 and Hs1pro-1 plants were slightly up-regulated, while its expression was considerably enhanced in cZR3Hs1pro-1 gene pyramiding plants. The expression of EDS1 gene did not change significantly among transgenic cZR3, Hs1pro-1 and cZR3Hs1pro-1 gene pyramiding plants and wild type. The expression of SGT1b gene was slightly up-regulated in transgenic cZR3 and Hs1pro-1 plants compared with the wild type, however, its expression was not changed in cZR3Hs1pro-1 gene pyramiding plant and had no interaction effect. RAR1 gene expression was significantly up-regulated in transgenic cZR3 and cZR3Hs1pro-1 genes pyramiding plants, but almost no expression was found in Hs1pro-1 transgenic plants. These results show that nematode resistance genes from sugar beet were functional in oilseed rape and conferred BCN resistance by activation of a CC-NBS-LRR R gene mediated resistance response. The gene pyramiding had enhanced resistance, thus offering a novel approach for the BCN control by preventing the propagation of BCN in oilseed rape. The transgenic oilseed rape could be used as a trap crop to offer an alternative method for beet cyst nematode control.
As a resource for structural and functional barley genome analysis, more than 140 000 ESTs (expressed sequence tags) were generated from 22 cDNA libraries that yielded 25 224 tentative unigenes. About 50% of them belong to gene families. The size of the complete transcriptome is estimated to comprise between 35 000 and 75 000 genes. The barley EST collection is a rich source for the development of novel markers including SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms). Several bioinformatic tools have been developed facilitating the computer-assisted analysis of EST databases for the presence of either SNPs or SSRs and the development of SNP-derived CAPS (cleaved amplified polymorphic sequences) markers. In an attempt to systematically map barley genes a high-density transcript map is under construction and presently comprises more than 1000 markers. This map is a gateway to comparative genomics with particular emphasis on the rice genome. 65% of the mapped ESTs showing a significant homology to rice ESTs were found to display a syntenic relationship between barley and rice. Thus, the barley EST resource facilitates the rapid and systematic transfer of genetic information from rice to barley and other Triticeae, which can readily be exploited for marker saturation of defined chromosome regions and their detailed comparison to rice. In the context of a functional genomics study, the complex trait “malting quality” is investigated using a barley cDNA array. By correlating the phenotypic malting trait data of selected barley lines with the corresponding expression profiles, a set of candidate genes was identified and further verified by genetic analysis.
A novel approach has been developed to allow for the efficient selection of loss-of-function wheat mutants in the M 1 generation, following either physical or chemical mutagenesis. This has generated an order of magnitude increase in the efficiency of identification of mutants, and also greatly increases the likelihood that selected individuals reflect mutation events at the target locus, rather than at genes acting elsewhere in the disease resistance pathway. The approach relies only on prior knowledge of the chromosomal location of the target gene, and uses the polyploidy of wheat to construct populations for mutagenesis in which large numbers of individuals are hemizygous for the target gene. The idea is illustrated with the mass identification of mutants at three independent genes for race-specific resistance to yellow rust, and one gene for resistance to powdery mildew.Key words: disease resistance mutant, hemizygotes, loss-of-function mutant.
Plant innate immunity relies on genetically predetermined repertoires of immune receptors to detect pathogens and trigger an effective immune response. A large proportion of these receptors are from the Nucletoide Binding Leucine Rich Repeat (NLR) gene family. As plants live longer than most pathogens, maintaining diversity of NLRs and deploying efficient ‘pathogen traps’ is necessary to withstand the evolutionary battle. In this review, we summarize the sources of diversity in NLR plant immune receptors giving an overview of genomic, regulatory as well as functional studies, including the latest concepts of NLR helpers and NLRs with integrated domains.
Advances made over the last two decades in techniques and approaches used in molecular cytogenetics research and the results obtained so far have been briefly reviewed. These include the use of chromosome banding, fluorescence in situ hybridisation (FISH) and multicolour FISH (McFISH), genomic in situ hybridisation (GISH), flow cytometry, pulse field gel electrophoresis (PFGE), microdissection and microcloning, among others. These tools have been used both for identification of individual chromosomes and for physical localization of DNA sequences on individual chromosomes. Construction of genetic and physical maps of crop plants involving molecular markers as well as genes for agronomic traits has been discussed briefly. The use of these maps in comparative genomics, both at the macro- and micro-colinearity levels and for map based cloning has been briefly reviewed. The development and use of bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and the future prospects of the development of plant artificial chromosomes (PACs) have also been discussed. The progress made in discovery of new genes through reverse genetics and functional genomics approach has also been presented.
Recent studies have indicated that many plant genomes have regions of common gene content and genetic map colinearity. This is particularly well documented in the grasses, where colinearity has been demonstrated both for recombinational maps using DNA markers and in the orthologous placement of comparable genes. Colinearity in regions of a centiMorgan or less (i.e., microcolinearity) has not been extensively investigated. Preliminary studies demonstrate that gene content, order and orientation are often conserved in small regions of related genomes, but there are frequent exceptions. Results indicate that grass genome colinearity will be a tremendously valuable tool that allows the synergistic pooling of knowledge and biological materials across a broad range of plant species. From this colinear perspective, genes from any grass species can be used for the improvement and understanding of any other grass. However, further investigations across a wide range of species and genomic locations will be needed to determine the limitations of the approach. In the interim, it is important to carefully investigate (rather than assume) the microcolinearity of the syntenic regions that are being analyzed.
With the rediscovery of Mendel’s laws in 1900, radical changes took place in plant breeding methods. The new science of genetics contributed new concepts: the gene, the role of chromosomes as gene carriers, gene linkage, the Mendelian basis of continuous variation, heterosis, maternal inheritance, experimental mutagenesis, polyploidy and gene-enzyme relationships. These scientific discoveries rapidly permeated breeding theory, to the point that plant breeding became synonymous with applied genetics. It should, nevertheless, be emphasized that the science of plant breeding has received and continues to assimilate relevant contributions from cytology, systematics, physiology, pathology, entomology, chemistry, statistics, and, more recently, from molecular biology.
We have constructed a high resolution rice genetic map containing 1,383 DNA markers at an average interval of 300 kilobases (kb). The markers, distributed along 1,575 cM on 12 linkage groups, comprise 883 cDNAs, 265 genomic DNAs, 147 randomly amplified polymorphic DNAs (RAPD) and 88 other DNAs. cDNAs were derived from rice root and callus, analysed by single-run sequencing and searched for similarities with known proteins. Nearly 260 rice genes are newly identified and mapped, and genomic DNA and cloned RAPD fragments were also sequenced to generate STSs. Our map is the first significant gene expression map in plants. It is also the densest genetic map available in plants and the first to be backed up comprehensively by clone sequence data.
The classical genetic map and molecular map of rice chromosome 11 were oriented to facilitate the use of these maps for genetic studies and rice improvement. Three morphological markers (d-27, z-2, and la) were crossed to a rice breeding line, IRBB21, which has the Xa-21 gene for bacterial blight resistance. Three F2 populations were analyzed with RFLP markers known to be located on chromosome 11. Segregation analysis of molecular markers and morphological markers was used to construct an RFLP map for each population. The recombination frequency between markers varied from population to population although the marker order on the maps was the same for all three populations. Based on a common set of markers mapped in the three populations, an integrated map was generated consisting of both RFLP and morphological markers. The genetic distance between markers on this map was determined by taking a weighted average of the data from the three populations. The oriented map serves as a bridge to understand the relationship between the classical and molecular linkage maps. Based on this information, the location of several genes on the classical map can be approximated with respect to RFLP markers without having to map them directly.
The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich
repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and
subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.
We describe here a protocol for obtaining clones containing sequences present in low copy-number from genomic DNA where moderately
and highly repeated sequences predominate. Specific chromosomal regions can be targeted by using deletion or addition line
material. We have used this protocol to identify a sequence which has been deleted in both the tetraploid and hexaploid wheat
mutants for the homoeologous chromosome pairing locus.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing
unique corresponding avirulence (avr) genes. The molecular basis of this gene-for-gene specificity is unknown. The Arabidopsis
thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes.
Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes.
Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.
Plant breeders have used disease resistance genes (R genes) to control plant disease since the turn of the century. Molecular
cloning of R genes that enable plants to resist a diverse range of pathogens has revealed that the proteins encoded by these
genes have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms
for the expression of resistance to a wide range of unrelated pathogens. Characterization of the molecular signals involved
in pathogen recognition and of the molecular events that specify the expression of resistance may lead to novel strategies
for plant disease control.
A molecular map has been constructed for the rice genome comprised of 726 markers (mainly restriction fragment length polymorphisms; RFLPs). The mapping population was derived from a backcross between cultivated rice, Oryza sativa, and its wild African relative, Oryza longistaminata. The very high level of polymorphism between these species, combined with the use of polymerase chain reaction-amplified cDNA libraries, contributed to mapping efficiency. A subset of the probes used in this study was previously used to construct an RFLP map derived from an inter subspecific cross, providing a basis for comparison of the two maps and of the relative mapping efficiencies in the two crosses. In addition to the previously described PstI genomic rice library, three cDNA libraries from rice (Oryza), oat (Avena) and barley (Hordeum) were used in this mapping project. Levels of polymorphism detected by each and the frequency of identifying heterologous sequences for use in rice mapping are discussed. Though strong reproductive barriers isolate O. sativa from O. longistaminata, the percentage of markers showing distorted segregation in this backcross population was not significantly different than that observed in an intraspecific F2 population previously used for mapping. The map contains 1491 cM with an average interval size of 4.0 cM on the framework map, and 2.0 cM overall. A total of 238 markers from the previously described PstI genomic rice library, 250 markers from a cDNA library of rice (Oryza), 112 cDNA markers from oat (Avena), and 20 cDNA markers from a barley (Hordeum) library, two genomic clones from maize (Zea), 11 microsatellite markers, three telomere markers, eleven isozymes, 26 cloned genes, six RAPD, and 47 mutant phenotypes were used in this mapping project. Applications of a molecular map for plant improvement are discussed.
Moroberekan, a japonica rice cultivar with durable resistance to blast disease in Asia, was crossed to the highly susceptible indica cultivar, CO39, and 281 F7 recombinant inbred (RI) lines were produced by single seed descent. The population was evaluated for blast resistance in the greenhouse and the field, and was analyzed with 127 restriction fragment length polymorphism (RFLP) markers. Two dominant loci associated with qualitative resistance to five isolates of the fungus were tentatively named Pi-5(t) and Pi-7(t). They were mapped on chromosomes 4 and 11, respectively. To identify quantitative trait loci (QTLs) affecting partial resistance, RI lines were inoculated with isolate PO6-6 of Pyricularia oryzae in polycyclic tests. Ten chromosomal segments were found to be associated with effects on lesion number (P < 0.0001 and LOD > 6.0). Three of the markers associated with QTLs for partial resistance had been reported to be linked to complete blast resistance in previous studies. QTLs identified in greenhouse tests were good predictors of blast resistance at two field sites. This study illustrates the usefulness of RI lines for mapping a complex trait such as blast resistance and suggests that durable resistance in the traditional variety, Moroberekan, involves a complex of genes associated with both partial and complete resistance.
The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing
indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered,
with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more
than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout
the gene-containing regions of the maize genome.
The tobacco N and Arabidopsis RPS2 genes, among several recently cloned disease-resistance genes, share highly conserved structure, a nucleotide-binding site (NBS). Using degenerate oligonucleotide primers for the NBS region of N and RPS2, we have amplified and cloned the NBS sequences from soybean. Each of these PCR-derived NBS clones detected low-or moderate-copy soybean DNA sequences and belongs to 1 of 11 different classes. Sequence analysis showed that all PCR clones encode three motifs (P-loop, kinase-2, and kinase-3a) of NBS nearly identical to those in N and RPS2. The intervening region between P-loop and kinase-3a of the 11 classes has high (26% average) amino acid sequence similarity to the N gene although not as high (19% average) to RPS2. These 11 classes represent a superfamily of NBS-containing soybean genes that are homologous to N and RPS2. Each class or subfamily was assessed for its positional association with known soybean disease-resistance genes through near-isogenic line assays, followed by linkage analysis in F2 populations using restriction fragment length polymorphisms. Five of the 11 subfamilies have thus far been mapped to the vicinity of known soybean genes for resistance to potyviruses (Rsv1 and Rpv), Phytophthora root rot (Rps1, Rps2, and Rps3), and powdery mildew (rmd). The conserved N- or RPS2-homologous NBS sequences and their positional associations with mapped soybean-resistance genes suggest that a number of the soybean disease-resistance genes may belong to this superfamily. The candidate subfamilies of NBS-containing genes identified by genetic mapping should greatly facilitate the molecular cloning of disease-resistance genes.
Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.
Analysis of viral and bacterial pathogenesis has revealed common themes in the ways in which plants and animals respond to pathogenic agents. Pathogenic bacteria use macromolecule delivery systems (types III and IV) to deliver microbial avirulence proteins and transfer DNA-protein complexes directly into plant cells. The molecular events that constitute critical steps of plant-pathogen interactions seem to involve ligand-receptor mechanisms for pathogen recognition and the induction of signal transduction pathways in the plant that lead to defense responses. Unraveling the molecular basis of disease resistance pathways has laid a foundation for the rational design of crop protection strategies.
Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve novel configurations and potentially new disease resistance specificities.
In many interactions between plants and their pathogens, resistance to infection is specified by plant resistance (R) genes and corresponding pathogen avirulence (Avr) genes. In tomato, the Cf-4 and Cf-9 resistance genes map to the same location but confer resistance to Cladosporium fulvum through recognition of different avirulence determinants (AVR4 and AVR9) by a molecular mechanism that has yet to be determined. Here, we describe the cloning and characterization of Cf-4, which also encodes a membrane-anchored extracellular glycoprotein. Cf-4 contains 25 leucine-rich repeats, which is two fewer than Cf-9. The proteins have > 91% identical amino acids. DNA sequence comparison suggests that Cf-4 and Cf-9 are derived from a common progenitor sequence. Amino acid differences distinguishing Cf-4 and Cf-9 are confined to their N termini, delimiting a region that determines the recognitional specificity of ligand binding. The majority of these differences are in residues interstitial to those of the leucine-rich repeat consensus motif. Many of these residues are predicted to form a solvent-exposed surface that can interact with the cognate ligand. Both Cf-4 and Cf-9 are located within a 36-kb region comprising five tandemly duplicated homologous genes. These results provide further insight into the molecular basis of pathogen perception by plants and the organization of complex R gene loci.
In "gene-for-gene" interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.
Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve novel configurations and potentially new disease resistance specificities.
In order to construct an RFLP map of barley, two populations were analyzed using 251 genomic and cDNA markers: one population comprised 71 F1 antherderived double haploid (DH) individuals of an intraspecific cross (IGRI x FRANKA), and the other 135 individuals of an interspecific F2/F3 progeny (VADA x H. spontaneum). The distribution of nonrepetitive clones over the seven barley chromosomes revealed a maximum for chromosome 2H and a minimum for 6H. The polymorphism of the interspecific progeny (76%) clearly exceeded that of the intraspecific progeny (26%) although, based on their pedigrees, IGRI and FRANKA are only distantly related. The contribution of individual chromosomes of the DH parents to the overall polymorphism varied between 8% and 50%. A significant portion (44% versus 10% of the interspecific progeny) of the markers mapped on the DH offspring showed distorted segregation, caused mainly by the prevalence of variants originating from the parent that better responded to in vitro culture (IGRI). In contrast to the interspecific map, probes displaying skewed segregation were clustered on the DH map on discrete segments. The colinear arrangement of both maps covers a distance of 1,453 cM and identifies regions of varying map distances.
Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F1 hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F1s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.
Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F2 population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08
750
was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).
Eighty-three third backcross lines which comprise a set of near isogenic lines (NIL's) of the barley cultivar ‘Clipper’ but each carrying a different chromosomal segment from Hordeum spontaneum, marked with a distinct isozyme, were tested for resistance to three races of the barley leaf rust pathogen (Puccmia hordei). Fourteen lines showed resistance to at least one race and three showed resistance to all three races. The resistance in two of these lines was controlled by separate, single partially dominant genes. In one case the resistance gene named Rph1O was on chromosome 3 and linked (r = 0.15 ±0.05) with the isozyme locus Est2. In the second case, the gene (Rph11) was on barley chromosome 6 and linked (r = 0.07±0.02) with the isozyme locus Acp3 and (r = 0.11±0.02) with Dip2.
In plants, resistance to pathogens is frequently determined by dominant resistance genes, whose products are proposed to recognize pathogen-encoded avirulence gene (Avr) products. The tomato resistance locus Cf-2 was isolated by positional cloning and found to contain two almost identical genes, each conferring resistance to isolates of tomato leaf mould (C. fulvum) expressing the corresponding Avr2 gene. The two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat (LRR) motifs. Of the LRRs, 20 show extremely conserved alternating repeats. The C-terminus of Cf-2 carries regions of pronounced homology to the protein encoded by the unlinked Cf-9 gene. We suggest that this conserved region interacts with other proteins involved in activating plant defense mechanisms.
An RFLP map was constructed from 99 doubled haploid lines of a cross between two spring barley varieties (Blenheim Kym) and used to map quantative trait loci (QTL) controlling ear emergence time and plant height using a marker-regression approach. Three QTL affecting plant height were identified. The largest effect was from the denso dwarfing gene, present in Blenheim, on chromosome 3(3H)L. Two other effects were found, one on 7(5H)L and one on 1(7H)L. The denso gene also had a major effect on ear emergence time. However, eight additional QTL for ear emergence were also identified. Two of these were in regions previously identified as carrying the Sh and Sh2 vernalization response genes, suggesting that allelic variation at these genes may affect ear emergence time in spring barley crosses. Comparisons with maps of related species show conservation and collinearity of markers and genes.Keywords: comparative mapping, flowering time, height, Hordeum vulgare, quantitative trait loci (QTL), RFLP-mapping
FLOWERING plants or angiosperms have dominated the Earth's flora since at least the late Cretaceous1 and were already highly diversified by Barremian times, about 120 million years (Myr) ago. However, because of the paucity of fossilized angiosperm reproductive structures from lower Cretaceous sediments2,3 and the absence of generally recognized angiosperm fossils from pre-Cretaceous strata4,5, their origins and early evolution remain obscure. Similarly, attempts to understand pre-Cretaceous angiosperm evolution4–11 have been impaired by difficulties in defining and interpreting angiospermous characters in fossil specimens8,12. We report here molecular evidence suggesting that angiosperm ancestors underwent diversification more than 300 Myr ago.
Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.
Bennett and Smith (Philosophical Transactions of the Royal Society of London B274: 227-274; B334: 309-345) and Bennett, Smith and Heslop-Harrison (Proceedings of the Royal Society of London, B216: 179-199) published lists of nuclear DNA amounts estimated for 1612 angiosperm species collected from 163 sources dated between 1951 and 1986. Subsequently, interest in genome size in angiosperms and its significance has continued, and many new DNA estimates were published during 1986-1994. Their inaccessibility, and the how of enquiries for such information, shows that a further compilation is needed. This paper presents a supplementary list of nuclear DNA C-values for 105 sources for 899 angiosperm species not listed in the above-mentioned compilations, plus 284 additional estimates for 208 species already listed by them. The data are assembled primarily for reference purposes, with species listed in alphabetical order, rather than by any taxonomic scheme. Some advantages and limitations of Bow cytometry, now increasingly used to quantify DNA C-values in plants, are reviewed. Recent reports regarding the occurrence and extent of intraspecific variation in genome size are also discussed. While some examples are real, others reflect technical shortcomings. Work has begun to combine the genome size data compiled in this and the above-mentioned papers into a unified data base, and to present the information in separate lists, with species in alphabetical and systematic orders, respectively. DNA C-values are now known for 1 % of the world's angiosperm flora, but improved representation of taxonomic groups, geographical regions and plant life forms is urgently needed. (C) 1995 Annals of Botany Company
The number of angiosperm species for which nuclear DNA amount estimates have been made has nearly trebled since the last collected lists of such values were published, and therefore, publication of a more comprehensive list is over due. This paper lists absolute nuclear DNA amounts for 753 angiosperm species. The dats were assembled primarily for reference purposes, and so the species are listed in alphabetical order, as this was felt to be more helpful to cyto- and biochemists whom, it is anticipated, will be among its major users. The paper also reviews aspects of the history, nomenclature, methods, accuracy and problems of nuclear DNA estimation in angiosperms. No attempt is made to reconsider those aspects of nuclear DNA estimation which have been fully revised previously, although the bibliography of such aspects is given. Instead, the paper is intended as a source of basic information regarding the terminology, practice and limitations of nuclear DNA estimation, especially by Feulgen microdensitometry, as currently practiced.
Ectopic recombination between interspersed repeat sequences generates chromosomal rearrangements that have a major impact on genome structure. A survey of ectopic recombination in the region flanking the white locus of Drosophila melanogaster identified 25 transposon-mediated rearrangements from four parallel experiments. Eighteen of the 25 were generated from females carrying X chromosomes heterozygous for interspersed repeat sequences. The cytogenetic and molecular analyses of the rearrangements and the parental chromosomes show: (1) interchromosomal and intrachromosomal recombinants are generated in about equal numbers; (2) ectopic recombination appears to be a meiotic process that is stimulated by the interchromosomal effect to about the same degree as regular crossing over; (3) copies of the retrotransposon roo were involved in all of the interchromosomal exchanges; some copies were involved much more frequently than others in the target region; (4) homozygosis for interspersed repeat sequences and other sequence variations significantly reduced ectopic recombination.
A method for rapid preparation of plant genomic DNA for PCR analysis was established. A small amount (4~6 mg) of leaf tissue of rice seedling, 200 滋L of TE buffer, and one tungsten bead were put into a 2 mL microcentrifuge tube. After vigorously shaking in a miller for 5 min, 1 滋L of the solution was directly applied to PCR amplification. This method is simple, rapid, high efficient, low cost, and reliable for PCR analysis, thus is es- pecially suitable for genotyping of large number of samples.
The recurring intrachromosomal rearrangements observed in an unstable X chromosome, designated Uc, of Drosophila melanogaster are shown to be mediated by hobo transposable elements. Each of 29 chromosome rearrangement breakpoints in 16 gross aberrations detected in the Uc-derived X chromosomes had a hobo element. In one particular unstable X chromosome line selected for detailed studies, a hobo element was found in each of the five hot spots for rearrangements. Furthermore, hobo elements at deletion hot spots were found to lie in the same orientation, whereas those hobo elements at inversion hot spots were in the opposite orientation. The restriction maps of two phage lambda clones containing rearrangement breakpoints indicated that a hobo element was inserted exactly at the breakpoints. Pairing of hobo elements in the same chromosome followed by recombination between the paired hobo elements is suggested as the explanation for the intrachromosomal aberrations observed in the Uc X chromosomes. A clear qualitative difference among the hobo elements in their ability to participate in rearrangement formation was noted. It was also found that each of the 11 recessive lethal mutations mapped in the 6F1-2 doublet had a hobo element in the doublet, whereas none of the 16 independent revertants of the mutation had a hobo element in the site. This observation indicates that hobo movement is responsible for production and subsequent instability of recessive lethal mutations in the 6F region of the Uc X chromosomes.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
We studied a collection of 746 chromosome rearrangements all induced by the activity of members of the P family of transposable elements in Drosophila melanogaster. The chromosomes ranged from simple inversions to complex rearrangements. The distribution of complex rearrangement classes was of the kind expected if each rearrangement came about from a single multibreak event followed by random rejoining of chromosome segments, as opposed to a series of two-break events. Most breakpoints occurred at or very near (within a few hundred nucleotide pairs) the sites of preexisting P elements, but these elements were often lost during the rearrangement event. There were also a few cases of apparent gain of P elements. In cases in which both breakpoints of an inversion retained P elements, that inversion was capable of reverting at high frequencies to the original sequence or something close to it. This reversion occurred with sufficient precision to restore the function of a gene, held-up-b, which had been mutated by the breakpoint. However, some of the reversions had acquired irregularities at the former breakpoints that were detectable either by standard cytology or by molecular methods. The revertants themselves retained the ability to undergo further rearrangements depending on the presence of P elements. We interpret these results to rule out the simplest hypotheses of rearrangement formation that involve cointegrate structures or homologous recombination. The data provide a general picture of the rearrangement process and its possible relationship to transposition.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
The genomes of six major grass species can be aligned by dissecting the individual chromosomes into segments and rearranging these linkage blocks into highly similar structures.
In order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric regions of barley chromosome 1P and
rice chromosome 6. We have also developed and applied a simple and efficient method for selecting useful probes from large
Insert genomic YAC and cosmld clones. The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared
wtth the most terminal 2.7 cM of rice chromosome 6. Nine rice probes, previously mapped In rice or Isolated from YAC or cosmld
clones from this region, were mapped In barley. All, except one, were In synteny with the rice gene order. The exception,
probe Y617R, was duplicated in barley. One copy was located on a different chromosome and the other in a non-syntenic position
on barley chromosome 1 P. The barley probes from this region could not be mapped to rice, but two of them were inferred to
be In a syntenlc location based on their position on a rice YAC. This work demonstrates the utility of applying the results
of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome
relatives.
DNA markers distributed over large chromosomal regions exhibit conservation of order (colllnearity) in different cereal species,
but it is not known whether this is maintained on a finer scale, i.e. ≥2 cM. To address this, sets of two or more genetically
linked DNA markers were localised to yeast artificial chromosomes containing rice DNA inserts. Linkage analysis of these DNA
markers in barley revealed complete correspondence with their genetic order in rice, the distance between linked sequences
on rice chromosomes being <1.6 cM or <1×10° bp (1 Mb). Thus, DNA markers separated in this range are collinear in rice, barley
and, by Inference, other members of the Triticeae. These results are discussed with respect to the use of rice as a key system
for the isolation of cereal genes.
Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. All proteins containing these repeats are thought to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features may be responsible for the protein-binding functions of proteins containing leucine-rich repeats.
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The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology.
More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/ threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.
Comparative genetic studies have demonstrated that gene content and orders are highly conserved, both at the map and megabase level, between different species within the grass family. Integration of the genetic maps of rice, foxtail millet, sugar cane, sorghum, maize, the Triticeae cereals and oats into a single synthesis reveals that some chromosome arrangements characterise taxonomic groups, while others have arisen during or after speciation. A detailed analysis of the comparative maps of seven species, belonging to three subfamilies, and their applications are described below.
Genes conferring host resistance to an obligate parasite, grouped together in complex loci provide opportunities to study their structure. By means of an appropriate operational definition of these genes, a modified cis-trans test was used to interpret the position effects of codominant genes mutually recombined within each of two complex loci of flax, with the use of a specially developed method of analysis among F(2) segregants. The different behavior of genes in the M and L groups may reflect a difference in their structure sufficient to raise important implications in the theory of specific host-parasite interactions.