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Simple sequence repeat map of the sunflower genome
Abstract and Figures
Several independent molecular genetic linkage maps of varying density and completeness have been constructed for cultivated
sunflower (Helianthus annuus L.). Because of the dearth of sequence and probe-specific DNA markers in the public domain, the various genetic maps of sunflower
have not been integrated and a single reference map has not emerged. Moreover, comparisons between maps have been confounded
by multiple linkage group nomenclatures and the lack of common DNA markers. The goal of the present research was to construct
a dense molecular genetic linkage map for sunflower using simple sequence repeat (SSR) markers. First, 879 SSR markers were
developed by identifying 1,093 unique SSR sequences in the DNA sequences of 2,033 clones isolated from genomic DNA libraries
enriched for (AC)n or (AG)n and screening 1,000 SSR primer pairs; 579 of the newly developed SSR markers (65.9% of the total) were polymorphic among
four elite inbred lines (RHA280, RHA801, PHA and PHB). The genetic map was constructed using 94 RHA280 × RHA801 F7 recombinant inbred lines (RILs) and 408 polymorphic SSR markers (462 SSR marker loci segregated in the mapping population).
Of the latter, 459 coalesced into 17 linkage groups presumably corresponding to the 17 chromosomes in the haploid sunflower
genome (x = 17). The map was 1,368.3-cM long and had a mean density of 3.1cM per locus. The SSR markers described herein supply a
critical mass of DNA markers for constructing genetic maps of sunflower and create the basis for unifying and cross-referencing
the multitude of genetic maps developed for wild and cultivated sunflowers.
Microsatellite Simple sequence repeat Helianthus Sunflower
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... There are numerous different molecular markers available which can be used in sunflower breeding. Pérez-Vich and Berry (2010) described three different generations of markers in sunflower research: firstly, anonymous deoxyribonucleic acid (DNA) markers like RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism) and genomic SSR (Simple Sequence Repeat) markers were developed (Al-Chaarani et al., 2002;Berry et al., 1995;Gentzbittel et al., 1995;Lawson et al., 1998;Lu et al., 2000;Quagliaro et al., 2001;Tang et al., 2002). Usage of several molecular markers combined with numerous linkage maps makes it possible to develop a hybrid line that provides required properties (Knapp et al., 2001). ...
... There are several linkage maps available to use for marker assisted selection programmes. Researchers completed the first linkage map of sunflower in 2002 (Tang et al., 2002) and this was improved by another research group in 2003 through usage of new recombinant inbred lines population with SSR markers (Yu et al., 2003). Another research group accomplished the genetic mapping of the fertility restoration gene by using SSR and TRAP (Targeted Region Amplified Polymorphism) markers (Yue et al., 2010). ...
The oomycete pathogen Plasmopara halstedii responsible for sunflower downy mildew (DM), that is a significant and important disease that greatly affects the economy. As of now, there is no non-race-specific resistance for this disease and breeders are depended on race-specific resistance to control DM disease. On the other hand, using conventional breeding procedure introgression of the DM resistance genes is a long-term task due to the highly virulent and aggressive nature of the P. halstedii pathogen. Molecular markers that can be applied at the seedling stage, offers rapid response for selection with higher precision as well as a lower cost. There are currently 36 downy mildew resistance genes (R genes), designated as Pl (Pl1-Pl36, Plhra, and PlArg, in sunflowers, each with a unique linkage group (LGs). The availability of DM resistance genomic data of sunflower, related to Single Nucleotide Polymorphisms (SNP) based markers with mine allelic diversity maximize the opportunity of utilizing Marker assisted selection (MAS) techniques for downy mildew resistance breeding. This review highlights the available genetic marker and their utilization at MAS techniques for enhancing downy mildew disease resistant breeding program of sunflowers.
... A set of fifteen simple sequence repeat (SSR) primer pairs (Table 3) were used for molecular analyses. Nine primer pairs were acquired from Tang et al. (2002) The PCRs were carried out in a final volume of 25 μl containing 5 μl of PCR buffer (1x), 1.25 μl of MgCl2 (2.5 mM), 0.25 μl of dNTPs (100 μM), 0.625 μl of each primer (0.25 μM), 0.2 μl of Taq DNA polymerase (1U) and 5 μl of template DNA (5 ng/μl). The PCR amplifications were carried out in a thermocycler (Boi_Rad C100 TM ). ...
... This was followed by 33 cycles of 94ºC for 30 seconds, 54ºC for 30 seconds and 72ºC for 45 seconds. The final extension was 20 minutes at 72ºC (Tang et al., 2002). Amplified PCR products were separated by electrophoresis using 2% agarose gel (1xTBE buffer), stained by ethidium bromide (0.5μg/ml) and visualized under ultraviolet light. ...
Sunflower (Helianthus annuus L.) is considered as one of the most important oilseed crops in the world. In Tunisia, sunflower is cultivated in the Northern region of the country using local populations which are genetically not well studied. This study was conducted to evaluate the genetic variation of 33 accessions (26 local and 7 introduced) using 23 agro-morphological traits and 15 simple sequence repeats (SSR) markers. The variance analysis of the agro-morphological traits showed a significant variation among sunflower accessions. The relationship among the sunflower accessions was also performed by using the principal component analysis; the two first axes explained 54.4% of the total variability and showed that the accessions spread into five groups. The groups (G3 and G4) could be used to improve sunflower varieties with high performance especially for the diameter of the head and the weight of 1000 seeds characters. Ten among the 15 SSR primers used revealed clear polymorphic bands and were able to amplify 29 alleles with an average of 2.9 alleles per locus. The percentage of total polymorphism, the polymorphism information content (PIC) and the dissimilarity coefficient values varied from 50 to 100% with an average of 91%, from 0.35 to 0.75 with an average of 0.50, and from 0.00 to 0.81 with an average of 0.41, respectively. Cluster analysis of the SSR markers grouped accessions into 3 distinct groups. A significant correlation was observed among SSR markers and morphological traits. These results found in this study may be helpful for improvement of sunflower breeding programs.
... Для изучения генетического разнообразия подсолнечника доступны различные виды молекулярных маркеров, наиболее популярными из которых являются микросателлиты (single sequence repeats, SSR) и однонуклеотидные полиморфизмы (SNP). Порядка 2000 SSR-маркеров были разработаны ранее на основе геномных (gSSR) и EST (EST-SSR) библиотек [4,5,6]. SNP-технологии бурно развиваются в последнее десятилетие [7,8,9]. ...
... На дендрограммах видно сходство некоторых генотипов. Минимальные генетические дистанции у линий ЦЭБ ниже (0,0), чем у образцов АОС (2,2) и ДОС (1,5). Для линий ЦЭБ, ДОС и АОС ВНИИМК была характерна группировка материнских и отцовских линий в отдельные кластеры или субкластеры. ...
The breeding of high-yielding sunflower varieties and hybrids requires the use of parent material with considerable genetic diversity. It can be identified using molecular genetic markers. The purpose of this study was the genotyping of sunflower lines bred by V. S. Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK), developed in different ecological areas of cultivation, and to evaluate their genetic diversity using microsatellite loci. For the studies conducted in 2020-2022, there were used 23 lines from the Central experimental base (СEB), 17 lines from L.A. Zhdanov Don experimental station (DES), and 10 lines from the Armavir experimental station (AES). DNA was isolated from seedlings by the CTAB method. Samples were genotyped using 12 SSR markers. PCR products were separated in 8 % denaturing polyacrylamide gel or by capillary electrophoresis in a genetic analyzer. The main indicators of genetic diversity for all collections did not differ significantly from each other, and were moderate and increased in the collections of VNIIMK: AES – DES – CEB -the joint collection. In total there have been identified 37 alleles, an average 3.083 alleles per locus. The effective number of alleles ranged from 1.094 to 3.290 with an average value of 2.154. The values of the polymorphic information content (PIC) index ranged from 0.084 to 0.651, with an average of 0.434. The values of observed and expected heterozygosity ranged from zero to 0.071, 0.030, on average, and from 0.086 to 0.696, 0.500, on average, respectively. The number of shared alleles between different collections was 23. Molecular variation analysis revealed that most of the total variance (91 %) was due to differences between lines within each collection and 3 % to differences between collections. According to the results of the cluster analysis, the maternal lines from the collections of the CEB, DES, and AES of VNIIMK were mostly grouped into a cluster or subcluster separate from the paternal ones. The obtained results indicate a moderate genetic diversity of the studied sunflower lines of the breeding of VNIIMK and the existence of small differences between the collections.
... Tracking polygenes with genetic markers can be traced back to the early 1920's when Sax (1923) reported the association of quantitatively inherited seed size with monogenes controlling seed coat pigmentation and pattern in bean (Dudley, 1993;Paterson and Tanksley, 1997). Effort to construct high-density linkage maps of molecular genetic polymorphism (marker loci) is currently underway for sunflower (Gentzbittel et al., 1995;Jan et al., 1998;Tang et al., 2002). Simple sequence repeats (SSR), also called microsatellites, are widely used as molecular markers. ...
The present investigation was carried out in sunflower F2:5 progenies of a cross TNHSF239-68-1-1-1 x 17B to identify the markers associated with seed quality traits. In this study, the QTL mapping work was based on the genotyping data obtained in the segregating population for the publicly available ORS markers previously mapped was used. In ICIM, the QTL analysis resulted into five QTLs viz., volume weight, seed colour, strips on margin, pollen colour and oil content. The QTL for volume weight had a LOD of 2.93 and 24.86 per cent of phenotypic variation with the flanking markers ORS471 and ORS1265 on chromosome 7. The QTL for seed colour was formed on chromosome 11 had a LOD of 36.58 and 96.46 per cent of phenotypic variation with the flanking markers ORS707 and ORS503. The QTL for strips on margin wasformed on chromosome 10 had a LOD of 3.23 and 63.88 per cent of phenotypic variation with the flanking markers ORS502 and ORS1037. The QTL for pollen colour had a LOD of 64.52 and 97.94 per cent of phenotypic variation with the flanking markers ORS707 and ORS503 on chromosome 11. The QTL for oil content was formed on chromosome 8 had a LOD of 2.51 and 28.99 per cent of phenotypic variation with the flanking markers ORS537 and ORS613. Hence the QTL identified for seed quality traits with closely linked markers may be exploited for the genetic improvement of sunflower genotypes through marker assisted selection.
... Another way is to search for markers genetically linked to the Rf-gene. A number of molecular markers linked to the Rf1-locus on the genetic map have been identified and published: microsatellite ORS224, ORS317, ORS511, ORS630, ORS799, ORS1030 [11], TRAP-markers and STS-marker STS115 [12], SCAR-markers HRG01 and HRG02 [13]. Also Horn et al. recently developed the Polymerase chain reaction (PCR) Amplification of Multiple Specific Alleles (PAMSA) marker 67N04 and two SCARmarkers, PPR621.5 M and PPR621.5R ...
Background
Ability to restore male fertility is important trait for sunflower breeding. The most commonly used fertility restoration gene in the production of sunflower hybrids is Rf1. The localization of Rf1 on the linkage group 13 has been previously shown, however, its exact position, its sequence and molecular mechanism for fertility restoration remain unknown. Therefore, several markers linked to Rf1 gene, commonly used for MAS, don’t always allow to identify the genotype of plants. For this reason, the search for new markers and precise localization of the Rf1 gene is an urgent task.Methods and resultsBased on previously identified single nucleotide polymorphisms (SNPs) at LG13, significantly associated with the ability to restore male fertility, two markers have been developed that have performed well after careful evaluation. These markers, together with other Rf1 markers, were applied for genotyping 72 diversity panel accessions and 291 individuals of F2 segregating population, obtained from crossing the cytoplasmic male sterility (CMS) AHO33 and restorer RT085HO lines. The analysis revealed no recombinants between Rf1 gene and SRF833 marker, the distance between Rf1 and SRF122 marker was 1.0 cM.Conclusions
Data obtained made it possible to specify the localization of the Rf1 gene and reduce the list of candidate genes to the 3 closely linked PPR-genes spanning a total of 59 Kb. However, it cannot be ruled out that analysis of the candidate region in the genome of fertility restorer lines can reveal new candidate genes in this locus that are absent in the cytoplasmic male sterility maintainer reference sequence.
... Several molecular markers linked to the Plarg locus have been published in the literature [13,14,15]. The closer the gene and the molecular marker are to each other in the genetic map, the higher the probability that, as a result of recombination in meiosis of an F1 hybrid, the gene and the marker fragment will fall into the same gamete and be simultaneously passed on to the progeny. ...
... The PCR amplification and genotyping followed Liu et al. (2012). The PCR amplification system was conducted following Tang et al. (2002) with minor modifications. The 15-µl PCR reaction mixture contained 1 × PCR buffer, 2 mM MgCl 2 , 0.2 mM dNTPs, 0.27 µM each of the forward and reverse primers, 40 ng DNA and Taq DNA polymerase (Qiagen). ...
Cytoplasmic male-sterile line CMS ANN3 was discovered from a wild annual Helianthus annuus L. accession PI 413180. A single dominant fertility restoration (Rf) gene for CMS ANN3 was identified in PI 413180, P 21, RHA 801, RCMG 1, and RHA 280, designated as Rf9. This study reports the mapping of the Rf9 locus using an F2 population of 160 individuals. Bulked segregant analysis with SSR primers identified two polymorphic SSR markers in linkage group 3 of the sunflower genetic map. A linkage map of Rf9 was constructed with 16 markers including 11 SSR/STS and five SNP markers, covering a genetic distance of 87.3 cM. The Rf9 gene is located between a co-dominant marker ORS1114 and a dominant marker NSA002021, with a distance of 4.1 and 1.0 cM, respectively. The physical distance between the two closest flanking markers corresponds to 1.44 and 2.05 Mb on the HA 412-HO and XRQ assemblies, respectively. The closely linked marker for Rf9 will facilitate marker-assisted selection in breeding programs, which provides a basis for further characterization of this gene, and for studying the mechanism between cytoplasmic male sterility and Rf genes. The mapping of the Rf gene will also be useful for studying Rf gene suppression and nuclear–cytoplasm interactions.
Sunflower is a staple oilseed crop ofthe world. Genetic diversity in the parental lines is key for hybrid breeding programmes in sunflower. In this study, genetic diversity in a set of 102 parental lines (100 restorer and twoCMS) of sunflower was assessed using 69 polymorphic SSR markers. The genetic diversity parameters: average number of alleles (3.07) per locus, gene diversity (0.356) and polymorphism information content (0.296) revealed low to moderate genetic diversity in the restorer lines. The SSR marker ORS447 located on linkage group 2 was more informative with high number of alleles (10) and high PIC value (0.89). Cluster analysis (neighbour-joining tree) revealed three major genotypic groups. Model based STRUCTURE analysis showed recognizable population structure; based on membership coefficients(>80%), 82 genotypes were classified into two populations (K=2) and the remaining 20 genotypes were classified into admixture group. The Fst value (0.278) suggested that the populations were differentiated. Analysis of molecular variance resultsshowed that maximum of genetic variation (72%) was observed between the individuals within the population suggesting that the population was weakly structured. These results would be useful for selecting SSR markers for genotype characterization as well as choosing diverse parents for hybrid development programme in sunflower.
Cultivated sunflower (Helianthus annuus L.) is a member of the subtribe Helianthinae of the Compositae (Asteraceae) family (Seiler and Rieseberg 1997). The genus is a polyploid complex, with diploid, tetraploid and hexaploid species, and a basic chromosome number of 17 (Heiser and Smith 1955). There are 12 annual diploid species and 37 perennial species. North America is the center of diversity for sunflowers. Sunflowers were first cultivated by Native Americans in 1000 BC, were introduced into Europe in the sixteenth century, and were first grown as a source of edible oil in nineteenth century Russia. Russian plant breeders increased sunflower seed oil concentrations from 330g/kg up to as much as 550g/kg between 1940 and 1965 and developed the first high oil cultivars (Putt 1997). These cultivars dramatically transformed sunflower as an oilseed crop and they have been widely used in the development of modern-day cultivars and hybrids — a significant fraction of the diversity in elite inbred lines traces to high-oil germplasm developed in Russia (Korell et al. 1992; Cheres and Knapp 1998).
Phylogenetic relationships among species representing 57 genera from 15 currently recognized tribes of Asteraceae were assessed by chloroplast DNA restriction site mapping. Phylogenetic analyses of 328 informative restriction site mutations were performed using both Dollo and Wagner parsimony and the bootstrap method. The results provide strong support for the monophyly of the subfam. Asteroideae, which includes the nine tribes Astereae, Anthemideae, Inuleae, Senecioneae, Calenduleae, Eupatorieae, Heliantheae, Coreopsideae, and Tageteae. The monophyly of the Lactucoideae (excluding the Barnadesiinae) is less strongly supported and this subfamily consists of the six tribes Mutisieae, Cardueae, Vernonieae, Liabeae, Lactuceae, and Arctoteae. The molecular phylogeny indicates that 13 of 15 tribes are monophyletic, with the Heliantheae and Tageteae being paraphyletic. Several groups of tribes are shown to be closely related, including the Liabeae and Vernonieae, and the Tageteae, Coreopsideae, Heliantheae, and Eupatorieae, but relationships among the other tribes remain unclear. Tribal placement of five morphologically anomalous genera (Blennosperma, Cotula, Echinops, Stokesia, and Ursinia) is also clarified. The chloroplast DNA phylogeny shows almost complete congruence with the less explicit phyletic scheme proposed by Thorne but differs markedly from the cladistically-based phylogeny of Bremer. The availability of a molecular phylogeny for the Asteraceae allows for an independent assessment of the patterns of morphological, chemical, and chromosomal evolution in the family. A much higher incidence of parallelism occurs in secondary chemistry than in morphological characters. The subfam. Asteroideae has a number of evolutionarily derived floral features and it exhibits a much more diverse and biosynthetically complex secondary chemistry.
A map of the barley genome consisting of 295 loci was constructed. These loci include 152 cDNA restriction fragment length polymorphism (RFLP), 114 genomic DNA RFLP, 14 random amplified polymorphic DNA (RAPD), five isozyme, two morphological, one disease resistance and seven specific amplicon polymorphism (SAP) markers. The RFLP-identified loci include 63 that were detected using cloned known function genes as probes. The map covers 1,250 centiMorgans (cM) with a 4.2 cM average distance between markers. The genetic lengths of the chromosomes range from 124 to 223 cM and are in approximate agreement with their physical lengths. The centromeres were localized to within a few markers on all of the barley chromosomes except chromosome 5. Telomeric regions were mapped for the short (plus) arms of chromosomes 1, 2 and 3 and the long (minus) arm of chromosomes 7.
Length variations in simple sequence tandem repeats are being given increased attention in plant genetics. Some short tandem repeats (STRs) from a few plant species, mainly those at the dinucleotide level, have been demonstrated to show polymorphisms and Mendelian inheritance. In the study reported here a search for all of the possible STRs ranging from mononucleotide up to tetranucleotide repeats was carried out on EMBL and GenBank DNA sequence databases of 3026 kb nuclear DNA and 1268 kb organelle DNA in 54 and 28 plant species (plus algae), respectively. An extreme rareness of STRs (4 STRs in 1268 kb DNA) was detected in organelle compared with nuclear DNA sequences. In nuclear DNA sequences, (AT)n sequences were the most abundant followed by (A)n · (T)n, (AG)n · (CT)n, (AAT)n · (ATT)n, (AAC)n · (GTT), (AGC)n · (GCT)n, (AAG)n · (CTT)n, (AATT)n · (TTAA)n, (AAAT)n · (ATTT)n and (AC)n · (GT)n sequences. A total of 130 STRs were found, including 49 (AT)n sequences in 31 species, giving an average of 1 STR every 23.3 kb and 1 (AT)n STR every 62 kb. An abundance comparable to that for the dinucleotide repeat was observed for the tri- and tetranucleotide repeats together. On average, there was 1 STR every 64.6 kb DNA in monocotyledons versus 1 every 21.2 kb DNA in dicotyledons. The fraction of STRs that contained G-C basepairs increased as the G+C contents went up from dicotyledons, monocotyledons to algae. While STRs of mono-, di- and tetranucleotide repeats were all located in non coding regions, 57% of the trinucleotide STRs containing G-C basepairs resided in coding regions.
cDNA and PstI genomic clones have been used to assess levels of restriction fragment length polymorphism (RFLP) in Helianthus annuus and to determine the inter-relationships between a diverse set of 24 inbred lines. Of the cDNA clones screened 45% were useful as RFLP probes, compared to less than 20% from the PstI library, which showed high levels of redundancy for high copy sequences. Fifty-seven low-copy DNA probes (23 PstI and 34 cDNA clones) were used to fingerprint 12 maintainer (B) lines and 12 restorer (R) lines. The average number of RFLP variants per probe was found to be 3.2, with a mean polymorphic index of 0.49, indicating that high levels of nuclear DNA polymorphism are to be found in cultivated sunflower. Cluster and principal coordinate analysis of the fingerprinting data clearly separated the maintainer and restorer lines, but there was a degree of association between 2 unbranched R-lines and the B-line germ plasm pool.
One-hundred-and-eighty-one nuclear DNA probes were used to examine restriction-fragment length polymorphism in inbred lines of the cultivated sunflower (Helianthus annuus L.). The probes were from six libraries: two genomic libraries - one made with PstI and the other with HindIII, and four cDNA libraries - from etiolated plantlets, green leaves, ovaries, petals and anthers. Total DNA from 17 inbred lines representing an overview of the genetic stocks of sunflower, including restorer and maintainer lines of the classical cytoplasmic male sterility, was digested with four different restriction enzymes and probed in 331 probe-enzyme combinations. Of 181 clones analysed, 73 probes were found to be polymorphic. Genetic distances between inbreds were calculated from the resultant proportion of shared bands and submitted to principal component analysis and the UPGMA 'tree-making' method. The RFLP analysis allowed a clear differentiation between restorer and maintainer lines of the cytoplasmic male sterility, together with a grouping of some of the genotypes from the same origin. The analysis of the accuracy of distance estimation as a function of the number of probe-enzyme combinations used, indicates that 40-50 combinations ensure a confidence level of near 95%. Considering the inbreds as representatives of the range of cultivated inbreds, estimates of gene diversity, as well as estimates of average gene diversity between and within the sets of restorer and maintainer lines, were calculated. Estimation of gene diversity showed that the available genetic variability in cultivated sunflower, based on allelic frequencies, is lower than that of other plants (H=0.20). Moreover, we show that the proportion of genetic variability due to the difference between maintainer and restorer lines (Dm) is about 2%.