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Overall characteristics of AS events. a The proportions for the three types of dinucleotides at the splicing border. b Number and frequency of the different AS types. (IR) Intron retention, (A3SS) alternative 3′ splice site, (A5SS) alternative 5′ splice site, (ES) exon skipping. c Four genes representing four types of AS events were validated by RT-PCR. The lanes from left to right are DNA ladder, leaf and root of 'NAU-RG', 'NAU-LB', 'NAU-YH', and genomic DNA of 'NAU-YH'. The RNA-Seq read density of the four genes was viewed by IGV browser (green peaks). The red arrows indicate alternative splice sites. The blue arcs indicate splice junction reads that support the junctions
Source publication
Background
Alternative splicing (AS) plays important roles in gene expression and proteome diversity. Single nucleotide polymorphism (SNP) and insertion/deletion (InDel) are abundant polymorphisms and co-dominant inheritance markers, which have been widely used in germplasm identification, genetic mapping and marker-assisted selection in plants. So...
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Citations
... Molecular marker is a valuable tool for genetics and breeding research in plant, such as fingerprinting genotypes, genetic map construction, QTL mapping, association analysis and marker-assisted selection (MAS) [1,2]. In the past three decades, a large number of molecular markers have been successfully developed in plant, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), insertion/Deletions (InDels) and single-nucleotide polymorphisms (SNPs) [3,4]. SSR and InDel are PCR-base markers with the advantage of bi-allelic, co-dominant, abundance and low-cost [5]. ...
... Radish (Raphanus sativus L.) is an important root vegetable belonging to cruciferous family. A large number of InDel markers have been characterized and applied for genetic and genomic studies in radish [3,11]. A total of 9,436 InDel were detected in three radish transcriptome and 40 InDel markers were used for genetic diversity analysis in 32 radish accessions [3]. ...
... A large number of InDel markers have been characterized and applied for genetic and genomic studies in radish [3,11]. A total of 9,436 InDel were detected in three radish transcriptome and 40 InDel markers were used for genetic diversity analysis in 32 radish accessions [3]. In total, 99 EST-SSR and InDel markers were used to construct linkage groups and QTL mapping, QTL for late-bolting trait was located a 1.1-cM region between InDel520 and InDel535 [12]. ...
Background
Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) are the most abundant genetic variations and widely distribute across the genomes in plant. Development of SNP and InDel markers is a valuable tool for genetics and genomic research in radish (Raphanus sativus L.).
Results
In this study, a total of 366,679 single nucleotide polymorphisms (SNPs) and 97,973 insertion-deletion (InDel) markers were identified based on genome resequencing between ‘YZH’ and ‘XHT’. In all, 53,343 SNPs and 4,257 InDels were detected in two cultivars by transcriptome sequencing. Among the InDel variations, 85 genomic and 15 transcriptomic InDels were newly developed and validated PCR. The 100 polymorphic InDels markers generated 207 alleles among 200 Chinese radish germplasm, with an average 2.07 of the number of alleles (Na) and with an average 0.33 of the polymorphism information content (PIC). Population structure and phylogenetic relationship revealed that the radish cultivars from northern China were clustered together and the southwest China cultivars were clustered together. RNA-Seq analysis revealed that 11,003 differentially expressed genes (DEGs) were identified between the two cultivars, of which 5,020 were upregulated and 5,983 were downregulated. In total, 145 flowering time-related DGEs were detected, most of which were involved in flowering time integrator, circadian clock/photoperiod autonomous, and vernalization pathways. In flowering time-related DGEs region, 150 transcriptomic SNPs and 9 InDels were obtained.
Conclusions
The large amount of SNPs and InDels identified in this study will provide a valuable marker resource for radish genetic and genomic studies. The SNPs and InDels within flowering time-related DGEs provide fundamental insight into for dissecting molecular mechanism of bolting and flowering in radish.
... The PCR conditions comprised initial denaturation at 94 ℃ for 2 min, followed by 35 cycles of 94 ℃ for 40 s, 55 ~ 60 ℃ (varying with the T m of the different primers) for 45 s, 72 ℃ for 1 min, and a final extension of 72 ℃ for 7 min. PCR products were separated on 8% non-denaturing polyacrylamide gels at 120 V for 2 ~ 2.5 h and visualized with a silver staining method (Luo et al. 2017). ...
Unlabelled:
Radish (Raphanus sativus L.), an important annual or biennial root vegetable crop, is widely cultivated in the world for its high nutritive value. Isolated microspore culture (IMC) is one of the most effective methods for rapid development of homozygous lines. Due to imperfection of the IMC technology system, it is particularly important to establish an efficient IMC system in radish. In this study, the effects of different factors on radish microspore embryogenesis were investigated with 23 genotypes. Buds with the largest population of late-uninucleate-stage microspores were most suitable for embryogenesis, with a ratio of petal length to anther length (P/A) in buds of about 3/4 ~ 1. Cold pretreatment was found to be genotype specific, and the highest microspore-derived embryoid (MDE) yield occurred for treatment of the heat shock of 48 h. In addition, the supplement of 0.75 g/L activated charcoal (AC) could increase the yield of embryoids. It was found that genotypes, bud size, as well as temperature treatments had significant effects on microspore embryogenesis. Furthermore, somatic embryogenesis-related kinase (SERK) genes were profiled by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which indicated that they are involved in the process of MDE formation and plantlet regeneration. The ploidy of microspore-derived plants was identified by chromosome counting and flow cytometry, and the microspore-derived plants were further proved as homozygous plants through expressed sequence tags-simple sequence repeats (EST-SSR) and genetic-SSR markers. The results would facilitate generating the large-scale double haploid (DH) from various genotypes, and promoting further highly efficient genetic improvement in radish.
Supplementary information:
The online version contains supplementary material available at 10.1007/s11032-022-01312-w.
... Genetic polymorphisms, such as SNPs and base insertions and deletions (InDels), which can all be called short variants, between the genotypes can be identified in target genes that are likely to affect the function of the gene product (such as a protein involved in a metabolic pathway) and, thereby, also affect the phenotype. It should be noted that short variants found through transcriptomebased analyses can also affect the function of the encoded protein by causing alternative splicing of mRNA (Luo et al., 2017). Markers have been developed based on transcriptome analyses for many crops (Kaur et al., 2012;Parra-González et al., 2012;Mantello et al., 2014;Webb et al., 2016;Nie et al., 2017;Tsehay et al., 2020) but not often fully exploiting the possibility of identifying interesting target genes through genotype comparisons. ...
Quinoa (Chenopodium quinoa Willd.) is a crop that has great potential for increased cultivation in diverse climate regions. The seed protein quality obtained from this crop is high concerning the requirements to meet human nutritional needs, but the seed protein content is relatively low if compared to crops such as grain legumes. Increased seed protein content is desirable for increasing the economic viability of this crop in order for it to be used as a protein crop. In this study, we characterized three genotypes of quinoa with different levels of seed protein content. By performing RNA sequencing of developing seeds, we determined the genotype differences in gene expression and identified genetic polymorphisms that could be associated with increased protein content. Storage nutrient analyses of seeds of three quinoa genotypes (Titicaca, Pasankalla, and Regalona) from different ecoregions grown under controlled climate conditions showed that Pasankalla had the highest protein content (20%) and the lowest starch content (46%). Our seed transcriptome analyses revealed highly differentially expressed transcripts (DETs) in Pasankalla as compared to the other genotypes. These DETs encoded functions in sugar transport, starch and protein synthesis, genes regulating embryo size, and seed transcription factors. We selected 60 genes that encode functions in the central carbon metabolism and transcription factors as potential targets for the development of high-precision markers. Genetic polymorphisms, such as single nucleotide polymorphisms (SNPs) and base insertions and deletions (InDels), were found in 19 of the 60 selected genes, which can be further evaluated for the development of genetic markers for high seed protein content in quinoa. Increased cultivation of quinoa can contribute to a more diversified agriculture and support the plant protein diet shift. The identification of quinoa genotypes with contrasting seed quality can help establish a model system that can be used for the identification of precise breeding targets to improve the seed quality of quinoa. The data presented in this study based on nutrient and transcriptome analyses contribute to an enhanced understanding of the genetic regulation of seed quality traits in quinoa and suggest high-precision candidate markers for such traits.
... Additionally, the present study showed that at least 9-12 repeats were detectable for monomer nucleotide SSRs, whereas the di-to hexanucleotide SSRs were explored mostly in the context of 5 to 8 repeats. These results indicated that more repeats were likely to be detected for repeat types with fewer nucleotides, which is consistent with reported studies in various plants [27], and the types of SSRs are affected by the selective pressure on different genes [28]. In summary, a large number of molecular markers were detected in the present study, indicating that high variation might exist in Z. armatum. ...
... The Perl script MISA was used to identify the simple sequence repeats (SSRs) according to the procedure described by Wang et al. [62]. Similar criteria to those employed in previous studies were used for the screening of high-quality SSRs in the five different samples collected in this study [20,27]. ...
Background:
Zanthoxylum armatum (Z. armatum) is a highly economically important tree that presents a special numbing taste. However, the underlying regulatory mechanism of the numbing taste remains poorly understood. Thus, the elucidation of the key genes associated with numbing taste biosynthesis pathways is critical for providing genetic information on Z. armatumand the breeding of high-quality germplasms of this species.
Results:
Here, de novo transcriptome assembly was performed for the five major organs of Z. armatum, including the roots, stems, leaf buds, mature leaves and fruits. A total of 111,318 unigenes were generated with an average length of 1014 bp. Additionally, a large number of SSRs were obtained to improve our understanding of the phylogeny and genetics of Z. armatum. The organ-specific unigenes of the five major samples were screened and annotated via GO and KEGG enrichment analysis. A total of 53 and 34 unigenes that were exclusively upregulated in fruit samples were identified as candidate unigenes for terpenoid biosynthesis or fatty acid biosynthesis, elongation and degradation pathways, respectively. Moreover, 40 days after fertilization (Fr4 stage) could be an important period for the accumulation of terpenoid compounds during the fruit development and maturation of Z. armatum. The Fr4 stage could be a key point at which the first few steps of the fatty acid biosynthesis process are promoted, and the catalysis of subsequent reactions could be significantly induced at 62 days after fertilization (Fr6 stage).
Conclusions:
The present study realized de novo transcriptome assembly for the five major organs of Z. armatum. To the best of our knowledge, this study provides the first comprehensive analysis revealing the genes underlying the special numbing taste of Z. armatum. The assembled transcriptome profiles expand the available genetic information on this species and will contribute to gene functional studies, which will aid in the engineering of high-quality cultivars of Z. armatum.
... However, it was found that the distributions of SNP and InDel at lncRNA loci were not uniform, where some were concentrated in certain regions, and some regions did not have a SNP or InDel footprint ( Fig. 5c; Fig. 6c). In addition, genomic DNA sequences typically have a higher SNP frequency than observed in transcribed regions [26]. Moreover, SNPs and InDels in coding regions are more likely to contribute to the phenotypes of plants, and the AS lncRNAs may only play a minor regulatory role, although the mechanism of action merits further investigation. ...
Alternative splicing (AS) is a key modulator of development in many eukaryotic organisms. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that play essential regulatory roles in various developmental processes and stress responses. However, the functions of AS lncRNAs during the initial flowering of tomato are
largely unknown. This study was designed to investigate the AS pattern of lncRNAs in tomato flower, leaf, and root tissues at the initial flowering stage. Using RNA-Seq, we found that 72.55% of lncRNAs underwent AS in these tissues, yielding a total of 16,995 AS events. Among them, the main type of AS event is alternative first exon (AFE), followed by retained intron (RI). We performed candidate target genes analysis on tissue-specific AS
lncRNA, and the results indicated that the candidate target genes of these lncRNAs may be involved in the regulation of circadian rhythm, plant immunity, cellulose synthesis and phosphate-containing compound metabolic process. Moreover, a total of 73,085 putative SNPs and 15,679 InDels were detected, and the potential relationship between the AS of lncRNAs and interesting SNP and InDel loci, as well as their numbers, revealed
their effects on tomato genetic diversity and genomic stability. Our data provide new insights into the complexity of the transcriptome and the regulation of AS.
... This dataset was primarily established as a reference transcriptome for gene mining in A. H11648, which also provided an important resource for molecular biology and genetic studies in Agave species. There were a series of potential uses with the dataset, such as meta-analysis, gene cloning, selection pressure analysis, molecular marker detection, alternative splicing analysis, multi-omics analysis, genome assembly, weighted gene co-expression network analysis, expression quantitative trait loci analysis, miRNA target site prediction, etc. [8,[11][12][13][14][15][16][17]]. ...
... This dataset was primarily established as a reference transcriptome for gene mining in A. H11648, which also provided an important resource for molecular biology and genetic studies in Agave species. There were a series of potential uses with the dataset, such as meta-analysis, gene cloning, selection pressure analysis, molecular marker detection, alternative splicing analysis, multi-omics analysis, genome assembly, weighted gene co-expression network analysis, expression quantitative trait loci analysis, miRNA target site prediction, etc. [8,[11][12][13][14][15][16][17]. ...
Sisal is widely cultivated in tropical areas for fiber production. The main sisal cultivar, Agave H11648 ((A. amaniensis × A. angustifolia) × A. amaniensis) has a relatively scarce molecular basis and no genomic information. Next-generation sequencing technology has offered a great opportunity for functional gene mining in Agave species. Several published Agave transcriptomes have already been reused for gene cloning and selection pressure analysis. There are also other potential uses of the published transcriptomes, such as meta-analysis, molecular marker detection, alternative splicing analysis, multi-omics analysis, genome assembly, weighted gene co-expression network analysis, expression quantitative trait loci analysis, miRNA target site prediction, etc. In order to make the best of our published transcriptome of A. H11648 leaf, we here represent a data descriptor, with the aim to expand Agave bio information and benefit Agave genetic researches.
... Next-generation sequencing (NGS) has been widely used to obtain an abundance of transcriptome sequences at greater depth for plant genome re-sequencing (van Dijk et al. 2014;Taheri et al. 2018;Luo et al. 2017). As one of the most central steps in gene expression, alternative splicing (AS) can generate multiple transcript variants from a single precursor mRNA (pre-mRNA). ...
Main conclusion
Alternative splicing (AS) events were identified and verified in cabbage by comparative transcriptome analysis. The corresponding markers were developed and the germplasm resources were identified.
Alternative splicing (AS) is a central regulatory mechanism that greatly contributes to plant gene expression and transcriptome diversity. A large body of evidence has shown that AS complexity is relevant for plant development, evolution, complexity, and adaptation. Both insertion/deletion (InDel) and single nucleotide polymorphism (SNP) are typically co-dominant inheritance markers and have abundant polymorphisms. These have been widely used for marker-assisted selection, genetic mapping, and germplasm identification in plants. However, little is known about the molecular mechanisms underlying AS events and the development of markers including SNP and InDel from the cabbage transcriptome. In this study, three cabbage transcriptome datasets were collected and aligned to the cabbage reference genome to analyze AS events and marker development. 31,524 AS events were identified from three cabbage genotypes, accounting for 20.8% of the total cabbage genes. Alternative 3′ splice site donor (A3SS) was the most frequent type of the four main AS events in cabbage. 70,475 InDels and 706,269 SNPs were identified with average frequencies of 1 InDel/6.9 kb and 1 SNP/0.7 kb, respectively. 71,942 potential SSRs were identified in 53,129 assembled unigenes with a density of 1 SSR/6.8 kb. The ratio of SNPs with synonymous/non-synonymous mutations was 1:0.65. 142 InDels and 36 SNPs were randomly selected and validated via Sanger sequencing and polymorphism was found among 66.2% of the InDels and 78.6% of the SNPs. Furthermore, 35 informative InDel markers were successfully used for genetic diversity analysis on 36 cabbage accessions. These results facilitate understanding of the molecular regulation mechanism underlying AS events in cabbage. They also provide molecular marker resource data for genetic mapping construction and germplasm identification, and facilitate the genetic improvement of cabbage via breeding.
... With the development of high-throughput transcriptome sequencing, it is possible to cap- ture the whole spectrum of transcripts and reveal their involvement in biological processes of a specific tissue or at specific stage of development of an organism (for review, see [37; 38-40]). The RNA-Seq analysis can also identify novel transcripts, as well as other variations in the genome, such as single nucleotide polymorphism (SNP), insertion and deletion (Indels) and alternative splicing (AS), which can not only provide information of the candidate genes, but can also be used for the development of molecular markers [41][42][43]. This information can be further utilized to understand the molecular mechanism or the biosynthetic pathways involved in the control of a trait [18,39,44]. ...
Yellow seed is a desirable trait in Brassica oilseed crops. The B. rapa var. Yellow Sarson carry unique yellow seed color genes which are not only important for the development of yellow-seeded oilseed B. rapa cultivars but this variant can also be used to develop yellow-seeded B. napus. In this study, we developed near-isogenic lines (NILs) of Yellow Sarson for the major seed coat color QTL SCA9-2 of the chromosome A9 and used the NILs to fine map this QTL region and to identify the candidate genes through linkage mapping and transcriptome sequencing of the developing seeds. From the 18.4 to 22.79 Mb region of SCA9-2, six SSR markers showing 0.63 to 5.65% recombination were developed through linkage analysis and physical mapping. A total of 55 differentially expressed genes (DEGs) were identified in the SCA9-2 region through transcriptome analysis; these included three transcription factors, Bra028039 (NAC), Bra023223 (C2H2 type zinc finger), Bra032362 (TIFY), and several other genes which encode unknown or nucleic acid binding protein; these genes might be the candidates and involved in the regulation of seed coat color in the materials used in this study. Several biosynthetic pathways, including the flavonoid, phenylpropanoid and suberin biosynthetic pathways were significantly enriched through GO and KEGG enrichment analysis of the DEGs. This is the first comprehensive study to understand the yellow seed trait of Yellow Sarson through employing linkage mapping and global transcriptome analysis approaches.
... Furthermore, apart from use of mutually exclusive first or last exons, we also observe use of alternative 5 and 3 splice site selection in DAL3, DAL4, DAL12, DAL21, DAL32, DAL4, DAL3, DAL33, and DAL38. Studies in the model plant Arabidopsis thaliana, and other angiosperm species, indicate that this form of alternative splicing is more prevalent than the use of mutually exclusive exons (Severing et al., 2012;Zhang et al., 2015;Luo et al., 2017;Verhage et al., 2017). Among the P. abies MADSbox genes, these alternative splice sites often result in frame shifts and premature stop-codons shortly after the MADS-box region. ...
Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister clade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this sub-clade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P. abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses reveal the use of mutually exclusive exons of MADS-box gene isoforms during early bud development in P. abies, and we find that the large number of identified MADS-box transcripts in P. abies results not only from expansion of the gene family through gene duplication events but also from the generation of numerous splice variants.
... Population genetic variation therefore includes variation in how transcripts are spliced. Luo et al. (9) identified thousands of differential splicing events among three radish samples. Although some studies report a high degree of splicing conservation among taxa (10), others report varying degrees of splicing differentiation among taxa, with more dissimilar groups having greater divergence in splicing (11). ...
Significance
Alternative splicing is a form of genetic regulation that enables the production of multiple proteins from a single gene. This study is one of the first to investigate variation in alternative splicing during a major evolutionary transition. We analyzed RNA from wild and domesticated sunflowers to examine differentiation in splice patterns during domestication. We identified divergent splice forms that may be involved in seed development, a major target of selection during domestication. Genetic mapping revealed that relatively few regulatory switches affecting many proteins have been altered in domesticated sunflowers. Our findings indicate that differences in splicing arose rapidly during a recent evolutionary transition and appear to contribute to adaptation and population divergence.
