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. 2012 Jul 16:13:316.
doi: 10.1186/1471-2164-13-316.

Development and validation of genic-SSR markers in sesame by RNA-seq

Haiyang Zhang  1 , Libin Wei, Hongmei Miao, Tide Zhang, Cuiying Wang
Affiliations

Development and validation of genic-SSR markers in sesame by RNA-seq

Haiyang Zhang et al. BMC Genomics. .

Abstract

Background: Sesame (Sesamum indicum L.) is one of the most important oil crops; however, a lack of useful molecular markers hinders current genetic research. We performed transcriptome sequencing of samples from different sesame growth and developmental stages, and mining of genic-SSR markers to identify valuable markers for sesame molecular genetics research.

Results: In this study, 75 bp and 100 bp paired-end RNA-seq was used to sequence 24 cDNA libraries, and 42,566 uni-transcripts were assembled from more than 260 million filtered reads. The total length of uni-transcript sequences was 47.99 Mb, and 7,324 SSRs (SSRs ≥15 bp) and 4,440 SSRs (SSRs ≥18 bp) were identified. On average, there was one genic-SSR per 6.55 kb (SSRs ≥15 bp) or 10.81 kb (SSRs ≥18 bp). Among perfect SSRs (≥18 bp), di-nucleotide motifs (48.01%) were the most abundant, followed by tri- (20.96%), hexa- (25.37%), penta- (2.97%), tetra- (2.12%), and mono-nucleotides (0.57%). The top four motif repeats were (AG/CT)n [1,268 (34.51%)], (CA/TG)n [281 (7.65%)], (AT/AT)n [215 (5.85%)], and (GAA/TTC)n [131 (3.57%)]. A total of 2,164 SSR primer pairs were identified in the 4,440 SSR-containing sequences (≥18 bp), and 300 SSR primer pairs were randomly chosen for validation. These SSR markers were amplified and validated in 25 sesame accessions (24 cultivated accessions, one wild species). 276 (92.0%) primer pairs yielded PCR amplification products in 24 cultivars. Thirty two primer pairs (11.59%) exhibited polymorphisms. Moreover, 203 primer pairs (67.67%) yielded PCR amplicons in the wild accession and 167 (60.51%) were polymorphic between species. A UPGMA dendrogram based on genetic similarity coefficients showed that the correlation between genotype and geographical source was low and that the genetic basis of sesame in China is narrow, as previously reported. The 32 polymorphic primer pairs were validated using an F2 mapping population; 18 primer pairs exhibited polymorphisms between the parents, and 14 genic-SSRs could be integrated into 9 main linkage groups.

Conclusions: 2,164 genic-SSR markers have been developed in sesame using transcriptome sequencing. 276 of 300 validated primer pairs successfully yielded PCR amplicons in 24 cultivated sesame accessions. These markers increase current SSR marker resources and will greatly benefit genetic diversity, qualitative and quantitative trait mapping and marker-assisted selection studies in sesame.

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Figures

Figure 1
Figure 1
Frequency distribution of the six perfect SSR unit types.
Figure 2
Figure 2
Polymorphism of the primer HS233 in 25 sesame accessions. 6% PAGE of 24 cultivar accessions and one wild species M: DNA marker; Lanes 1 ~ 25: Samples M1 ~ 25 (Additional file 2).
Figure 3
Figure 3
UPGMA dendrogram of the genetic relationships among 24 cultivated sesame accessions. The dendrogram was generated using the Jaccard similarity coefficient based on 32 polymorphic primer pairs.
Figure 4
Figure 4
Distribution of 14 new polymorphic SSR markers across the 9 linkage groups of the F2 backbone genetic linkage map. * new sesame genic-SSR markers.
See this image and copyright information in PMC

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