Large-scale development of expressed sequence tag-derived simple sequence repeat markers by deep transcriptome sequencing in garlic (Allium sativum L.)
Garlic (Allium sativum L.) is an economically important crop and has significant value as a food, spice, and medicine. The lack of simple sequence repeat (SSR) markers is a major obstacle in genetic studies of garlic. In order to develop SSR markers on a large scale, we performed transcriptome analysis using Illumina pair-end sequencing. Approximately 69.7 million clean sequence reads were generated, and these reads were eventually assembled into 135,360 unigenes. Of these, 56,953 (42.1 %) unigenes were annotated for their function. Examination of SSR loci in these 135,360 ESTs identified 2446 SSRs. Because 940 of the loci were located on the end of the EST, only the residual 1506 SSRs were flanked by designing primer pairs complementary to regions, and these regions were designated as SSR markers. Within these markers, the trinucleotide repeat motif was the most abundant type (66.1 %), with the AGA/TCT and GAA/TTC motifs occurring most frequently. Using 200 randomly selected EST-SSRs, 194 markers were successfully amplified in garlic and 155–186 SSRs in five other Allium species. This suggests that the markers were good quality and had high cross-species transferability. The EST-SSRs developed in this study represent the first large-scale development of SSR markers for garlic. These SSRs could be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping, and cultivar fingerprinting.
KeywordsGarlic Transcriptome Illumina sequencing SSR marker Transferability
We kindly thank the Novogene Bioinformatics Institute for its assistance in original data processing and related bioinformatics analysis. This work was supported by grants from The Agricultural Science and Technology Innovation Program (ASTIP) and National Modern Agro-industry Technology Research System.
- Cunha C, Hoogerheide E, Zucchi M, Monteiro M, Pinheiro J (2012) New microsatellite markers for garlic, Allium Sativum (Alliaceae). Am J Bot e17–e19Google Scholar
- Ipek M, Ipek A, Simon PW (2003) Comparison of AFLPs, RAPD markers, and isozymes for diversity assessment of garlic and detection of putative duplicates in germplasm collections. J Am Soc Hortic Sci 128:246–252Google Scholar
- Kamenetsky R, Shafir I, Zemah H, Barzilay A, Rabinowitch H (2004) Environmental control of garlic growth and florogenesis. J Am Soc Hortic Sci 129:144–151Google Scholar
- Kamenetsky R, Faigenboim A, Mayer E, Michael T, Gershberg C, Kimhi S, Esquira I, Shalom S, Eshel D, Rabinowitch H et al (2015) Integrated transcriptome catalogue and organ-specific profiling of gene expression in fertile garlic (Allium sativum L.). BMC Genomics 16:12PubMedCentralCrossRefPubMedGoogle Scholar
- Luro FL, Costantino G, Terol J, Argout X, Allario T, Wincker P, Talon M, Ollitrault P, Morillon R (2008) Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other Citrus species and their effectiveness for genetic mapping. BMC Genomics 9:287PubMedCentralCrossRefPubMedGoogle Scholar
- Wang HB, Jiang JF, Chen SM, Qi XY, Peng H, Li PR, Song AP, Guan ZY, Fang WM, Liao Y, Chen FD, Chen FD (2013) Next-generation sequencing of the Chrysanthemum nankingense (Asteraceae) transcriptome permits large-scale unigene assembly and SSR marker discovery. PLoS One 8:e62293PubMedCentralCrossRefPubMedGoogle Scholar
- Zhang D, Choi D, Wanamaker S, Fenton R, Chin A, Malatrasi M, Turuspekov Y, Walia H, Akhunov E, Kianian P et al (2004) Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.). Genetics 168:595–608PubMedCentralCrossRefPubMedGoogle Scholar