Global Transcriptome Sequencing Using the Illumina Platform and the Development of EST-SSR Markers in Autotetraploid Alfalfa

Abstract

BackgroundAlfalfa is the most widely cultivated forage legume and one of the most economically valuable crops in the world. The large size and complexity of the alfalfa genome has delayed the development of genomic resources for alfalfa research. Second-generation Illumina transcriptome sequencing is an efficient method for generating a global transcriptome sequence dataset for gene discovery and molecular marker development in alfalfa.Methodology/Principal FindingsMore than 28 million sequencing reads (5.64 Gb of clean nucleotides) were generated by Illumina paired-end sequencing from 15 different alfalfa tissue samples. In total, 40,433 unigenes with an average length of 803 bp were obtained by de novo assembly. Based on a sequence similarity search of known proteins, a total of 36,684 (90.73%) unigenes were annotated. In addition, 1,649 potential EST-SSRs were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 100 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 82 were able to amplify sequences in initial screening tests, and 27 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 10 alfalfa accessions.Conclusions/SignificanceThe present study provided global sequence data for autotetraploid alfalfa and demonstrates the Illumina platform is a fast and effective approach to EST-SSR markers development in alfalfa. The use of these transcriptome datasets will serve as a valuable public information platform to accelerate studies of the alfalfa genome.

De novo sequencing and characterization of seed transcriptome of the tree legume Millettia pinnata for gene discovery and SSR marker development

“…In the present study, 5710 putative EST-SSRs were identified from the transcriptome dataset. The distribution density of one SSR per 7.39 kb was higher than those reported for alfalfa (1/12.06 kb) (Liu et al 2013) and black gram (1/11.90 kb) (Souframanien and Reddy 2015), but was lower than those in chickpea (1/ 5.80 kb) (Garg et al 2011) and common bean (1/ 4.70 kb) (Wu et al 2014). Though the transcriptomes of these legume species were all generated by Illumina sequencing, the differences in SSR abundance might still be caused by inconsistency in genome structure or composition, dataset size, search method and criteria.…”

“…Transcriptome sequencing and de novo assembly resulted in 53,586 Pongamia unigenes with a mean length of 787 bp. Compared with the results from other legume species generated by the Illumina sequencing platform, the mean length of unigenes for Pongamia was longer than those for chickpea (523 bp) (Garg et al 2011), peanut (619 bp) , common bean (777 bp) (Wu et al 2014) and black gram (443 bp) (Souframanien and Reddy 2015), but shorter than those for alfalfa (803 bp) (Liu et al 2013), adzuki bean (1213 bp) (Chen et al 2015a) and mung bean (874 bp) (Chen et al 2015b). Moreover, the average sequencing depth of all unigenes was up to 130.7-fold.…”

“…For those EST-SSR markers with amplified products, only 17 exhibited polymorphism among 12 Pongamia germplasms. The polymorphic ratio of these markers was relatively low, yet it was still comparable to the ratios for some EST-SSR markers and genomic-SSR markers in legumes (Liu et al 2013;Chen et al 2015a;Wang et al 2015). Besides, the polymorphic ratio of SSR markers might also enhance with the number of germplasms tested.…”

“…In recent years, the NGS-based transcriptome analyses have already been applied in more than a dozen legume species (Garg et al 2011;Zhang et al 2012;Garg and Jain 2013;Liu et al 2013;Hiz et al 2014;Chen et al 2015a, b;Souframanien and Reddy 2015). In a previous study, we employed the Illumina sequencing platform to produce a large-scale collection of Pongamia transcripts from root and leaf tissues for studying their transcriptome changes under salt treatments (Huang et al 2012).…”

Genetic diversity studies of alfalfa germplasm (Medicago sativa L. subsp. sativa) of United States origin using microsatellite analysis

“…In the current study, to select suitable SSR primers, 27 primer pairs were screened using 9 individual genotypes from Arc, Orca, and Saranac cultivars (Liu et al, 2013). Amplification reactions were performed in final volumes of 10.0 µl as follows: 5.0 µl of 2× Taq MasterMix (BioTeke, Beijing, China), 2.0 µl of primer, 50 ng of DNA template, and 2.0 µl of doubled-distilled water.…”