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.
Highlights
Alfalfa (Medicago sativa L. subsp. sativa), a member of the Fabaceae family, is the most widely cultivated forage legume in the world and is the third most valuable crop in the United States, after corn (Zea mays L.) and soybean (Glycine max (L.) Merr.) [1]
Several framework genetic linkage maps have been constructed for diploid and tetraploid alfalfa using Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeat (SSR), Amplified Fragment Length Polymorphism (AFLP), and Singledose Restriction Fragments (SDRFs) markers
To globally and comprehensively cover the alfalfa transcriptome, total RNA was extracted from 15 different alfalfa tissues
Summary
Alfalfa (Medicago sativa L. subsp. sativa), a member of the Fabaceae family, is the most widely cultivated forage legume in the world and is the third most valuable crop in the United States, after corn (Zea mays L.) and soybean (Glycine max (L.) Merr.) [1]. 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/Significance: The 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
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