title: Global transcriptome sequencing using the Illumina platform and the development of EST-SSR markers in autotetraploid alfalfa.
abstract: BACKGROUND
  Alfalfa 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.
  
  
  RESULTS
  More 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
  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.
authors.affiliation: State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
authors.forename: Tianlong
authors.initials: T
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
authors.forename: Lichao
authors.initials: L
authors.lastname: Ma
authors.affiliation: Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou, China.
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, United States of America.
authors.forename: Patrick X
authors.initials: PX
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
authors.forename: Zhibiao
authors.initials: Z
authors.lastname: Nan
authors.affiliation: State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
authors.forename: Yanrong
authors.initials: Y
authors.lastname: Wang
mesh_terms: D030561:Databases, Nucleic Acid; D020224:Expressed Sequence Tags; D018745:Genome, Plant / Q000502:physiology*; D000455:Medicago sativa / Q000235:genetics* / Q000378:metabolism; D011123:Polyploidy; D059467:Transcriptome / Q000502:physiology*