Liu Zhipeng, Chen Tianlong, Ma Lichao, Zhao Zhiguang, Zhao Patrick X, Nan Zhibiao, Wang Yanrong
State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou, China.
PLoS One. 2013 Dec 12;8(12):e83549. doi: 10.1371/journal.pone.0083549. eCollection 2013.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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/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.
苜蓿是世界上种植最广泛的豆科牧草，也是最具经济价值的作物之一。苜蓿基因组的庞大规模和复杂性延缓了苜蓿研究基因组资源的开发。第二代Illumina转录组测序是一种为苜蓿基因发现和分子标记开发生成全球转录组序列数据集的有效方法。
方法/主要发现：通过Illumina双末端测序从15个不同的苜蓿组织样本中产生了超过2800万个测序读数（5.64Gb的纯净核苷酸）。通过从头组装总共获得了40433个单基因，平均长度为803bp。基于对已知蛋白质的序列相似性搜索，总共注释了36684个（90.73%）单基因。此外，从长度超过1kb的单基因中鉴定出1649个潜在的EST-SSR作为潜在的分子标记。随机选择总共100对PCR引物来验证组装质量并从基因组DNA开发EST-SSR标记。在这些引物对中，82对在初步筛选试验中能够扩增序列，27对引物成功扩增了DNA片段并在10个苜蓿种质中检测到大量多态性。
结论/意义：本研究为同源四倍体苜蓿提供了全球序列数据，并证明Illumina平台是苜蓿中开发EST-SSR标记的快速有效方法。这些转录组数据集的使用将作为一个有价值的公共信息平台，以加速苜蓿基因组的研究。
