Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China.
Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China; Department of Grassland Science, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
PLoS One. 2014 Mar 18;9(3):e92029. doi: 10.1371/journal.pone.0092029. eCollection 2014.
Sufficient codominant genetic markers are needed for various genetic investigations in alfalfa since the species is an outcrossing autotetraploid. With the newly developed next generation sequencing technology, a large amount of transcribed sequences of alfalfa have been generated and are available for identifying SSR markers by data mining. A total of 54,278 alfalfa non-redundant unigenes were assembled through the Illumina HiSeqTM 2000 sequencing technology. Based on 3,903 unigene sequences, 4,493 SSRs were identified. Tri-nucleotide repeats (56.71%) were the most abundant motif class while AG/CT (21.7%), AGG/CCT (19.8%), AAC/GTT (10.3%), ATC/ATG (8.8%), and ACC/GGT (6.3%) were the subsequent top five nucleotide repeat motifs. Eight hundred and thirty- seven EST-SSR primer pairs were successfully designed. Of these, 527 (63%) primer pairs yielded clear and scored PCR products and 372 (70.6%) exhibited polymorphisms. High transferability was observed for ssp falcata at 99.2% (523) and 71.7% (378) in M. truncatula. In addition, 313 of 527 SSR marker sequences were in silico mapped onto the eight M. truncatula chromosomes. Thirty-six polymorphic SSR primer pairs were used in the genetic relatedness analysis of 30 Chinese alfalfa cultivated accessions generating a total of 199 scored alleles. The mean observed heterozygosity and polymorphic information content were 0.767 and 0.635, respectively. The codominant markers not only enriched the current resources of molecular markers in alfalfa, but also would facilitate targeted investigations in marker-trait association, QTL mapping, and genetic diversity analysis in alfalfa.
苜蓿是一种异花授粉的同源四倍体植物，因此在进行各种遗传研究时需要足够的共显性遗传标记。随着新一代测序技术的发展，大量苜蓿转录序列已经产生，并可通过数据挖掘来鉴定 SSR 标记。通过 Illumina HiSeqTM 2000 测序技术共组装了 54278 条苜蓿非冗余基因。基于 3903 条基因序列，鉴定出 4493 个 SSR。三核苷酸重复（56.71%）是最丰富的基序类别，而 AG/CT（21.7%）、AGG/CCT（19.8%）、AAC/GTT（10.3%）、ATC/ATG（8.8%）和 ACC/GGT（6.3%）是随后的前五个核苷酸重复基序。成功设计了 837 对 EST-SSR 引物。其中，527 对（63%）引物产生了清晰可辨的 PCR 产物，372 对（70.6%）表现出多态性。在 M. truncatula 中，ssp falcata 的转移率高达 99.2%（523）和 71.7%（378）。此外，在 527 个 SSR 标记序列中，有 313 个在 M. truncatula 的 8 条染色体上进行了计算机定位。用 36 对多态性 SSR 引物对 30 个中国苜蓿栽培品种进行遗传关系分析，共产生 199 个可得分等位基因。观测杂合度和多态信息含量的平均值分别为 0.767 和 0.635。这些共显性标记不仅丰富了苜蓿分子标记的现有资源，而且还将有助于标记-性状关联、QTL 作图和苜蓿遗传多样性分析等靶向研究。
