International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Hyderabad, 502324, India.
BMC Plant Biol. 2011 Mar 29;11:56. doi: 10.1186/1471-2229-11-56.
Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea.
A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme.
In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea.
木豆[菜豆属（Cajanus cajan （L.）Millsp.]是雨养农业中一种重要的豆科作物。尽管人们为了提高木豆的产量进行了协同研究，但在过去几十年中，木豆的生产力一直停滞不前，这可能是由于存在各种生物和非生物限制，而且可用的基因组资源不足以开展任何分子育种计划来加速作物改良，情况因此而恶化。本研究旨在为木豆提供更多的基因组资源，首次从 BAC 末端序列中大量开发 SSR 标记，并随后将其用于木豆的遗传作图和杂种鉴定。
用 HindIII（34560 个克隆）和 BamHI（34560 个克隆）两种内切酶构建了两个 BAC 文库，共产生了 88860 个 BAC（细菌人工染色体）末端序列（BES）。基于 BES 序列的同源性聚类产生了一组 >52K 的非冗余序列，包含 35Mbp 或 >4%的木豆基因组。对这些序列进行分析，以生成注释列表，并将 BES 划分为基因组部分（例如基因、反转录元件、转座子和未注释序列）。对 BES 进行微卫星或简单序列重复（SSR）的平行分析，鉴定出 18149 个 SSR，从中选择了 6212 个 SSR 进一步分析。共合成了 3072 对新的 SSR 引物，并在由 13 个分离感兴趣性状的作图群体组成的 22 个亲本基因型中测试了其长度多态性。总共鉴定出 842 个多态性 SSR 标记，这些标记将在木豆改良中具有应用价值。基于这些标记，为这个以前未被描述的基因组开发了第一张包含 239 个位点的基于 SSR 的遗传图谱。开发的 SSR 标记的实用性还通过鉴定每个杂种（ICPH 2671 和 ICPH 2438）的 42 个标记，用于商业杂交种育种计划中的遗传纯度评估得到了证明。
总之，虽然 BAC 文库和 BES 应该对基因组学研究有用，但 BES-SSR 标记和遗传图谱对于将遗传图谱与未来的物理图谱联系起来以及在木豆中进行分子育种也非常有用。
