Joobeur T, Gusmini G, Zhang X, Levi A, Xu Y, Wehner T C, Oliver M, Dean R A
Department of Plant Pathology, Fungal Genomics Laboratory, North Carolina State University, Raleigh, NC 27965, USA.
Theor Appl Genet. 2006 May;112(8):1553-62. doi: 10.1007/s00122-006-0258-6. Epub 2006 Apr 8.
A bacterial artificial chromosome (BAC) library was constructed for watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly distributed on the genomes of melon or Arabidopsis. Twenty eight probes (representing 66% of the tested probes) from melon and 30 probes (65%) from Arabidopsis identified positive BAC clones. Two methods were implemented to identify SSRs from the positively hybridizing BAC clones. First, analysis of BAC end sequences revealed 37 SSRs. For the second method, pooled DNA of BACs identified by the melon probes was used to develop a shotgun library. The library was then screened with synthetic SSR oligonucleotides by hybridization. Sequence analysis of positively hybridizing shotgun clones revealed 142 different SSRs. Thirty eight SSRs were characterized using three watermelon cultivars, five plant introduction (PI) accessions of C. lanatus var lanatus and four PIs of C. lanatus var citroides. Of these, 36 (95%) were found to be polymorphic with up to six alleles per marker. Polymorphism information content values for polymorphic markers varied between 0.22 and 0.79 with an average of 0.53. The methods described herein will be valuable for the construction of a watermelon linkage map with SSRs evenly distributed on its genome that is anchored to the genomes of melon and Arabidopsis.
构建了西瓜（Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus）的细菌人工染色体（BAC）文库，其平均插入片段大小为106 kb，相当于21个单倍体基因组。该文库用于鉴定与均匀分布在甜瓜或拟南芥基因组上的探针锚定的BAC克隆。来自甜瓜的28个探针（占测试探针的66%）和来自拟南芥的30个探针（65%）鉴定出了阳性BAC克隆。采用两种方法从阳性杂交的BAC克隆中鉴定简单重复序列（SSRs）。首先，对BAC末端序列的分析揭示了37个SSRs。对于第二种方法，将由甜瓜探针鉴定出的BAC的混合DNA用于构建鸟枪法文库。然后用合成的SSRs寡核苷酸通过杂交筛选该文库。对阳性杂交的鸟枪法克隆进行序列分析，揭示了142个不同的SSRs。使用3个西瓜品种、5个西瓜栽培变种（C. lanatus var lanatus）的植物引进系（PI）和4个西瓜野生变种（C. lanatus var citroides）的PI对38个SSRs进行了特征分析。其中，36个（95%）被发现具有多态性，每个标记最多有6个等位基因。多态性标记的多态信息含量值在0.22至0.79之间，平均为0.53。本文所述方法对于构建一个基因组上均匀分布有SSRs且与甜瓜和拟南芥基因组锚定的西瓜连锁图谱将具有重要价值。
