Coyne C J, McClendon M T, Walling J G, Timmerman-Vaughan G M, Murray S, Meksem K, Lightfoot D A, Shultz J L, Keller K E, Martin R R, Inglis D A, Rajesh P N, McPhee K E, Weeden N F, Grusak M A, Li C-M, Storlie E W
USDA-ARS Plant Germplasm Introduction and Testing Unit, Washington State University, Pullman, WA 99164-6402, USA.
Genome. 2007 Sep;50(9):871-5. doi: 10.1139/g07-063.
Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.
豌豆（Pisum sativum L.）的基因组约为4Gb，尽管存在广泛的基因间扩展，但似乎与基因组为0.2 - 0.4Gb的模式豆科植物具有保守的同线性。豌豆植物种质资源（PI）编号269818已被用于将遗传多样性渗入栽培种质库中。此处的目的是构建豌豆细菌人工染色体（BAC）文库，以便能够分离参与植物抗病性或控制经济重要性状的基因。这些BAC文库包含约3.2个单倍体基因组当量，由平均大小为105kb的部分经HindIII酶切的DNA片段组成，这些片段插入到2种载体中的1种。基于低拷贝oriT的T-DNA载体（pCLD04541）文库包含55680个克隆。基于单拷贝oriS的载体（pIndigoBAC-5）文库包含65280个克隆。通用叶绿体探针的菌落杂交表明，文库中约1%的克隆来自叶绿体。通过白/蓝菌落平板计数推断约有0.1%的空载体存在。通过两种重复方法测试了文库的实用性。首先，用低拷贝数序列探测高密度滤膜。其次，BAC平板池DNA成功用于PCR扩增9个已发表的豌豆抗病基因类似物（RGA）中的7个以及其他几个低拷贝数豌豆序列。鉴定出了编码特定序列的单个BAC克隆。因此，基于种质资源PI 269818构建的豌豆HindIII BAC文库将有助于分离抗病性及其他经济重要性状的潜在基因。
