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Cloning DNA markers, mapping and transformation in the tribe Triticeae crops

小麦族作物中 DNA 标记的克隆、定位和转化

基本信息

批准号：
01480038
负责人：
YASUMURA Yoshimasa
金额：
$ 4.16万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

Rye genome- and D genome-specific repeated sequences were cloned for the use of chromosome identification in newly reconstructed triticale genomes.The strategy for the cloning method of rye genome- or rye chromosome specific repeated DNA sequences was based on the selective reannealing of rye specific sequences by using rye chromosome addition wheat lines as follows : Rye chromosome addition wheat genomic DNA was digested with Ybo I and mixed with five times of sheared wheat genomic DNA, and then, the DNA mixture was denatured and annealed in phenol emulsion. So that only reannealed cohesive fragments enriched in rye specific sequences were legated into pUCi9 Bav HI sites. Recombinants of 145 and 77 clones were obtained from 5R and 6R chromosome addition wheat lines, respectively. Reassociation rates of Afbo I fragments were estimated to be 0.1-1.2%. Fourteen highly repetitive rye-specific clones were selected by differential dot blot hybridization of total rye and wheat DNA to Ybo I library. These clones were classified into six different rye genome-specific repeated sequences by southern hybridization. Clone pTS5R-l from 5R could probe a tandemly repeated 380bp-sequence family which was defined by Eco 0109I and consisted of various numbers of polymers caused by the mutation at the ends of Eco 0109I palindrome sites. RFLPs among inbred rye lines (S. cerea-l&) were detected with three rye specific clones.A tandemly repeated 2010bp-sequence family was cloned by Kpn I from Ae. Sq, va. rrosa. AB genomic DNA fragments was subtracted from Ybo I-digested common wheat genomic DNA by hybridizing sheared Tdzirus genomic DNA, and the rests were cloned to pUC19 Rain HI sites. Among 253 recombinants seven D genome-specific repetitive sequences were obtained.Above genome-specific repeated clones are useful DNA markers which identify chromosome fragments of R and D genomes.

为了在新构建的小黑麦基因组中进行染色体鉴定，克隆了黑麦基因组特异性重复序列和D基因组特异性重复序列，黑麦基因组特异性重复序列或黑麦染色体特异性重复DNA序列的克隆方法的策略是基于黑麦特异性序列的选择性再退火，利用黑麦染色体附加小麦系如下：黑麦染色体附加小麦基因组DNA经Ybo Ⅰ酶切后，与5倍剪切的小麦基因组DNA混合，然后在苯酚乳液中变性退火。因此，只有富含黑麦特异性序列的再退火的粘性片段才能连接到pUC 19 Bav HI位点。从5 R和6 R染色体附加系中分别获得145个和77个无性系。Afbo I片段的再结合率估计为0.1- 1.2%。利用黑麦和小麦总DNA与Ybo Ⅰ文库进行差异斑点杂交，筛选出14个高重复的黑麦特异性克隆。通过Southern杂交将这些克隆分为6个不同的黑麦基因组特异重复序列。从5 R中克隆的pTS 5 R-1可以探测到一个由Eco 0109 I定义的串联重复的380 bp序列家族，该家族由Eco 0109 I回文位点末端突变引起的不同数量的聚合物组成。黑麦自交系（S. cerea-l&）的2010 bp序列，并利用Kpn I技术克隆了一个2010 bp序列的串联重复家族。弗吉尼亚州，rrosa。通过与剪切后的Tdamus基因组DNA杂交，从Ybo I消化的普通小麦基因组DNA中减去AB基因组DNA片段，并将剩余片段克隆到pUC 19 Rain HI位点。在253个重组体中，获得了7个D基因组特异性重复序列，这些重复序列是鉴定R和D基因组染色体片段的有用DNA标记。