摘要
用距离水稻抗白叶枯病基因Xa230.8 cM的EST标记C189,扩增Xa23的近等基因系CBB23的基因组片段(0.8 kb)为探针,筛选水稻明恢63的TAC文库和广陆矮4号的PAC文库,对获得的7个阳性克隆用酶切法和Tail-PCR法进行末端片段分离,获得15个末端片段,用于感病亲本金刚30和抗病亲本CBB23之间的多态性检测,发现7个末端片段在双亲间有多态性,分别为69B、70N、81N、45B、45N、84N和84B。用这些末端片段作RFLP标记,对金刚30/CBB23的F2群体进行检测和连锁分析,结果表明这些标记与Xa23的遗传距离依次为0.4、0.4、0.4、0.5、0.6、0.6和1.1 cM。虽然69B、70N和81N与Xa23的遗传距离均为0.4 cM,但序列比对揭示69B与70N的物理距离为35 kb,与81N为95 kb,69B距Xa23最近。三者与Xa23的遗传距离虽然相同,但物理距离存在很大差异。这些末端片段标记加密了Xa23基因一侧的遗传图谱,并使其遗传距离缩短到0.4 cM,加速了Xa23的定位进程,为Xa23的分离克隆奠定了重要基础。讨论了这种染色体步移方法的适用条件。
To develop closely linked markers for the rice bacterial blight resistance gene Xα23, a TAC library of Minghui 63 and a PAC library of Guanglu＇ai 4 were screened using the 0.8 kb PCR-product from rice near iso-genic line CBB23 as probe. The probe DNA was amplified with the primer of the EST marker C189 that is linked to Xα23 gene with a genetic distance of 0.8 cM. Fifteen insert-end-fragments from the obtained seven positive TAC and PAC clones were isolated by enzyme digestion or Tail-PCR. RFLP-Southern blotting analysis showed that seven of the 15 end-fragments displayed polymorphism between CBB23 and the susceptible recurrent parent Jingang 30. Consequently, individual plants from F2 population of Jingang 30/CBB23 were further RFLP-surveyed using the seven polymorphic end-fragments 69B, 70N, 81N, 45B, 45N, 84N and 84B as probes. The results showed that 69B, 70N, 81N, 45B, 45N, 84N and 84B were linked to the Xα23 gene with genetic distances of 0.4, 0.4, 0.4, 0.5, 0.6, 0.6 and 1.1 cM, respectively. However, BLASTN analysis against the genome sequence of Nipponbare showed that the physical distances between Xα23 and 69B, 70N and 81N were different even though the genetic distances were the same. The Xα23 gene locus was successfully fine mapped with the 7 end-fragment markers. The applieahility of this chromosome walking strategy for other crops was discussed.
出处
《中国水稻科学》
CAS
CSCD
北大核心
2006年第4期355-360,共6页
Chinese Journal of Rice Science
基金
农业部948项目(2001-203)
国家863计划资助项目(2002AA2Z1001-05
2001AA211061)
