摘要
为了获得3种大白菜细胞质雄性不育系Ogu CMS,Pol CMS,CMS96和保持系间的多态性以及定位大白菜CMS96不育系所属的不育类型,利用设计的atp6,orf222单一和混合引物PCR扩增3组11份同核异质大白菜细胞质雄性不育系和保持系mtDNA。结果表明,atp6引物在大白菜Ogu CMS不育系扩增的200 bp片段为其特异带;orf222引物仅在大白菜Pol CMS和CMS96不育系有扩增产物,但二者有3点完全不同:大白菜Pol CMS不育系扩增产物为675bp,CMS96不育系扩增产物为669 bp,二者相差6个核苷酸,后者定名为大白菜CMS96-orf222。大白菜CMS96-orf222与甘蓝型油菜Nap CMS的nad5c基因和Nap-orf222基因同源性均为99%,E值为0.0;大白菜Pol CMS的675 bp序列具有ORF224开放阅读框,没有保守结构域,而大白菜CMS96的669 bp序列具有ORF222开放阅读框和保守结构域YMF19。另外,atp6和orf222混合引物多重PCR扩增产物存在明显多态性:800 bp为大白菜保持系的差异带型;2 300 bp和1 500 bp为大白菜Pol CMS不育系特异带型;200 bp为大白菜Ogu CMS不育系特异带型;690 bp为大白菜CMS96不育系特异带型。该方法仅用一次PCR反应快速地将3种大白菜细胞质雄性不育系和保持系一次性全部区分开,为大白菜分子育种和常规育种更好地相结合提供了简单、快速、准确和重复性好的方法和手段。
To study specific fragments linked to Pol CMS, Ogu CMS, CMS96 and to identify sterile system of CMS96 in Chinese cabbage （ Brassica campestris L. ssp. pekinensis）, primers ofatp6, orf222 and their primer combinations were designed for PCR analysis in 11 Chinese cabbage mtDNAs. Results showed 200 bp fragments could only be amplified in all Ogn CMS by atp6 primer; Fragments of 669 bp in CMS96 and 675 bp in Pol CMS could only be amplified by orf222 primer. Further study showed that ORF（open reading frame）in Pol CMS 675 bp fragments was ORF224, while it was ORF222 in CMS96 669 bp fragments .669 bp fragment in CMS96 was named CMS96-orf222. Alignment with nucleotide acids showed CMS96-orf222 in Chinese cabbage was 99 % homological to nad6c and oqe222 of Nap CMS in Brassica hapus. The E-value was 0.0. CMS96-ORF222 owned a putative conserved domain YMF19 which was similar to the mitochondrial membrane protein YMF19, while 675 bp fragments in Pol CMS did not. Meanwhile, by muhi-PCR technique with atp6 and orf222primer combinations, polymorphic results were obtained： 200 bp fragments were specific for Ogu CMS; 800 bp fragments were different bands for maintainer; 1 500 and 2 300 bp fragments were specific for Pol CMS;690 bp fragments were specific for CMS96. A rapid molecular technique for distinguishing 3 CMS types in Chinese cabbage were founded. Therefore, 3 CMS systems in Chinese cabbage were different in the rearrangement of mtDNAs. Now the chimeric rearrangement of mtDNA in CMS96 is obtained and studied.
出处
《华北农学报》
CSCD
北大核心
2007年第6期53-59,共7页
Acta Agriculturae Boreali-Sinica
基金
北京市自然基金项目(6012008)
北京市委组织部优秀人才项目(200503)资助
