丹参雄性不育与可育近等基因系差异片段的发掘

Screening of different AFLP fragments between near-isogenic lines of male sterile and fertile Salvia miltiorrhiza and their comparison analysis

利用雄性不育系培育新品种和生产杂种一代,在产量、品质等方面均具有独特优势。前期课题组已通过多代连续杂交获得丹参雄性不育与可育近等基因系。该文在前期工作基础上采用AFLP和BSA技术对378对引物进行筛选,发现7对引物和26个标记与丹参育性调控基因紧密连锁,由此构建出连锁遗传图谱。以前期发现的E11/M4-208标记为模板,运用染色体步移技术,在不育和可育系中分别扩增出2 028,2 027 bp的差异片段,发现4个突变碱基位点均处于内含子中。在所有差异标记中发现E01/M09-418,E05/M13-308,E05/M04-750,E01/M01-204 4个与丹参育性调控基因紧密连锁的标记,与拟南芥1,3,5号染色体相似性达100%。其中与育性调控基因距离很近(2.1 c M)的E01/M09-418与同处于拟南芥第3号染色体的核不育基因MS2相似度高达100%。不育系中获得的2 028 bp片段与MS2基因在两处相似度达100%。与拟南芥第5号染色体相似度100%的E05/M04-750,与杨树POP085-M05基因和拟南芥中低亲和力钙逆向转运蛋白序列具有较高相似性,且E非常低。该文遗传图谱的构建和功能性差异序列的发现,为后续准确锚定丹参基因组中育性调控基因及揭示其功能奠定了坚实基础。

There is distinctive advantage of using male sterile lines to breed new cultivar and produce hybrids,when compared with general breeding method on yield and quality. In our previous work,near-isogenic lines（ NILs） of male sterile and fertile Salvia miltiorrhiza have been obtained through continuous hybridization in many years. In this investigation,378 primer combination were screened by using AFLP and BSA technique,in which 26 markers amplified from seven primers were found to tightly link to male sterile gene.Based on these markers,two linkage genetic maps were constructed. A 2 027,2 028 bp fragment was amplifed from NILs of fertile and sterile S. miltiorrhiza,respectively,using genome walking technique and previous E11 / M4-208 marker as template. Four base mutations were found in intron when comparing both fragments. Among all different markers between NILs of male sterile and fertile S. miltiorrhiza,four was found to have 100% identities to chromosome 1,3 and 5 of Arabidopsis,namely,E01 / M09-418,E05 / M13-308,E05 / M04-750 and E01 / M01-204. The E01 / M09-418 marker was very close to male sterile gene of S. miltiorrhiza with distance of 2. 1c M,which also had 100% identities to male sterile gene MS2 in Arabidopsis. Both were distributed in chromosome 3 of Arabidopsis.The 2 028 bp fragment also had 100% identities to MS2 gene. Another E05 / M04-750 marker that had 100% identities to chromosome5 of Arabidopsis was found to have high identities to POP085-M05 gene of poplars and low affinity calcium antiporter CAX2 of Arabidopsis with very low E-value. The constructed genetic map and differential fragments with potential functions found in this study provide a solid foundation to lock male sterile genes in S. miltiorrhiza genome and to discover their functions.