Improvement Of The New Recessive Genic Male Sterile Line In Brassica Napus By Molecular Marker-Assisted Selection

The key problem in utilizing heterosis is that of producing potential hybrid seeds economically,which contains two aspects of meaning.The first one is the effective pollination system,such as male sterility.Currently,the main approaches for rapeseed hybrid production are cytoplasmic male sterility(CMS) and genic male sterility(GMS) in China. There are some disadvantages in CMS system,such as limited availability of restorer lines, sensitivity to temperature.On the other hand,GMS has more advantages,such as rich resources of restorers,stable and complete male sterility.Now GMS is widely accepted and utilized.Although the recessive genic male sterility(RGMS) 7-7365A (Bnms3ms3ms4ms4RfRf) has been used in rapeseed production,yet for its very complex genetic basis,the traditional methods of three-line improvement are involved in a great deal of test cross analyses.The second aspect is genetic difference of parents.Many breeding practices showed that appropriate genetic difference of parents was helpful to F₁ hybrid production.Professor Meng' lab created the new-typed Brassica napus 7-749(A^rA^rC^cC^c), which had A^r genome from B.rapa and C^c genome from Brassica carinata.There existed some differences between 7-749 and common Brassica napus,therefore the hybrids of them showed strong intersubgenomic heterosis.In order to take advantage of this RGMS line and the new-typed B.napus,we mapped the recessive genic male sterile genes in 7-7365A,and transferred the genetic background of the new-typed B.napus to 7-7365A,so as to improve the 7-7365A.The main results were as follows:1 Fine mapping of BnMs3 gene1.1 From a survey of 1,024 primer combinations,we identified 17 AFLP markers linked to BnMs3 gene in an NIL(near-isogenic lines) population through AFLP analysis combined with BSA(bulked segregation analysis).By integrating the previous markers reported by Ke (2005) linked to the BnMs3 gene,four of which showed polymorphlism.The NIL population consisting of 2000 individuals was used to analyse the linkage of all markers,as a result,they were located on either side of the BnMs3 gene.EA01MC12 and P05MC11₃₅₀,the nearest markers,were at a distance of 0.1 and 0.5 cM,respectively.All markers were sequenced and primers were designed,five of which(P05MG05,P11MG02,P03MG04,P05MC11₂₅₀, EA09P06) were converted into SCARs successfully.1.2 In order to determine the location of the BnMs3 gene in the published B.napus genetic maps,we used two DH populations derived from Tapidor×Ningyou7 and Quantum×No.2127-17.Finally the gene was mapped to the N19 of B.napus genetic maps,and a co-dominant marker sR12384 was identified at a distance of 2.0 cM from the BnMs3 gene.1.3 The sequences of all the markers were delivered to NCBI web site for BLASTn analysis and the genes corresponding to the marker sequences were all located on the chromosome 5 of Arabidopsis.The homologous region of BnMs3 in Arabidopsis was between At5g13020 and At5g17020.2 Mapping of BnMs4 geneWe constructed an NIL population 7-736512AB.From a survey of 1,792 primer combinations,we identified 12 AFLP markers linked to BnMs3 gene in the NIL population 7-736512AB through AFLP analysis combined with BSA.All of markers were used to screen the 7-736512AB population including 1986 individuals,as a result,they were located on either side of the BnMs4 gene,AF6 and AF8,the nearest markers,were at a distance of 0.9 and 0.8 cM,respectively.All of markers were sequenced and three markers(P16MC08, P16MC12,AF6) were converted into SCARs successfully,among which AF6 was a co-dominant marker.Through PCR-walking,AF8 was also converted into a SCAR marker.3 Improvement of new recessive genie male sterile lines in B.napus by molecular marker-assisted selection(MAS)By MAS along with phenotype selection and quality analysis,we successfully transferred the genic male sterility genes of 7-7365A into the new-typed B.napus 7-749 using cross-self-backcross methods,Through three times of background selection and one foreground selection,we selected the sterile individuals and their sibmates,of which the exogenous genome content(A^rand C^c) ranged from 44.2%to 49.8%.We obtained a stable sterile line through continuous sibmating for two times.3.1 The combining ability analyses of yeild and yeild-related characters were done in the crosses from the new sterile line and 10 restorers within a random block design.The results demonstrated that the improved sterile line showed positive effects of value in the general combining ability(GCA) of silique numbers on main inflorescence,siliques per plant,seed yield per plant and yield per plot,compared with the original sterile line;the difference was very significant.For seed number per silique,the improved sterile line showed negative GCA value,and the difference reached a significant level.For 1000-seed weight,the improved sterile line showed positive GCA value,but the difference was not significant.In the 10 crosses,seven crosses showed positive heterosis compared with the control in the yield per plot character,and the heterosis advantage of three crosses from the improved sterile line with 7-1260,7-107 and 7-138 was up to more than 10%.3.2 Correlation analysis between GD and heterosis showed no significant correlation between the GD based on SSR markers,F₁ traits performance,SCA and GCA.4 Improvement of new recessive genie male temporary maintainer in B.napus by molecular-marker assisted selectionFifteen AFLP primer combinations were used to perform background selection on the fertile plants of the two segregating populations in two generations for two times, respectively.The results showed that the exogenous genome content of them ranged from 14.8%to 22.3%and 27.2%to 31.4%in two populations,respectively.Combined with agronomic characters and quality analysis,we selected two individuals which had high exogenous genome content in the two populations respectively for the next selection procedure.