Breeding Of Recessive Genic Male Sterility Line And Its Temporary Maintainers In Raperseed (Brassica Napus) By Marker-assisted Selection

Recessive GMS(RGMS) systems have playing an increasingly important role in rapeseed hybrid production,due to the stable and complete male sterility,no limitation of restorers and no negative cytoplasmic effect.9012AB is a recessive epistatic genic male sterility(REGMS) line derived from a natural mutant found in the Brassica napus accessions '9012'(Chen et al.,1993).This system overcomes the defect of traditional RGMS because a 100%male-sterile population(ms₃ms₃ms₄ms₄RfRf) could be produced and no need is required for removing 50%fertile plants of maternal line.It's the key step to select homozygous two-type line and its homologous temporary line because of the fact that the vast majority of breeding lines are restorers of 9012A,. Nevertheless,it is labour-cost and inefficient to breed male-sterile lines and temporary maintainer lines by traditional phenotype selection,since the male sterility is conditioned by the interaction of three pairs of recessive genes.Marker-assisted selection(MAS) is therefore the preferred solution to this problem.In this research,a backcross breeding procedure was applied in combination with MAS to breed new male sterile lines and TAMs, in which a spring type canola temporary maintainer line TAM was used as the donor parent o and another elite semi-winter type line Zhongsuang No.9(ZS9) as the recipient parent. The main results are as follows:1.Fertility investigation on the 845 plants of the F₂ population(TAM×ZS9) indicated that the ratio of fertile/sterile is 13:3,so it can be deduced that the genotype of ZS9 is Ms₃Ms₃ms₄ms₄RfRf or ms₃ms₃Ms₄Ms₄RfRf in the fertility related loci of RGMS.Analysis with the molecular markers linked with ms₃ allele further confirmed that the genotype of ZS9 is Ms₃Ms₃ms₄ms₄RfRf2.Using the SCAR markers P6 and WSC1 which linked with ms₃ and Rf,we identified 155 individuals genotyped ms_3ms₄ms₄rfrf in the F₂ population of 845 plants.75 individuals with good figure were further selected to backcross with ZS9 and testcross with 9012A.Twenty-three individuals were identified as the temporary maintainer line genotype (ms₃ms₃ms₄ms₄),from which four individuals having closest genetic background with ZS9 were picked out and selfed to obtain BC₁F₂ seeds.3.Four BC₁F₂ families consisting of 384 individuals were grown.Two different methods are applied to continue selection.Method No.1:we continueed using the two SCAR markers to select the population,and obtained 74 individuals which had the genotype of ms₃ms₃ms₄ms₄rfrf.After background analysis,10 individuals which have closest genetic background with ZS9 were picked out to backcross with ZS9 and testcross with 9012A.Based on the result of testcross F₁ generation in summer propagation,four individuals genotyped as temporary maintainer line and their related BC₂ individuals were selfed to obtain the BC₂F₂ populations.Method No.2:the codominant SSR marker was used to select heterozygous sterile plants(ms₃ms₃ms₄ms₄Rfrf) in BC₁F₂ population,from which some individuals with good figures were backcrossed with ZS9 and testcrossed with TAM.The fertile plants heterozygous in Rf locus from the BC₂ population were selected and selfed to obtain the BC₂F₂ generations.4.One hundred and forty-two individuals genotyped as ms₃ms₄ms₄rfrf have been selected in the BC₂F₂ populations.Twenty-two individuals having good figure have been selected to testcross with 9012A and selfed for obtaining BC₂F₂ individuals which have the genotype of temporary maintainer line.Simultaneity,the fertile individuals genotyped as Ms₃ms₃ms₄ms₄ were selected to hybridize with the homozygous sterile plants (ms₃ms₃ms₄ms₄RfRf).By investigation on fertility segregation ratio in summer propagation, homozygous sterile lines(ZS9AB) will be identified from the F₁ populations.The temporary maintainer line individuals from the BC₂F₂ populations will be used as the homologous maintainers of ZS9AB.The backcross procedure in combination with MAS established in this research not only set a good example for the improvement of recessive genic male sterility lines,but also provided new RGMS lines and their homologous temporary lines sharing similar genetic background with elite cultivar ZS9.