Development Of Molecular Markers Linked To Sterility Genes Of A Recessive Genic Male Sterile Two-Type Line 9012AB In Brassica Napus L.

The utilization of heterosis in rapeseed is an efficient way to increase yield,to solve the problem between yield and quality,and to improve resistance/tolerance.The study and utilization of high efficient pollination control system is the key step in hybrid seed production.The genic male sterility(GMS) systems had great potential for hybrid seed production in virtue of the characteristics of complete and stable male sterility,no negative cytoplasmic effects on plant growth,and ease of transference of the male sterility gene(s) to diverse genetic backgrounds.However,the production of F₁ hybrid seed requires the removal of 50%of segregating fertile plants in the sterile female line which limits its utilization for production of hybrid seed.In 1993,Chen et al.reported a recessive genic male sterility(RGMS) line with an epistatic inhibitor gene,a 100%sterile population can be obtained via temporary maintainer,which made three-line systems available for hybrid seed production,and set up condition for the widely utilization of RGMS lines.The Brassica napus oilseed rape line 9012AB is a recessive epistatic GMS two-type line in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms3 and ms4) interacting with one pair of a recessive epistatic inhibitor gene(rf). Homozygosity at the rf locus(rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms3ms3ms4ms4 plants.In order to make the RGMS system a wider application,it is necessary to initiate further study on them.In this research,RGMS two-type lines 9012AB were used to identify AFLP markers linked to the Ms3/ms3 genes of RGMS lines 9012AB,construct genetic linkage map surrounding the Ms3/ms3 loci,and initiate comparative analysis between Arabidopsis and Brassica napus surrounding the Ms3/ms3 locus of 9012A.Main results of the present study are as follows:1.Crosses were made between the fertile plants and sterile plants of 9012AB,and then 40 F₂ plants from the F₁ plants were selfed and harvested.The investigation on fertility of the 40 F₂-derived F_2:3 populations showed that 11 F_2:3 populations were completely fertile with no fertility segregation,and the other 29 showed fertility segregation incordance with the ratio 3:1.It suggested that the fertile plants of 9012AB were heterologous in Ms3/ms3 locus.2.Two sterile bulks(BS1,BS2) and two fertile bulks(BF1,BF2) DNA samples prepared from 16 male sterile plants with the genotype of ms3ms3ms4ms4RfRf and 16 male fertile plants with the genotype of Ms3Ms3ms4ms4RfRf from the F₃s of the two-type line 9012AB were subjected to AFLP analysis.From the survey of 512 AFLP primer combinations,19 AFLP fragments were identified as being tightly linked to the Ms3/ms3 locus.Among them,only one co-dominant marker(YWE);8 markers(YWA,YWB,YWC,YWD,YWE,YWF,YWG,YWH,YWI) are in coupling phase with ms3 gene, and 9 markers(YWJ,YWK,YWL,YWM,YWN,YWO,YWP,YWQ,YWR) are in repulsion phage with ms3 gene.The closest marker linked to ms3/Ms3 was 1.3 cM and 3.1 cM respectively.A linkage map surrounding the Ms3/ms3 locus was constructed. All the AFLP fragments were cloned,sequenced.But only four fragments,YWB,YWD, YWJ and YWK were successfully converted into SCAR markers.3.753 SSR primer combinations were conducted to screen the markers linked to the Ms3/ms3 locus,and two out of them tightly linked to the target locus.4.A NIL population including 1240 individuals of 9012AB was developed for further study of the 4 SCAR markers,.The 4 markers(SYWB,SYWD,SYWJ,andSYWK) were located at one side of the target locus(Ms3/ms3) with a genetic distance of 5.1 cM,1.2 cM,0.1 cM,and 0.3 cM respectively.5.Four SCAR markers were used to breed new sterile lines and temporary maintaimer, only SYWD showed good polymorphism in many types of materials.The marker SYWD has been utilized for marker-assisted selection,and showed a good effect.