AFLP Markers For The Dominant Genic Male Sterility Gene And SSR Fingerprint Of Cultivars (Lines) In Brassica Napus L.

Rapeseed is one of the major oilseed crops in China. Breeders still face the common and main goal of improving the production level of rapeseed. Li et al (1985) had proposed for the first time that the sterility of dominant genic male sterility (GMS) followed a digenic mode of inheritance with epistatic interaction, and established two-line system and three-line system as GMS breeding models. This hypothesis and models made heterosis utilization of GMS to be a primary strategy in Brassica napus L. breeding.The dominant GMS is an excellent system in hybrid breeding for its stable and complete sterility, impervious to environment, vigorously growth and easy to transform sterile and fertile genes. In three-line system, homozygous GMS two-type line is a foundation material selected from hybrid of restorer and sterile plant of heterozygous two-type line. The selection depends on analyzing of segregation rate between fertile and sterile hybrids. To make this system a wider use, it is necessary to develop molecular makers of its sterile genes and analyze the diversity of parental lines to make the most optimizes hybrids.The present study includes (1) breeding of a homozygous GMS two-type line from B. napus heterozygous two-type line 70056AB and a restorer line 9548; (2) developing AFLP (Amplified Fragment Length Polymorphism) molecular markers of male sterility gene based on 70056AB sib-mating population by BSA (Bulked Segregant Analysis) method; (3) analyzing the SSR fingerprint of parental lines including restorer 9548 in order to gain the genetic distance data for breeding program. Main results of the present studies are as following:1. The genotype of restorer line was identified as msmsRfRf after crossed sterile plant of heterozygous two-type 70056AB with restorer 9548 and investigated the sterility of F1, F2 and F3 populations. The investigation results showed that Fl plants were 100% fertile, F2 populations were tested to fit the segregation rate of 1 : 1 by x~2 test, and F3 population had the ideal fertility segregation. These results would lead to successful selection of homozygous GMS two-type line in the near future.2. Fertile bulk (A) and sterile bulk (B) were constructed in the sib-mating population 70056AB (70056A×70056B) by BSA method and were used in the following optimization. The protocol of AFLP molecular marker approach was optimized in restriction endonucleases digestion, ligation and dilution of templates. The two-step method, i.e. separating the digestion and ligation steps, was confirmed to be better for the digestion activity of restriction enzymes and result in legible and repeatable bands. The template dilution was not an important parameter in two-step method and had little influence to amplification results. Whereas in one-step method, main differential fragments appeared in the same sample only with different dilution grades of templates, which misled the primer screening result.An optimized AFLP protocol were then confirmed as following: genomic DNA were incubated for 5 hr at 37℃ with restriction enzymes, then transferred to 65℃ bath for 45min; after 10hr of...