| Clubroot an obligate parasite specific infects Brassica species is a worldwide soil-borne disease.This disease spreads rapidly these years in China.Except for founded in some province such as Hubei,Anhui,Yunnan,the disease forcibly occupies more than three million mu fertile farms in Sichuan.Now it spreads faster than ever before following the development of agricultural mechanization.Clubroot threatens to destroy the industry of canola in China,because there are no effective control methods.Clubroot resistant breeding is considered to be the best methods to control the disease.Considering the lack of resistant varieties and resistant resources,it is significant to keep canola production security with the methods of clubroot resistant breeding.To solve the problems above we focus on such researches as following:1.Transfer the new resistant locus located in R genome to canola with distant hybridization methodAllotetraploid material RRCC generated through artificially synthesized with radish(RR=18;Raphanus sativus)and cabbage(CC=18;Brassica alboglabra Bailey)is resistant to physiological races No.2,4,7 and 10 of clubroot.The resistant locus was demonstrated located in R genome.In order to transfer this locus to canola we successfully got the F1(ARCC)hybrids with embryo rescue technique after the cross between RRCC with Huashuang No.5.Then we got the BC1 and BC2 generations after cross with the backcross parent Huashuang NO.5.By observing the character of chrosmos pairing during meiosis with GISH,BAC-FISH technique and got the results as following:(1)The level of intergenomic pairing between A and R genomes in the F1 hybrid was significant high,allosyndetic bivalents formed in 73.53% PMCs indicative of a significant level of homologous recombination between two genomes and high probability of incorporating chromosomal segments/genes from R-genome into A/C-genomes.(2)The frequence of pollen fertility of generations increased gradually.We even screened resistant individuals with highest pollen fertility(98.70%),which had the normal chromosome number(2n=20A+19C).(3)F1 hybrid was immunity to Plasmodiophora brassica NO.4.The resistant percentage of BC1 and BC2 progeny was 77%,13.51% respectively.Furthermore,we had got the normal resistant canola in BC2 generation,which proved the feasibility of this method.2.Development of closely linked markers of Pb Ba8.1 conferring canola resistance to Plasmodiophora brassicaeThere were 5 resistant locus in turnip ECD04(Brassica rapa)resistant to different Plasmodiophora brassica races.Pb Ba8.1 locus was specifically resistant to physiological races No.4 which is the dominant Plasmodiophora brassicae race in China.In order to develop new resistant variety with expected locus,marker-assistant selection technique(MAS)was used in the breeding process in which ECD04 was used as a donor for crossing with an elite canola Huashuang No.5.During this process linkage markers were developed based on next-generation sequencing and comparative genome studies and got the results as following:(1)We developed 36762 In Del markers based on the comparative between the ECD04 and Huashuang No.5 genomes.The average marker density was 154.0/MB.There were 4181 markers located in the 5 reisitant locus.Further more,two marker A08-300 and Caps-134 close linkage to Pb Ba8.1 were developed.(2)Pb Ba8.1 is probably the allele of Crr1 a.A part sequence of Pb Ba8.1was cloned based on the sequence of Crr1 a with homology-based cloning method.A marker named Caps-134 was developmented according to the result of sequence difference.(3)A new resistant variety named Huashuang No.5R was obtained with A08-300 and Caps-134 markers selection.Field test results showed the new line was immunity to the pathogen in Anhui,Hubei and Sichuan provinces. |
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