Mapping Of TuMV Resistance Gene Retrcs03 In Chinese Cabbage

Chinese cabbage（Brassica rapa L.ssp.pekinensis,AA,2n=20）belongs to the Brassica genus and is native to China.It has important economic and edible value,is one of the most important vegetable crops in Asia,especially in countries such as China,South Korea and Japan.Virus disease is one of the most harmful diseases in the production of Chinese cabbage,and it occurs in all parts of the country.Especially in recent years,with the scale of vegetable production and the deterioration of the ecological environment,virus disease have become more serious,reducing crop yields and product quality,which has seriously affected the healthy and sustainable development of China”s vegetable industry.In terms of harm to Chinese cabbage,Turnip mosaic virus（Tu MV）is the most important pathogen.Chemical control is not long-lasting and seriously pollutes the environment.At present,the most effective and sustainable control measure is to cultivate disease-resistant varieties,and the breeding of disease-resistant varieties requires good technical methods.Marker-assisted selection（MAS）has emerged at the historic moment,compared with traditional breeding methods,it is more accurate and efficient,which can reduce the workload and greatly speed up the breeding process of new varieties.First,we selected“73”（the high-generation inbred line Tu MV resistant material）as the resistant parent（P₁）and“06-247”（Tu MV susceptible inbred line material）as the susceptible parent（P₂）to construct in the F₁,BC₁ and F₂ generation populations,Tu MV-C₄ was inoculated and identified to all individual plants,and the genetic laws of resistance were analyzed based on the results.Taking 180 strains of BC₁ population as the research object,using the Bulked Segregation Analysis（BSA）and SSR（Simple Sequence Repeats）marker technology to locate the Tu MV resistance gene carried by the parent“73”and 14 linkage markers obtained from the new screening,in which In Del marker Br ID101449 was co-segregated with gene retrcs03.Secondly,we used 8 high-generation inbred lines（inbred 8 or more）of Chinese cabbage Tu MV resistance materials（“73”,“8407”,“zao 219”,“07-372”,“07-14”,“322”,“826”and“07-55”）were the parents,and F₁ and F₂ populations were constructed by crossing and selfing as experimental materials,using the susceptible material“Guan 291”as the control material for Tu MV inoculation,the similarity and similarity study of the Chinese Tu MV resistance site was carried out.The resistance site of“8407”is allelic.The specific results are as follows:1.In the previous research of this research group,we used the Chinese cabbage Tu MV resistant material“73”and the susceptible material“06-247”as parents to construct F₂ and BC₁segregated populations,and identified a recessive Tu MV resistance gene retrcs03 and preliminary screening three molecular markers Br ID90143（4.2 c M）,Br SSR4068（4.2 c M）and Br ID10645（10.1 c M）linked to the gene were identified.Based on this study,using parental“73”and“06-247”as controls,16 strains of F₁,180 strains of BC₁ and 529 strains of F₂ were inoculated with Tu MV C₄ strain.The resistance identification results showed that all the single plants in F₁ group were susceptible,and BC₁ group showed resistance and susceptibility separation.Among them,89 strains were resistant（R）,91 strains were infected（S）,the number of resistant and susceptible plants In accordance with the 1:1 separation ratio(χ_c²=0.02<χ²_0.05=3.84),the number of resistant strains in the F₂ population was 122,and the number of susceptible strains was 407.The number of resistant and susceptible strains was in accordance with the 1:3 separation ratio(χ_c²=1.05<χ²_0.05=3.84),which was consistent with the previous research results.2.Takingthe resistant parent“73”and the susceptible parents“06-247”and 180 individual plants of the BC₁ population as the research object,using the BSA method to amplify the genomic DNA of the two parents using the designed primers,and screening the molecular markers linked to the Tu MV resistance gene retrcs03,and 300 pairs of primers were successfully expanded.There were 75 pairs of primers showing polymorphism between the parents.There were 26 primer pairs with polymorphism in the parent,resistance,and sensation pool.3.Further use the 26 pairs of primers selected above to amplify a single plant of the BC₁population.There are 14 markers that have a linkage relationship with the gene retrcs03.Combined with the results of disease resistance identification,use Join Map4.0 software to analyze and calculate the linkage distance of each marker,and the linkage distances of the markers were SAAS＿m Br4091（10.1 c M）、Br ID101487（4.2 c M）、SAAS＿m Br4161（4.7 c M）、SAAS＿m Br 4077（3.9 c M）、SAAS＿m Br4274（1.0 c M）、SAAS＿m Br4131（0.7 c M）、SAAS＿m Br4112（0.7 c M）、Br ID10766（0.6 c M）、SAAS＿m Br4174（0.6 c M）、SAAS＿m Br4183（0.6 c M）、Br ID10694（0.6 c M）、SAAS＿m Br4285（0.1 c M）、SAAS＿m Br4298（0.1 c M）,the In Del marker Br ID101449 was co-isolated with the gene retrcs03.Through the implementation of this project,14 molecular markers linked to the Chinese cabbage recessive Tu MV resistance gene retrcs03were newly selected.4.Among the 14 molecular markers obtained by the new screening,the In Del marker Br ID101449 was co-separated with retrcs03.Sequencing this marker showed thatthe amplified band of primer Br ID101449 in the resistant parents was 96bp,the size of the expanded band in the susceptible parents was 100bp,and the resistance marker deleted the base sequence ATTA at54-57.The sequence of the resistance marker Br ID101449 was further compared with the sequence of the cabbage genome B.rapa（Chromosome v1.5）,the results show that the physical location of the marker is A04:5560777...5560876（v1.5）.Searching for genes around the marker,the results showed that the gene Bra032679 is located near the marker,and gene annotations indicate that the gene is a interferonγinducible protein,which may be related to resistance.5.Using 8 high-generation inbred lines of Chinese cabbage Tu MV resistance materials（“73”,“8407”,“zao219”,“07-372”,“07-14”,“322”,“826”,and“07-55”）as parents,by crossing and inbreeding,F₁ and F₂ populations were constructedby hybridization and selfing as experimental materials,and the susceptible material“guan 291”was used as a control material for Tu MV inoculation.The study of Tu MV resistance alleles in Chinese cabbage was conducted to clarify the resistance allelic relationship between them.The results showed that the resistance sites in“322”,“07-55”and“73”were allelic;the resistance sites in“Zao 219”,“07-372”,“07-14”,“826”and“8407”were allelic;the resistance sites in“73”,“322”,and“07-55”were linked to that in“8407”but not allelic.At least two different resistance genes exist in the eight resistance materials.