Gene Fine Mapping And Analysis To Common Bacterial Blight Of Common Bean

Common bean is one of the most important legumes for direct human consumption.However,the production of common bean is limited by common bacterial blight（CBB）around the world.It is urgently to plant new resistant varieties instead of susceptible lines.Mapping new resistance genes through molecular biology methods is a better way to breed new green resistance varieties.The objective of this study was to fine mapping resistance genes from HR45 and Longyundou5 based on germplasm resources screening of common bean.Relative candidate gene was analyzed in this study.The main results were obtained as follows.1.Genetic analysis of resistance genes from HR45The F₁,F₂ and F_2:3 derived from a cross between HR45 and Bilucaidou were evaluated using needle method in greenhouse.The disease severity ratings of F₁ reciprocal crosses were 4.27 and 4.33 at14 days after inoculation（DAI）,4.67 and 4.73 at 21 DAI,respectively.So the disease severity ratings of F₁ reciprocal crosses were basically consistent.It showed that the resistance of HR45 is nuclear inheritance.In addition,F₁ plants showed moderate resistance to Xap isolate,which located between resistance and suspectible parents.The result indicates CBB resistance in HR45 is controlled by genes with incomplete dominance.The phenotype of F₂ population was continual and had quantitative character inheritance.Similarly,the segregation ratio of F_2:3 families did not meet the Mendel’s law of segregation.These results indicate that CBB resistance in HR45 was controlled by multiple genes.2.QTL mapping of HR45537 polymorphism markers were identified in parents from 2,802 different SSR motifs,and then detected the resistance and susceptible pools of F₂ plants.Polymorphism markers were found on chromosome Pv06 and Pv08,which means CBB resistance gene located at Pv06 and/or Pv08 of common bean.Combined with F₂ phenotype,we detected CBB resistance genes on Pv06 and Pv08.For Pv06,the gene located at between SSR markers p6s53 and p8s243 at both 14 and 21 DAI.For Pv08,we found two loci between markers p8s189 and p8b24,p8b27 and p8b28,the physical position of them was about8.5 kb.3.Gene fine mapping of HR4574 SSR markers and 125 In/Del markers were developed based on the results of initial mapping for target regions with genomic sequences,44 markers showed polymorphism between parents.Then constructed genetic linkage map and combined the results of phenotype,we detected resistance loci on Pv06 and Pv08,named qH6 and qH8.qH6 was located at Pv06 between SSR markers p6s260 and p8s243,the physical position of marker interval was 326 kb,LOD score was 7.8 and 12.2,explained 6.4%and 7.5%of phenotypic variance at 14 and 21 DAI,respectively.qH8 was located at Pv08 between markers p8s433 and p8b17,the physical position of marker interval is about 18.5 kb,LOD score was 15.5 and 16.1,explained46.6%and 39.9%of phenotypic variance at 14 and 21 DAI,respectively.The effect of qH8 was higher than qH6.Three genes were predicted in the region of qH8.Gene1 encoding AAA-type ATPase、Gene2encoding PPR protein、Gene3 encoding anthocyanidin 3-O-glucosyltransferase 1-like protein.4.Candidate gene analysis of HR45Structures prediction of these genes revealed that Gene1 belongs to UBA_like protein family,Gene2 belongs to PPR family,Gene3 belongs to UDPGT family.Blast analysis for three candidate genes showed that the CDS and promoter sequence of Gene2,Gene3 were polymorphic between resistant and susceptible parents.The expression of candidate genes was analyzed using RT-PCR,the relative expression levels of Gene1 was basically consistent between resistant and susceptible parents;Gene2 in HR45 was slightly less than Bilucaidou,while Gene3 in HR45 was a little more than Bilucaidou.Three genes’expression was induced by pathogen in resistant and susceptible parents.Of them,the relative expression levels of Gene3 changed significantly in resistance parent after inoculation.In resistant parent,the expression level of Gene3 reached peak at 48 h,then stable expression.The results of VIGS to reduce candidate gene silence showed that Gene3 may be a candidate gene for CBB resistant of HR45.5.Gene mapping of Longyundou5Longyundou 5,a Chinese cultivar in Heilongjiang,displays resistance to the Xff strain.To identify the genetic mechanisms behind this resistance,we crossed Longyundou5 with a susceptible genotype to develop a mapping population of F₂ plants.A major QTL at 14 and 21 DAI was mapped on chromosome Pv10 with LOD scores of 6.41 and 5.35,respectively.This locus was in a 270 kb interval between new SSR markers p10s174 and p10s244.The candidate region including ten candidate genes could encode defence response proteins responding to CBB pathogens,such as PKs,oxidase,transcription factors,etc.Four pairs each of epistatic QTL for CBB resistance were detected at 14 and21 DAI.Phenotypic variation explained by the epistatic QTL ranged from 7.2%to 12.2%and 7.7%to8.8%at 14 and 21 DAI,respectively.These results confirmed the importance of epistasis in CBB resistance in common bean.