Inheritance Of Chlorophyll-deficient Mutant L638-y In Brassica Juncea L. And Molecular Markers For Chlorophyll-deficient Gene Gr1

Photosynthesis is the basis for living beings need to survive, and it is mainly carried outin the leaves, of which the chloroplasts play the key role in the process of photosynthesis. Theleaves present some color by the comprehensive influence of various pigments of chloroplast.Generally, the leaves present green. But some factors directly or indirectly affect the synthesisor degradation of chlorophyll, alter the chlorophyll content of leaves, and finally lead to theleaves' color of mutants have significant variation compared with the normal leaves' color. Sothese mutants are known as chlorophyll-deficient mutants. So far, chlorophyll-deficientmutants are found in almost all higher plants. Chlorophyll-deficient mutants are idealmaterials for studying the inheritance of leaf color, chloroplast ultra structure anddevelopment, chlorophyll biosynthesis, photosynthetic physiology, pigment-proteincomplexes, location of leaf color mutant gene, identification of gene function, thenucleocytoplasmic gene interactions, and provide new elite germplasm resources for breedingof high light efficiency, and also serve as marker traits at seedling stage for identification ofthe purity of the hybrid seeds.Chlorophyll-deficient mutant L638-y was discovered from the normal line L638-g inBrassica juncea L. by our research group, and selfed for five generations. In this study, thechlorophyll-deficient mutant L638-y was crossed with the two normal lines,Weiyuandahuangjie and2598, respectively. The resulting F₁, F₂and BC₁population were usedto investigate inheritance of chlorosis character by checking leaf color phenotype in theseprogenies. The220plants of the NIL (near-isogenic line) derived from the progeny of thecross between L638-y and2598were used to map one of the chlorophyll-deficient genes-gr1by using SRAP, SSR, RAPD and AFLP with bulk segregant analysis strategy. The mainresults obtained are as follows:1. The chlorophyll-deficient mutant L638-y was crossed reciprocally with the two normallines, Weiyuandahuangjie and2598, respectively. Their F₁showed green, so the mutant wascontrolled by recessive nuclear gene, and wasn't influenced by cytoplasm. Their F₂and BC₁showed separation, including both green and yellow phenotype. Through χ_c²test, the greenand yellow phenotype of reciprocal F₂complied with the15:1segregation ratio and the greenand yellow phenotype of BC₁from L638-y×(L638-y×Weiyuandahuangjie), L638-y× (Weiyuandahuangjie×L638-y), L638-y×(2598×L638-y) complied with the3:1segregation ratio, which showed that chlorosis character of L638-y was controlled by twopairs of recessive nuclear genes, namely gr1and gr2. So the genotype of L638-y wasgr1gr1gr2gr2, and the genotypes of Weiyuandahuangjie and2598were Gr1Gr1Gr2Gr2.2. Two each of DNA Pools of green leaf plants and yellow leaf plants were constructedand screened by45pairs of SRAP primers,560pairs of SSR primers,822RAPD primers,and380pairs of AFLP primers. Finally we found two pairs of AFLP primers EA4TG4(EA-CA/TG-TC) and EA7MC1(EA-CT/MC-AA), which could stablely detect differentbands between the green pool and the yellow pool. Then we used these two pairs of AFLPprimers to amplify other200plants of NIL. We calculated the genetic distances betweenmarkers and gr1, and drew the linkage map showing the AFLP markers surrounding gr1locusin B. juncea by using Mapmaker3.0software. The result showed that the two markers wereon both sides of gr1, and the genetic distance was33.6cM and21.5cM, respectively.3. We cloned and sequenced the two polymorphic bands, and analyzed homology usingBLAST in NCBI and TAIR database. We preliminarily deduced that the homologoussequence of target gene region located at the5th chromosome of Arabidopsis thaliana, thenwe could develop molecular markers linked to the chlorophyll-deficient genes by utilizing thesequence of5th chromosome of Arabidopsis thaliana in the future.