Investigation On The Genetic Diversity And Leaf Slow-greening Mutation Of Pummelo

Pummelo(Citrus maxima)is one of the three basic species of citrus and occupies an important economic position in the citrus industry.Pummelo is native in China and has been cultivated for over 4,000 years according to the record of Tribute of Yu.The seed-propagation mode and different geographical conditions contribute to the diversity of pummelo to day.However,the investigation and protection of pummelo germplasm was lagging behind.Some old varieties are missing for all kinds of causes.There are few reports about the diversity and the population structure of comprehensive pummelo germplasm from China.Most studies about pummelo focused on fruits phenotypes.Nevertheless,the study about pummelo leaves was limiting.In this study,pummelo accessions were collected nationwide.This germplasm collection contains local cultivars and landraces.Genetic diversity and population structure were analyzed by n SSR markers.The results provide genetic evidence for the dispersal of pummelo in China.Basing on the re-sequencing study of Guanxi pummelo flesh mutant,a SNP that may be related to flesh color was identified.A filial generation of leaf slow-greening Guanxi pummelo was used to mapping the candidate gene that leads to the leaf slow-greening phenotype.It also laid a good foundation for the breeding of pummelo.The main results of this study are as follows:1.Genetic diversity,population structure of pummelo and color mutation analysisIt takes four years,from year 2010 to 2013,to collecte almost three hundred accessions of pummelo from 13 provinces of China.There are 60 commercial accessions,212 landraces and 2 hybrids.Phenotypes of this natural population were abundant,which 109 accessions are red flesh and 113 accessions are white flesh.Furthermore,several accessions are green flesh.Other phenotypes of fruit,such as fruit size,fruit shape,the number of seed and the thickness of pericarp,indicate the diversity of pummelo germplasm.It shows that the typical accessions of pummelo have been collected.Thirty-one n SSR markers that separately locate in the 9 chromosomes of citrus were used to analyze the diversity of this natural population.Totally 147 polymorphism alleles were identified,the average number of alleles for each locus was 4.7,the average Observed Heterozygosity(Ho)was 0.325,the average Expect Heterozygosity(He)was 0.430,and the average Polymorphic Information Content(PIC)of these 31 markers was 0.414.Population structure analysis indicated that there were two basic gene pools(K=2)of the natural population,i.e.,subpopulation A and subpopulation B.The pummelo accessions of these two gene pools showed that the two gene pools were related to geographical distribution.Pop-A included the accessions from the Southeast China and part from the central region of South China,and Pop-B included the accessions from the Southwest China and part from the central region of South China.Ho of subpopulation A was 0.307,He was 0.368,the Ho and He of subpopulation B were 0.345 and 0.465,respectively.It suggested that the heterozygosity of pummelo from Southwest China was higher.According to the results of population structure(K=3),Principal Coordinate Analysis(PCo A)and phylogenetic analysis,there were three subgroups of this germplasm.i.e.,Pop-a(included the accessions from Southeast China),Pop-b(included the accessions from the central region of South China)and Pop-c(included the accessions from the Southwest China).NSSR markers analysis indicated that the Pop-b may be the results of the hybrid zone between two basic gene pools.Bottleneck analysis showed that there was a weak bottleneck during the domestication of the pummelo from China.There was not a significant reduction in the effective population size of pummelo.The collected white flesh Guanxi pummelo and mutant red flesh Guanxi pummelo were re-sequencing.Five candidate different SNPs were identifid by bioinformatic analysis.One SNP was positive and presented only in the juice sac of red flesh Guanxi pummelo.The SNP was homozygous T in the white flesh Guanxi pummlo,but it was T/G heterozygosis in red Guanxi pummelo.Resulting that the gene(Ci PR)can encode 81 more amino acids.The express pattern of gene Ci PR was higher in red flesh Guanxi pummlo at 150 DAF(Days after flowering),when the lycopene was abundant accumulation in flesh.Ci PR gene was overexpressed in citrus callus.The express pattern of genes in carotene pathway of the transgenic callus were no significant difference,which suggested that the SNP may be a polymorphism locus in pummelo.2.Leaf slow-greening mutation of Guanxi pummeloLeaf slow-greening Guanxi pummelo(CP),which pericarp was orange and leaf was slow-greening,was a mutant of wild type Guanxi pummelo.A 551 first filial generation was derived from leaf slow-greening Guanxi pummelo × Wanbai pummelo(WBY).The phenotype of 135 offsprings were leaf slow-greening(MCW)and 416 offsprings were normal green(WCW).The chlorophyll and carotene content of young leaf of MCW was significantly lower than that of WCW(P < 0.05).DNA of CP,WBY,bulk of 50 leaf slow-greening offsprings and bulk of 50 green offsprings were extracted for re-sequencing,and sequencing depth were 27.73 ×?31.32 ×?60.18 × and 57.06 ×,respectively.Genome coverage were approximate 75% for parents and 80% for bulks.BSA(bulk segregant analysis)indicated a 1.45 Mb region in chromosome 6 that may be related to the slow-greening phenotype.Six In Del markers were designed to narrowdown the candidate region into 0.2 Mb which contained 22 genes.The express pattern of these 22 genes showed that there were four differentially expressed genes between MCW and WCW.Analysis of the c DNA sequences of these four genes showed that there were not difference loci which may relate to the phenotype between MCW and WCW.These four genes were transiently expressed in tobacco(Nicotiana benthamiana)leaves.Overexpressed gene Chl660 results in the content of chlorophyll significantly lower than that of empty vector in the tobacco leaves(P < 0.05).Gene function of Chl660 was predicted to encode protein YLS9(Yellow-leaf specific gene 9).Subcellular localization showed it was located in chloroplast,indicating that gene Chl660 was the candidate gene that related to the leaf slow-greening phenotype.