| This study is composed of two parts,one on genetic analysis of heading trait in lettuce and the other on genetic and molecular mechanisms of the color traits in Brassica vegetables.1.Genetic analysis of heading trait in lettuceHeading is found in several leafy vegetables,including lettuce（Lactuce sativa）,chicory（Cichorium intybus）,cabbage（Brassica oleracea）and Chinese cabbage（Brassica rapa）.Heading is a complex trait,controlled by several loci.The genetic and molecular mechanisms underlying heading remain unclear.In this study,a F2 segregating population was constructed through a cross between a crisphead lettuce and a romanie lettuce.BSR-seq in combination with linkage analysis was used to genetically dissect this complex trait.Three major QTLs controlling heading trait in lettuce were identified,named as Lettuce Heading Locus 1,2 and 3（LHL1,LHL2and LHL3）,respectively.Families,in which only one of the three loci segregated,were obatained from the F2 or advanced progenies,and were used to map and clone of the genes controlling heading in lettuce.Using 577 heading individuals of a segregating family,LHL2 was mapped to the region between 18.54 Mb and 18.99 Mb（with an approximately 450 kb interval）on LG7.A STM（SHOOT MERISTEMLESS）gene,Ls STM2（LG7600774）,was considered as the candidate gene.The function of Ls STM2 gene was subsequently confirmed using overexpression and knockout experiments.Sequence analysis reveals that a CACTA-like transposon（3,935 bp）inserted in the first exon of the Ls STM2 gene,which shortened the gene and upregulated the expression in leaves and veins of crisphead lettuce leading to heading phenotype.Using 589 individuals from another family that segregate the gene of LHL3,the heading associated gene was mapped to the region between 76.69 Mb and 76.86 Mb（with an approximately 165 kb interval）on LG9 of lettuce.A homologue of AGL47,Ls AGL47（LG9783807）,was considered as the candidate gene for LHL3,which showed a down-regulation of expression in the heading parent.The LHL1 gene was genetically mapped to the region between 345.33 Mb and 353.97Mb on LG4 of lettuce using a population of 207 idividuals.Summary:Three genes controlling the heading trait in lettuce were identified.Two of them were cloned through map-based cloning.2.Genetic and molecular mechanisms of color traits in B.oleracea vegetablesThis study also investigated the genetic and molecular mechanisms of color variation in vegetables of B.oleracea,including the purple trait associated with anthocyanin and the albino trait in kale.2.1 Genetic and molecular mechanisms of purple traits in B.oleracea vegetablesPrevious study showed that the purple color of cauliflower was controlled by Bo MYB2 gene,which was activated by an insertion of Harbinger transopon in its promoter region.However,such insertion was not found in this gene of purple kale“Red Kamome”,purple kohlrabi,purple cabbage,and Chinese kale,suggesting novel genetic mechanisms on the purple traits.Segregating populations were constructed for each of the purple vaieties through crosses between purple genotypes and green genotypes.BSR-seq and linkage analysis was used to genetically map and clone the causal genes.The results showed that the color variation in Chinese kale was controlled by a frameshift deletion in the Bo DFR gene,while gene controlling purple traits in kale,kohlrabi and cabbage were mapped to the same locus as the Bo MYB2 gene.Sequence analysis discovered that a7,606 bp CACTA-like transposon rather than the Harbinger inserted-1,847 bp upstream of start codon of the Bo MYB2 gene in purple kale and purple kohlrabi.One the other hand,a point mutation and/or 1-bp insertion in promoter region of Bo MYB2 were identified in purple cabbage.Studied of promoter activity revealed that above sequence variations up-regulate the expression of the Bo MYB2 genes.Our comprehensive studies on genetics of the purple traits in B.oleracea vegetables provide useful information for future breeding programs.2.2 Genetic and molecular mechanisms of albino leaves in kaleSegregation of albino and green leaves were discovered in the BC1and BC1F2populations derived from a cross between purple kale“Red Kamome”and green cabbage（DH line）.Segregation ratio suggests the albino was controlled by a single gene,named as Albino Kale（AK）.BSR-seq analysis mapped the AK gene to chromosome C03,and it was subsequently mapped to the region between 0.75 Mb and 0.81 Mb（with an approximately 60 kb）.An unknown function gene,CYP708A5（Bo3g003230）,from this region with homology to CYP708A4 in Arabidopsis is differentially expressed between albino and green progenies,and considered as the candidate gene for AK.The function of this gene was confirmed transgenic expriments.Physiological experiments showed that the albino phenotype was induced by low temperature rather than photoperiod.Our experiments suggested that the high expression of the CYP708A5 gene may activate the brassinosteroid pathway,which in turn causes the albino phenotype in kale. |
