Genetics Analysis And Fine Mapping Of Leaf Color Gene Ygl-1 And BoPr In Brassica Oleracea L.

Brassica oleracea L.contains several different subspecies,including cabbage(var.capitata),kale(var.acephala),broccoli(var.italica),cauliflower(var.botrytis),Brussels sprout(var.gemmifera),etc.For all the subspecies of Brassica oleracea,leaf colors are mainly green,while cabbage and ornamental kale have more leaf color variation,e.g.green,gray-green,dark green,purple in cabbage and purple,red,green,pink,white in ornamental kale.In order to discover the genetic basis of color variation,Brassica oleracea lines with different leaf colors were used to construct segregation population and fine mapping of two leaf-color genes(yellow-green and purple)in this study,then candidate genes in the fine-mapping interval were predicted and cloned based on the gene annotations of the reference genome.The results are as follows:1.Genetics and mapping of a yellow-green leaf gene(ygl-1)in cabbageSegregation populations were generated using two cabbage lines,i.e.,yellow-green leaf inbred line YL-1 and normal green leaf inbred line 01-20.Genetic analysis further demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene,named ygl-1.Positional mapping revealed that ygl-1 gene is flanked by In Del markers ID2 and M8,with genetic distances of 0.4 c M and 0.35 c M,respectively.The interval distance between two markers is 167 kb based on 02-12 reference genome.However,when the sequence of markers ID2 and M8 was aligned to the TO1000 reference genome,the interval between these two markers was 11.41 Mb.Therefore,considering the result of inconsistency between linkage map and physical map,we proposed that assembly error might exist in the 02-12 reference genome.New markers were designed based on TO1000 reference genome.The ygl-1 gene is further mapped and flanked by In Del markers T1-36 and T1-58,with genetic distances of 0.4 c M and 0.35 c M,respectively.The interval between the two markers is 11.47Mb(reference genome TO1000).The recombination rate was almost twenty times lower than the normal level for the cabbage genome(~600 kb/c M),suggesting that suppressed recombination was still present in this region.Thus we inferred that suppressed recombination in this mapping region was not caused by structure variation between two subspecies,which could possibly caused by the interval variation of mutant YL-1.2.Genetics and fine mapping of a purple leaf gene,Bo Pr,in ornamental kaleA backcross population and an F2 population were constructed using two parental lines: W1827(with white leaves)and P1835(with purple leaves).Genetic analysis indicated that the purple leaf trait is controlled by a single dominant gene,named Bo Pr.Using the markers developed based on the reference genome ‘02–12',the Bo Pr gene was preliminarily mapped to a 280-kb interval of chromosome C09,with flanking markers M17 and BOID4714 at genetic distances of 4.3 c M and 1.5 c M,respectively.The recombination rate within this interval is almost 12 times higher than the usual level,which could be caused by assembly error in reference genome ‘02–12' at this interval.Then primers were designed based on ‘TO1000',another B.oleracea reference genome.Among the newly designed In Del markers,BRID485 and BRID490 were found to be the closest to Bo Pr,flanking the gene at genetic distances of 0.1 c M and 0.2 c M,respectively;the interval between the two markers is 44.8 kb(reference genome TO1000).Seven annotated genes are located within the 44.8 kb genomic region,but only Bo9g058630,highly homology to AT5G42800(dihydroflavonol reductase),is an important enzyme in anthocyanin synthesis pathway,so Bo9g058630 is considered as the candidate gene for Bo Pr.Further sequence analysis of the gene revealed that large fragments of Insert and Deletions were present in the white leaf line W1827.So we inferred that the In Dels could cause the loss of gene function and white leaf phenotype.According to the sequence difference of parents,three primers were designed and added in one PCR reaction simultaneously,which could detect the genotype of two parents,F2 and BC1P1 populations.This marker is equal to a co-dominant marker,named 630-F-ZBR.