Fine Mapping Of The Major QTL QOilA5 For Oil Content And Identification Of Candidate Genes In Brassica Napus

Rapeseed is one of the most important oil crops in China.It has very important economic value to increase the oil content of rapeseed.In this study,QTL mapping of oil content in Brassica napus was performed by using the doubled haploid population ZD-DH.A novel locus qOilA5 was identified on chromosome A5.On this basis,near-isogenic lines were constructed for fine mapping,and candidate genes in the QTL region were predicted.The main findings are as follows:1.ZD-DH population was genotyped with the 60K SNP array of rapeseed,and QTLs were mapped by combining oil phenotypes for two consecutive years.Three main QTLs for oil content were identified to locate on chromosomes A1,A5 and A7,respectively.The main QTL which was located on chromosome A5 is a novle locus found by our laboratory.Its confidence interval is between 153.5-155.3cM,which can explain 20.44%of the phenotypic variation.Based on the re-sequencing results of ZD-DH parent,polymorphism molecular markers were developed,and 22 indel markers used to construct partial genetic linkage maps.QTL of qOilA5 loci was anchored between the two markers ZDM293 and ZDM339.8.6 cM with 638Kb of physical distance based on the“Darmor-bzh”reference genome.2.According to the data of array,we selected four DH homozygous lines with similar backgrounds in the qOilA5 locus as the parents of the fine mapping population and constructed two crosses.Among them,the BC₁F₂ population used for fine localization was derived from the cross combination ZD6×ZD56.Whole genome re-sequencing was performed on the four parental materials to obtain differential information on the whole genome of the localized population and the target region molecular markers were developed.3.Genotypic analysis was performed on 281 individuals from the BC₁F₂ population using the newly developed InDel marker,a local genetic linkage map of the qOilA5 locus was constructed,QTL scanning was performed in combination with the BC₁F₂ population phenotype data,and finally the qOilA5 locus target was constructed.The section is anchored between the ZDM 306 and the ZDM 428 with a physical distance of 178 Kb.4.The target segment of qOilA5 locus has a 178Kb physical distance in the"Darmor-bzh"genome and 46 genes in the segment.The physical distance in the"ZS11"genome is242Kb and 51 genes in the segment.Based on the sequence differences of the candidate genes in the parents and annotation information of Arabidopsis thaliana,we found three genes that may be involved in lipid metabolism.We tentativley identified these three genes as candidates for the qOilA5 locus.5.When Hemoglobin-2?HB2?gene is overexpressed in Arabidopsis seeds,it can significantly increase the oil content of Arabidopsis plants.A homologous copy of the HB2gene located on the A5 chromosome was cloned,and a constitutive overexpression vector p35S:Hb and seed-specific expression vector pNapin:Hb were constructed,respectively,which were used to transform rapeseed through Agrobacterium-mediated transformation.The HB2 transgene was detected over-expressed in oilseed rape but the oil content of the transgenic plants did not change significantly.The expression level of HB2 gene in seeds was increased in transgenic plants with pNapin:Hb,and the oil content of one line transgenic plants was significantly higher than that of the control plants.