Genetic Dissection Of Pod Shattering Resistance In Brassica Napus L.

Rapeseed pod dehisces to spread seeds after maturity,which causes serious yield loss,hampering mechanical harvest in rapeseed production.Breeding cultivars with high pod shattering resistance has been one of the most important objectives in rapeseed breeding.Till now,only few pod shattering resistant germplasms have been identified in rapeseed.However,these germplasms could not meet the requirement of long mechanic harvest period due to relatively low shatter resistant effect.Identification of elite pod shatter resistant lines and genetic dissertation of these lines is the key step for breeding shattering resistant varieties.After the assessment of a large number of germplasm resources and breeding materials,a breeding line OR88 with high pod shatter resistance was identified.In addition,a shattering resistant QTL qSRI.A06 was identified in multiple environments using ZS11×R11 DH population.In this study,anatomical analysis,genetic analysis,genome sequencing and transcriptome sequencing were performed to dissect shattering resistant trait in OR88.At the same time,mapping of qSRI.A06 was also forwarded.The major results obtained in this study are as follows:1.OR88 has high pod shattering resistance index in multiple environments(SRI > 0.8).Anatomical analysis showed that there is a unique lignified layer bridge(LLB)between two pod valves.After maturity,the sliliques could not dehisce because the LLB links valves tightly.LLB and high SRI were co-segregated in segregating populations.These results indicated that the high pod shattering resistance in OR88 is associated with LLB structure.2.By BSA-seq,a candidate region for LLB spanning 0-36.14 Mb was obtained.This LLB locus was further mapped into S12-S12.6 region using OR88×No.87 BC1 and OR88×OR5 F2 populations.The candidate region is about 688 kb referring to ZS11 genome.3.After Illumina sequencing,Pacbio sequencing and Hi-C sequencing,the whole genome and annotation information of C09 chromosome in OR88 were obtained.The results of collinearity analysis indicated that the 0-14 Mb of C09 chromosome in OR88 was replaced by the 24-38 Mb of radish R9 chromosome.The radish gneome introgression was reverse inserted in C09 chromosome.According to genome and annotation information of C09 in OR88,the LLB candidate region is about 165 kb,containing 26 ORFs which derived from Radish.4.The LLB structure is differentiated at stage 12 of gynoecium development based on the comparison of gynoecium development in OR88 and No.87.Stage11-12 gynoeciums were collected from OR88 and No.87 respectively and subjected to transcriptome sequencing.The results showed that cell cycle pathway was significantly downregulated in OR88.BnTCP8.C09 was identified in the candidate region.TCP8 is a regulator of cell cycle pathway in Arabidopsis.The BnTCP8.C09 of OR88 was derived from radish,and the expression of BnTCP8.C09 was severely surpressed in OR88,indicating BnTCP8.C09 is a key candidate gene for LLB.5.In ZS11×R11 DH population,a pod shattering resistance QTL qSRI.A06 which was stably detected in four different environments with R2 values of 4.8-15% was identified.ZS 11 contributed positive effect for pod shattering resistance.By comparing the physical position of this QTL with qSRI.A06 a and qSRI.A06 b which were previously obtained in R1×R2 DH and GWAS populations,it was found that these three qSRI.A06 s were overlapped and were actually the same QTL.6.Using R11 as the recurrent parent,a ZS11×R11 BC3F2 population was contructed.In the population,SRI values distributed in a triple peak pattern.The order of SRI values in BC3F2 was BC3F2-qSRI.A06R11 < heterozygotes < BC3F2-qSRI.A06 ZS11,indicating that qSRI.A06 near isogenic lines(NIL)were successfully constructed.Using this NIL population,qSRI.A06 was mapped into a 93.5kb region between S19.1-S19.2.7.There was BnERF040.A06(Bna A06g27900D)which is a homologs of ERF040 in the qSRI.A06 candidate region.Semi-quantitative RT-PCR analysis showed the expression level of BnERF040.A06-ZS11 was significant higher than BnERF040.A06-R11.ERF040 could surpress lignin deposition in secondary wall in Arabidopsis.Thus,it is hypothesised that BnERF040.A06 has effect on secondary wall of valves and regulates pod-shatter resistance in rapeseed.