Fine-mapping And Cytological Mechanism Of A Major Seed Number Per Pod QTL-qSN.A6 In Rapeseed (Brassica Napus L.)

Rapeseed is the important oil crops as well as the only winter oil crop in China.Seed number per pod?SNPP?is one of the important yield components,which is a typical quantitative trait?phenotypic data follow continuous distribution and easily influenced by the environment?and the genetic mechanism is relatively complex.To the present,many QTLs of SNPP have been identified,however only a few have been fine-mapped and only one has been cloned.Our previous research has identified a major QTL of SNPP?qSN.A6?on the A6 chromosome both screened in the BnaZN-F₂ and BnaZN-RIL population derived from two sequenced parental cultivars Zhongshuang11?de novo sequencing?and No.73290?re-sequencing?that showed significant difference on seed number per pod.Then,we successfully constructed the near-isogenic lines?NILs?for this major QTL-qSN.A6 and fine-mapped it to marker interval between BrSF47-10 and BrSF46-167?physical distance 267kb?,using the QTL-NILs of BC₄F₂.Meanwhile,we performed the genetic experiments of reciprocal cross and preliminary cytological observation on the NIL-q SN.A6 and the recurrent parent zhongshuang11.Finally,and ovule abortion was identified as the underlying cytological mechanism of qSN.A6 after excluding the biological processes associated with pollen development,fertilization and embryo development.Based on these results,we plan to further fine-mapping qSN.A6 using a larger QTL-NIL population.In addition,we plant to determine the cytological mechanism leading to ovule abortion caused by qSN.A6 by further cytological observation on the NIL-q SN.A6,including the structure of the mature embryo sac as well as its early developmental stages.The main results and conclusions were as follows:1.A total of 347 recombinant individuals were screened from the 37976 individuals of large population BC₅F₂,using two molecular markers?BrSF47-10 and BrSF46-167?flanking qSN.A6.Then,we developed 20 specific and polymorphic markers?17 SSR and 3 InDel?in target QTL region.The 16molecular makers were used to seek the breakpoints with the recombinants,which were then subjected to progeny test.Finally,we further narrow qSN.A6 to a smaller marker interval between BrSF4*-*8 and marker BrSF4*-*9,after the comparison of the phenotype of recombinant progenies with different recombinant breakpoints.Of This marker interval corresponded to approximate 40kb BAC physical map of the two parents Zhongshuang11 and No.73290,which both harboured only 9 predicted ORF.Two candidate genes had been identified through comparative sequencing and bioinformatics analysis,whose genetic complementation test vector had also been constructed and transformed into the corresponding NILs.2.We discovered that the number of ovules had no significant difference in different stage both Zhongshuang11 and NIL-qSN.A6,also had no significant difference in same satge between them.Through the observation of mature embryo sac morphology,we found the proportion of abnormal embryo sac coincides with the proportion of aborted embryo,which both showed significant difference between NIL-qSN.A6 and Zhongshuang11.Furthermore,embryo sac fertility of Zhongshuang11 and NIL-qSN.A6,which were also found to have significant difference between them;and the proportion of embryo sac abortion was consistent with that of abnormal tetrad,which both had significant difference between NIL-qSN.A6 and Zhongshuang11.In summary,the differences of the ratio of the abnormal tetrad,aborted embryo sac and aborted embryo were generally consistent,which suggested that qSN.A6caused the abnormal tetrad,which then leaded to the abnormal embryo sac morphology and the unfertilized ovule and aborted embryo,and finally reduced seed number.