Cytological Study And Genetic Analysis Of Seed Number Per Silique In Brassica Napus L.

Oilseed rape is one of the most important oil crops in Brassicaceae species.Increasing rapeseed yield is an important responsibility for breeders and researchers.Three major factors,seed number per silique,silique number per plant and seed weight,affect seed yield.In the present study,DH46 mutant was crossed with the open-pollination cultivar ZS11 to constract the genetic analysis population F₂ and BC₃F₃,believed that ovule fertility and silique length were important factors affecting seed number per silique.The critical region of female sterile related locus controlling seed number per silique was found by marker identification,and the candidate genes were analyzed.At the same time,to determine the cause of ovule abortion,pollen tube extension,callose accumulation and ovule development of female sterile mutant DH46 were investigated through multiple cytological methods.In addition,a single gene that could regulate both silique length and seed weight was found and cloned in the near isogenic population BC₃F₃,the function of the candidate gene was also analyzed in the natural population.The main findings were as follows:1. Phenotypic identification of the DH46 mutant.The siliques of DH46 mutant stopped to elongate after the petals fell off and yellowed after 5 days,then dropped off later.Anther paraffin section and pollen acetic acid magenta staining showed that the anther development was normal and the pollen could be dyed normally.The DH46 pollen was pollinated to the normal material ZS11 stigma,meantime,the ZS11 pollen was applied to the DH46 stigma,the pollen tube germination and extension were all observed.Only the pollen tube in DH46 could not enter the micropyle.These indicated that the pollen development of DH46 mutant was normal,and the abnormal development of ovule resulted in the phenotype of female sterility and few seed number per silique.2. Identification the period of female sterility.The developing ovules were observed by aniline blue staining,it was found that the callose produced during meiosis in DH46 ovule could not be degraded,and the fluorescent signal could even be observed in the mature embryo sac.It indicated that ovule abortion was due to undegraded callose in time,and the period of abortion may be happened in meiosis.Through paraffin section and ovary whole transparent DIC observation,it was found that megasporocytes could start meiosis in the early stage of ovule development,but multiple small nuclei,not mononuclear or binuclear,were found in the embryo sac,the nucellus could not differentiate into the central vacuole,or form a normal multinuclear embryo sac.The above results indicated that the cause of DH46 ovule abortion was the callose could not be degraded at the end of megaspore mother cells meiosis,the functional megaspores failed to develop and the nucellus could not continue to differentiate into multinuclear embryo sacs.3. Genetic analysis of the female sterile mutant with few seed number per silique.The seed number per silique of DH46 mutant was 1.13.There was significant difference between the F1 generations derived from reciprocal crossing with ZS11,which showed partial female sterility.A genetic population was constructed by crossing DH46 as the male parent with the cultivar ZS11 as the female parent to analyze the seed number per silique.The statistical analysis of seed number per silique in the F₂ population indicated that the segregation could not be fitted for any single genetic models.It is speculated that the phenotype might be interfered by other related QTL.The BC₃F₄ segregation population was constructed through multiple generations by backcrossing and selfing,with DH46 as the recurrent male parent.In this population,the near-isogenic line of N-DH46 was still growing new flower buds in the main inflorescence at the end of the flowering period.Combining the phenotype of the seed number,one population with a phenotypic segregation ratio of 3:1 was identified.4. Identification of the locus related to the seed number per silique.The rapeseed 60K chip was used to analyze the DNA pools of extreme phenotype plants in the BC₃F₄segregation population,the differential SNPs were mainly concentrated on C09chromosome from 12.5Mb to 20.1Mb,and the locus controlling female sterility was called Bn SSC09.Using the DH46 resequencing results,multiple pairs of In Del markers were designed to analyze the 259 individuals in BC₃F₄ population,and the Bn SSC09 locus was finally narrowed to a region of about 205 kb from 21.0 Mb to 21.2 Mb.This interval contained 19 annotated genes in the Dermor-bzh reference genome,through expression detection and comparison sequencing,Bna C09g23670D or Bna C09g23590D were predicted to be the candidate genes for the Bn SSC09 locus.5. Inheritance of the silique length phenotype.One population contained 236 plants derived from a line showed 1:1 separation in silique length was observed in the BC₃F₁population constructed to locate the Bn SSC09 locus.In the long silique plants,the beak was sharper and longer,while in the short silique plants,the beak was shorter.The genetic analysis of BC₃F₂ population found that the long silique was controlled by a single dominant nuclear gene,the segregation of the silique length was 3:1 in mapping population.6. Location of the silique length gene.The DNA pools of long and short siliques in the BC₃F₁ population was screened by rapeseed 60K chip,and the differential SNPs were concentrated in A09 chromosome with the interval of 2.21 Mb in ZS11 reference genome,this locus was named Bn SLA09.Mapping with the differential SSR primers further confirmed the chip results,and the candidate interval was constricted to 490 kb using the BC₃F₁ population.The BC₃F₃ population were fine mapped by the developed In Del markers and the candidate interval was further narrowed to 98.47 kb.7. Biological function of Bn SLA09 locus and identification of the candidate gene.In the BC₃F₃ population,the epidermal cells of long siliques were significantly longer and wider,and the 1000-grain was also larger than that of short ones.In the candidate interval,Bna A09g55530D is homologous to Arabidopsis gene CYP78A9（AT3G61880）,and its expression level in the long developing siliques was significantly higher than that in the short ones.This results suggested that this gene could be the candidate gene of Bn SLA09.8. Functional marker development and the distribution of Bn SLA09 locus in natural populations.A truncated CACTA transposon inserted at the 3.9 kb upstream of the Bna A09g55530D promoter in the long silique plants was found by sequencing,and it led the downstream gene to overexpress.A specific primer was designed according to the transposon segment,and it was used to screen a natural population containing 521 rapeseed accessions.The accessions containing the transposon fragment were found to have a larger silique length and 1000-grain weight.This haplotype could also promote the increase of silique length and 1000-grain weight in the absence of the Bna A.ARF18.a gene,which was related to the silique length and seed weight as the first map-based clone QTL.