Map-based Cloning And Functional Analysis Of The Trilocular Gene Mc2 In Brassica Juncea

Rapeseed is one of the important conventional oil crop in our country,and rapeseed oil production accounted for more than half of the domestic edible vegetable oil.How to further improve the yield of rapeseed plays a very important role in ensuring a safe supply of edible vegetable oil and promoting the strategy of rural revitalization and healthy China.However,the silique traits can directly affect the yield of rapeseed,mainly because the silique is not only an important storage organ,but also an important photosynthetic organ during the silique mature period.Therefore,silique character is one of the most critical factor.Since the 1950 s,there have been reports on the germplasm resources of multilocular rapeseed with natural variation.As the number of seeds per multilocular silique was significantly higher than that of bilocular rapeseed,the potential yield increased benefits of multilocular rapeseed germplasm resources have attracted the attention of many researchers.J163-4 was a stable natural variation material with trilocular silique in B.juncea.Under the same genetic background,the yield of trilocular plants was significantly more than that of bilocular plants,mainly due to the significant increase in seeds number per silique in trilocular rapeseed compared with bilocular rapeseed.This trilocular silique trait was controlled by two independently inherited recessive nuclear genes,named mc1 and mc2.We constructed a high-generation backcross population of trilocular parent J163-4 and bilocular parent J268-1 to map the mc2 gene.The functional analysis was carried out by expression pattern analysis,transgenic complementary verification,promoter activity analysis,CRISPR gene editing technology,subcellular localization,and in vitro polypeptide treatment.The main research results are as follows:1.The BC5 generation backcross population was confirmed to be the near-isogenic line of Mc2 locus by allelic analysis,and the genotypes of bilocular plants and trilocular plants were mc1mc1Mc2mc2 and mc1mc1mc2mc2,respectively.14 pairs of molecular markers closely linked to Mc2 were developed using a total of 553 individuals from the BC5F1 and BC6F1 mapping population.For fine mapping Mc2,genotyping 6207 individuals from the BC7F1 mapping population identified 45 recombinants between two flanking markers ZX4 and ZX10.Sequencing sequences of BAC positive clones were used to develop new markers to further narrow the localization interval.Finally,Mc2 was finely mapped within the 0.388 c M range between markers ZX17 and Bac SR96,with 3 and 21 recombinants,respectively,and markers SSR344,Bac SR73 and Bac SR74 were co-segregated with Mc2.2.Phenotypic observation of homozygous bilocular and trilocular plants in NIL-BC7F2 population at Mc2 locus showed that mc2 affected the development of carpel margin meristem(CMM),but did not affect the development of shoot apical meristem(SAM)and flower meristem(FM).During early trilocular pistil development,every CMM in the middle region expanded and developed into two CMM,each of which can differentiate into a septum and ovule,eventually developing into a trilocular silique consisting of four carpels,four rows of seeds,and a "II" type false septum.3.The fine mapping interval of Mc2 locus was placed into sequencing sequences of BAC positive clones,covering a 34.9 kb physical region,which was basically the same as that of the B.juncea reference genome.Through comparative sequencing,it was found that there was a deletion of 914-bp sequence in the promoter regulation region of mc2 in the trilocular parent.Therefore,Bj A7.CLV1 was selected as the candidate gene of Mc2.4.Through phylogenetic tree and promoter collinearity analysis,it was found that this promoter deletion fragment was very conserved in Cruciferous species,just like the CDS sequence.Complementation tests showed that the Bj CLV1 sequence with its complete promoter containing the deletion region can restore the Bjclv1 and clv1 mutant phenotypes in B.juncea and Arabidopsis,respectively,otherwise it cannot restore.5.Expression patterns results of Bj A7.CLV1 and Bj B3.CLV1 showed that the two genes were widely expressed in B.juncea by q RT-PCR analysis,and the expression of Bj A7.CLV1 was significantly different in the early ovary of bilocular and trilocular plants.In addition,the promoter activity analysis also showed that the deletion of this conserved region of Bj CLV1 promoter did not affect GUS expression in most tissues of transgenic Arabidopsis except in CMM,indicating that this deletion sequence of mc2 promoter may contained cis-regulatory elements specifically expressed Bj CLV1 in CMM.6.Bj A7.CLV1 or GUS reporter gene driven by a series of progressive 5′-deletion Bj A7.CLV1 promoters in Arabidopsis to identify the core regulatory region,respectively.The results showed that when the Bj A7.CLV1 promoter was truncated to-3511-bp from 5′–3′,it did not affect GUS expression in the CMM and would not increase the number of carpels in the silique.When it was truncated to-3467-bp,GUS expression in CMM decreased,increasing the number of carpels in some siliques.When truncated to-3409-bp,GUS expression in CMM was undetectable,increasing the number of carpels in more siliques and trilocular plant formation.Therefore,the-3512-to-3409-bp region of Bj A7.CLV1 promoter was necessary for Bj A7.CLV1 expression in CMM,and this region may contain the core cis-regulatory elements specifically expressed Bj CLV1 in CMM.7.We obtained four lines with one-to seven-bp deletions in the protein-coding region that likely disrupted Bj A7.CLV1 functionality by using CRISPR gene editing technique.Phenotypic observation showed that this null allele affected the development of IM and FM during the plant life,resulting in enlargement of both IM and FM and fasciation of stem and increasing the number of flower organs in each whorl.Moreover,the null Bj A7.clv1 allelic type cannot effectively terminate the flower meristem,resulting in a great mass of undifferentiated meristem in the center of gynoecium,which affects the ovule development.8.The results of subcellular localization of Bj A7.CLV1 showed that it was localized on the cell membrane,suggesting that it also functions as a conserved receptor kinase in B.juncea.The results of in vitro polypeptide treatment showed that the development of SAM and root apical meristem(RAM)of trilocular seedlings could be inhibited by CLV3 and CLE26 polypeptides suggesting Bj A7.CLV1 in SAM and RAM had normal function,which was consistent with the above research results.However,the expression of CLV3 and WUS homologous genes could not be detected in the ovary at stage 8 of flower development in B.juncea,while the expression of CLE26 and STM homologous genes were upregulated in the trilocular ovary compared with that in the bilocular ovary,suggesting that mc2 gene affected the expression of related genes that maintain CMM homeostasis.Therefore,Bj CLV1 may also act as a conserved receptor in a negative feedback regulatory pathway to maintain CMM homeostasis.When Bj CLV1 promoter lacked the cis-regulatory sequence specifically expressed it in CMM,the stem-cells in CMM divided more rapidly and then an enlargement of the CMM,and eventually the development of trilocular siliques with more seeds.