Screening And Functional Analysis Of Morphological Differentiation Genes In Different Brassica Rapa Ecotypes

The genus Brassica has been widely used as oilseed,condiments,fodder or vegetable crops in the world,mainly including three diploid species(Brassica rapa,Brassica nigra and Brassica oleracea)and three allotetraploid species(Brassica napus,Brassica juncea and Brassica carinata).Further,the Brassica species had highly diverse morphotypes,such as leafy heads,enlarged organs(roots,stems and inflorescences)and extensive axillary branching.Among them,Brassica rapa might be the best example for displaying the more abundant morphological characteristics than the other Brassica species,containing the extreme morphological characteristics.Such as the leafy vegetables(Chinese cabbage),swelling root(Turnip),and oilseed(B.rapa).Therefore,based on the extremely high morphological diversity within Brassica rapa,by mining more genes related to important agronomic traits,revealing the molecular mechanism of Brassica rapa growth,development patterns and differentiation of diverse organs will greatly promote Brassica rapa and other genetic improvement of Brassica species.This study was based on the resequencing data of 131 different types of Brassica rapa resource materials(46 Chinese cabbage,32 Turnip,and 53 B.rapa).Through the select sweep analysis of whole genome,a number of important candidate genes were screened,which related to diversification and convergent domestication of morphotypes in B.rapa.And the biological functions of the candidate genes were initially verified in the experiment.The results are as follows:1.Variation analysis of different types of Brassica rapaThis study completed the simplified genome sequence for 53 B.rapa(YC),combined with the published reference genomes of Chinese cabbage(CC)and Turnip(LB),and a Brassica rapa group consisting of 131 materials.The total resequencing data of the 131 accessions reached 667.84 Gb,the average depth is about 14.44×.By performing alignment analysis and variant detection on the resequencing data,we identified 2.39 M genome-wide SNPs and 0.28 M genome-wide In Dels.And the 131 Brassica rapa groups are divided into three branches,and the genetic distance between YC and LB types is relatively close,and the genetic distance between CC and YC,between CC and LB types is far.2.Screening of morphological diversity genes of different types of Brassica rapaBy whole genome selection sweep analysis,this study detected 2364 signal sites in three morphological types of Brassica rapa,including 1188 in CC,632 in LB and 544 in YC,respectively.The results indicate that the CC type has been domesticated in evolution.The SNPs and In Dels analysis and function associated with these candidate genes,7candidate genes are involved in the heading of Brassica rapa,including Br P2C56(Bra A01g016800.3C),Br NPR2(Bra A01g016870.3C),Br TCP17(Bra A03g003300.3C),Br YAB1(Bra A03g023630.3C)and Br ARF4(Bra A10g018230.3C),Br SAUR36(BraA03g023650.3C).And 2 candidate genes are involved in the tuber of Brassica rapa,including Br MYC2(Bra A05g023030.3C)and Br PYL1(Bra A09g022320.3C).3.Cloning of candidate genes for morphological development of different types in Brassica rapaUsing three different types of 5 materials(CC type Chinese cabbage CC2,YC type B.rapa type YC2,LB type Turnip LB2 and B.napus 20K132 and 20K204),the 9candidate genes were cloned alignment analysis.The results showed that 3 genes had consistent mutations between CC type and other ecotype gene sequences,two sense mutations were found in CC2 and LB2 in Br TCP17,and one sense mutation in CC2 and LB2 in Br ARF4 and Br NPR2.This mutation results in differences in amino acid sequences,as an important candidate gene for verification analysis.4.Preliminary verification of candidate genes function for morphological development of different types in Brassica rapaThe CDS sequences of 3 genes(Br TCP17,Br ARF4 and Br NPR2)amplified from CC2 and LB2 materials were selected.By constructing the overexpression vectors(LBTCP17,CC-ARF4,LB-ARF4 and CC-NPR2)transforming the A.thaliana,and CRISPRCas9 gene editing vector(Bn CLASP)transformable B.napus for basic biology functional analysis.The results are as follows:(1)Compared with wild-type,overexpression of LB2-TCP17 produced erect and curly rosette leaves in A.thaliana,the pollen fertility also decreased.At the same time,it promoted the growth of the main root and increased the number of lateral roots.These indicated that Br TCP17 also controls leaf and root development.(2)Compared with wild-type,overexpression of CC2-ARF4 and LB2-ARF4 in A.thaliana leads to significantly different root morphological characteristics,but a stronger effect with LB2-ARF4.At the same time,the number of rosettes increased and the flowering time was significantly advanced.These results show Br ARF4 had a biological function of regulating the growth and development of plants,blade morphology and root growth.(3)Compared with wild-type,overexpression of CC2-NPR2 in A.thaliana led to leaves becoming smaller and the number of heart rosette leaves increasing,indicating that Br NPR2 has a regulatory effect on leaf development.(4)Compared with control plants(ZS11),knockout of homologous Bn CLASP in B.napus showed that the main root thickened but the quantity and length of lateral roots decreased.In addition,there is also a significant change in the morphological characteristics of petals,doctrine and plant dwarf.The initial explanation of the Bn CLASP gene has a significant role in promoting the growth and development of plants,and the specific molecular mechanisms have been further studied.