| Rapeseed(Brassica napus L.)is one of the important oil crops worldwide,the planting area and production of rapeseed in China are at the front ranks of the world.Plant architecture is vital for crop yield and the branch angle is one of the key factors determining plant architecture.Furthermore,planting density and mechanical harvesting are also influenced by branch angle.Stem lodging,one of the two lodging types,is closely related with plant architecture.Lodging also severely reduces yield and impairs seed quality in rapeseed.In consideration of the significance of branch angle and stem lodging for rapeseed,it is absolutely essential to understand the molecule mechanism and excavate the candidate genes for genetic improvement in B.napus.In the present study,the genetic analyses on branch angle and stem breaking resistance in two segregation populations were implemented.Subsequently,genome-wide association studies for branch angle and stem lodging-related traits(stem breaking resistance,stem diameter,stem strength and lodging coefficient)among 472 B.napus accessions were carried out.Transcriptome analyses were also carried out on different B.napus accessions,which were significantly different in branch angle or stem breaking resistance.The major results are as follows: 1.Genetic analysis for branch angleThe major gene and polygene mixed genetic model was used to analyze the branch angle of F2 and F2:3 in two segregation populations.The analysis results indicated that,the optimal model for F2 and F2:3 were 1MG-AD and 1MG-A,respectively.These meaned that the branch angle of B.napus was under the control of one pair of major genes that with additive effects or additive-dominant effects.2.Genome-wide association study for branch angleThe population structure was investigated based on Brassica 60 K SNP array,the results implied that 472 rapeseed accessions could be clustered into three groups,which consisted of 64,199 and 209 accessions,respectively.A total of 2423 conserved haplotype blocks were detected,which spanned 181.53 Mb and covered 28.28% of the assembled B.napus genome.Additionally,the large haplotype blocks(?1Mb)were mostly distributed on the C-subgenome and were enriched around the centromere regions.The branch angle increased with the elevation of branch position in individual plant.Furthermore,three middle branches of individual plants were selected to measure the branch angle because they exhibited the most representative phenotypic values.As a result of association mapping,46 and 38 significantly-associated loci were detected using a mixed linear model(MLM)and a multi-locus random-SNP-effect mixed linear model(MRMLM),which explained up to 62.2% and 66.2% of the cumulative phenotypic variation,respectively.Numerous highly-promising candidate genes were identified,including some first found in rapeseed,such as TAC1,SGR1,SGR3 and SGR5.3.Transcriptome analysis for branch angleTranscriptome analysis on two rapeseed accessions that exhibited extremely large and small branch angle respectively was implemented.As a result,49 common up-regulated differentially expressed genes(DEGs),which were related with auxin biosynthesis and transport,were identified between upper and lower of branch in two different accessions.Of these genes,Bna.YUCCA6(Bna C09g03930D)and Bna.IAA19(Bna C03g39170D)were two promising candidate genes for branch angle.Furthermore,32 common down-regulated DEGs,including two promising candidate genes Bna.AXR3(Bna Cnng41350D)and Bna.SGR5(Bna C07g48900D)were also identified.Additionally,five TCP1(Bna A02g13010 D,Bna A07g26270 D,Bna A07g25110 D,Bna C06g26860 D,Bna C06g28330D)and two YAB2(Bna A06g04870 D and Bna C08g13560D)were common up-regulated and down-regulated respectively on upper of branch between two accessions.Integrating GWAS and RNA-Seq,22 candidates were identified,including one Bna.TAC1(Bna A05g01220D)and two Bna.LA1(Bna A10g19550 D and Bna C03g06250D).4.Genetic analysis for stem breaking resistanceThe major gene and polygene mixed genetic model was used to analyze the stem breaking resistance trait of six generations(P1,P2,F1,B1:2,B2:2 and F2:3)in two segregation populations.The results indicated that the stem breaking resistance was fitted the genetic model of MX2-ADI-ADI,i.e.,two pairs of additive –dominant-epistatic major gene plus additive-dominant-epistatic polygene model.Stem breaking resistance was mainly controlled by two pair major genes and the average heritability of major gene were 19.46% and 69.93% in two segregation populations,respectively.The additive effects and dominant effects of two major genes were in opposite directions in one population,but in same direction in another population.Moreover,multiple epistatic effects also have influence on two major genes.The proportion of environmental variance to phenotypic variance in two populations were 54.68% and 13.23%,respectively,meaning that environment factors have rather larger impact on stem breaking resistance in rapeseed.5.Genome-wide association studies for stem lodging-related traitsFour stem lodging-related traits,including stem breaking resistance(SBR),stem diameter(SD),stem strength(SS)and lodging coefficient(LC)were evaluated and extensive phenotype variations were found.Correlation analysis of the traits showed that SBR was positively correlated with SD(r=0.73 to 0.82,P<0.05)and SS(r=0.48 to 0.75,P<0.05)across each environment.While,the negative correlations were observed between SS and LC(r=-0.52 to-0.40,P<0.05)across each environment.Here,26,18,17 and 13 QTLs significantly associated with SBR,SD,SS and LC were detected using association mapping.In these QTLs,29 candidate genes,including Bna.ESK1(Bna C08g26920D)and Bna.CESA6(Bna A09g06990D),for SBR,19 candidate genes,including Bna.EXPA16(Bna C08g26400D)and Bna.BRX(Bna A08g07050D),for SD,18 candidate genes,including Bna.FRA2(Bna C02g23170D)and Bna.ARF2(Bna A05g14370D),for SS and 25 candidate genes,including Bna.CEL5(Bna A07g10240D)and Bna.IRX14-L(Bna A07g12000D),were identified.6.Transcriptome analysis for stem breaking resistanceOf the DEGs identified between high-SBR and low-SBR,1,679 DEGs were up-regulated and 2,586 DEGs were down-regulated during flowering,while 706 DEGs were up-regulated and 414 DEGs were down-regulated during silique developing.Moreover,361 common up-regulated DEGs,including Bna.TBL1(Bna C01g23560D),and 253 common down-regulated,including Bna.ARF2(Bna A05g14370D),were identified between two development stages.Seven differentially expressed transcription factors(MYB26,MYB43,MYB61,MYB85,WRKY47 and KNAT7)were also identified.WGCNA and GO analysis illustrated that,these genes in the ‘green' module mainly played roles during flowering and were mainly enriched in function of cellulose biogenesis and metabolic process.In conclusion,three high-promising candidate genes Bna.ARF2(Bna A05g14370D),Bna.CESA6(Bna A09g06990D)and Bna.FRA8(Bna C04g39510D)were mined by integrating GWAS,RNA-Seq and WGCNA. |
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