| During the growth and development,every plant organ needs to grow to a specific size to meet the growth needs of life cycle.Floral organs vary entensively in size and shape under natural environment.Although the interspecies diversity is very high,among individuals growing under constant conditions,the petal characteristics of specific species are usually highly consistent,and the petal characteristics of specific species are usually highly consistent.The size of the floral organs is regulated by strict genetic mechanisms and also affected by the external environment.This study mainly combined GWAS(genome-wide association analysis)and transcriptome data to dissect key candidate genes related to regulating petal development in B.napus.A multi-site mixed linear model was used to perform GWAS analysis on the petal area of the natural populations,and 17 SNPs(single nucleotide polymorphism)significantly related to petal area were identified.The observation of petal epidermal cytology of a pair of extremely different accessions in natural groups showed that the difference in petal area is mainly result from the difference in cell number.Gene Ontology enrichment analysis of differentially expressed genes in early petal transcriptomes of extremely different accessions revealed that regulatory pathway such as nucleic acid anabolic metabolism and cell cycle were significantly up-regulated.Combining the candidate genes obtained by GWAS,epidermal cytology observations of extremely differed accessions and transcriptome analysis results,it is speculated that there may be a way to affect petal development in B.napus:the key candidate gene Bna.A05.RAP2.2a may be inhibiting cell division through reducing cell cycle,thereby making petals smaller.1.Whole genome association analysis of petal areaIn order to dissect the genetic structure of petal area in B.napus and excavate SNP loci and candidate genes closely related to petal area,we selected 588 B.napus accessions with extensive diversity from worldwide for genomic sequencing and obtaining the petal area data from 2016 to 2018.To comprehensively and accurately detect SNPs related to petal area,we used the mr MLM package based on R program to perform GWAS analysis on the natural population petal area and filtered SNP data.Statistics analysis of petal phenotype indicate that extensive phenotypic variation of petal area(PA)were detected in 588 B.napus inbred lines.The size of individual petals in 2016,2017,and 2018 are 34.41-109.19,32-122,and 36-103 mm~2;the mean is 69.73,72.23 and 65.96 mm~2;the coefficients of variation(CV)are 20.06%,22.92%and 20.08%.Association analysis was performed using the phenotypic data of each year from 2016 to 2018 and the BLUP results data,and a total of 17 significantly related SNPs were identified.These SNPs are distributed on the A05,A07,A09,A10,C01,C06 and C07 chromosomes.The contribution of a single SNP to the phenotype is 1.59%-14.42%.In order to verify the effect of SNP on petal area,natural populations were classified according to the most significant locus of SNP:Bna-A05-p18392058.The population petals with G genotype bases at this SNP site are larger than the population petals with A bases as a whole.2.Plant phenotypic investigation and transcriptome analysisIn order to comprehensively understand the development of petals in B.napus,we selected a pair of inbred lines(Spe and Bpe)with significantly different petal area size for phenotypic observation and petal transcriptome analysis.Observation of the petal epidermal cells under a SEM(scanning electron microscope)revealed that no significant difference in the number of cells between the large petals and the small petals except in the base of the petals.It is estimated that there are 350,000 to 400,000epidermal cells in a single large petal and less than 90,000 epidermal cells in a single small petal.Thus,it is the extreme cell number difference mainly caused petal area difference.The original data obtained by sequencing the transcriptome was subjected to differential expression analysis,and a total of 20817 DEGs(differentially expressed genes)were finally obtained,of which 8601 were up-regulated and 12216 were down-regulated.DEGs were analyzed by GO enrichment,and the up-regulated genes were significantly enriched in biological processes such as cell biosynthesis,nucleic acid metabolism,and cell cycle pathways.The down-regulated genes are mainly enriched in biological processes such as cell growth and cell size regulation,as well as cell components such as plasma membrane,chloroplast thylakoid membrane and plant cell wall.From the results of observation of the petal epidermal cells and the transcriptome DEGs analysis,it can be seen that the cell number of the large petal accession(Bpe)is greater than that of the small petal accession(Spe),and the cell proliferation-related events of Bpe are significantly more than that of Spe in the early stage of petal development.Therefore,in this pair of materials,the difference between the petal areas is mainly caused by the cell number of petals.3.Screen candidate genes and build regulatory networksThe key candidate gene Bna.A05.RAP2.2a.was found by combing genes within the 500kb interval flanking Bna-A05-p18392058 and transcriptome differentially expressed genes.Analysis of transcriptome data showed that Bna.A05.RAP2.2a was significantly up-regulated in small petal accession.Its expression in small petals was more than 10 times that in large petals.Bna.A05.RAP2.2a contains the AP2 conserved domain of the AP2/ERF gene family.Many genes in the AP2/ERF gene family can affect the growth and development of various parts of the flower organ,such as At ANT,At AIL6,Os MFS1,and so on.At RAP2.2,At RAP2.3 and At RAP2.12 belong to the same clade in phylogeny.The Arabidopsis mutants of At RAP2.3 and At RAP2.12 promote leave size.Combined with the above results,Bna.A05.RAP2.2a may be a response factor of the ethylene signal pathway and regulate the petal area size of B.napus by inhibiting cell division. |
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