Gene Mapping And Candidate Gene Analysis Of Orange Flower In Brassica Napus L.

Oliseed rape as a Brassica crops,belongs to the cruciferous family.It’s one of the main oil crops in China and even the world.It is the main source of vegetable oils in China and occupies an important position in the oil markets.Oliseed rape has multiple functions,not only can be used as edible oil for human use,but also can be used as feed for animals.In addition,oliseed rape can be as vegetables,fertilizer,honey and so on.Generally,the flower color of oliseed rapa is yellow,while white flowers,pink flowers,orange flowers and other mutants begin appear,making oliseed rape color more colorful.So the ornamental function of oliseed rape has also been paid more attention.Therefore,studying the genetic rules of oliseed rape flower color,mining candidate genes and analyzing the function of candidate genes can lay a foundation for the research on the breeding and formation mechanism of oliseed rape flower color.In this study,yellow flower Y05 and orange flower R08,as well as the constructed F₁and F₂ populations,were used to reveal the genetic rules of orange flower traits.Based on the data of petals RNA transcriptome sequencing and leaves DNA resequencing,MMAPPR and QTL-seq were used to identify the candidate regions of orange genes in Brassica napus L,and the molecular markers closely linked to orange genes were developed in the candidate regions.According to the routine transcriptome sequencing analysis of petals of yellow and orange flowers,the candidate genes of orange flower in Brassica napus L.were screened.Then the candidate genes were quantified,cloned and analyzed by bioinformatics.The main research results are as follows:1、Genetic analysis of orange flowerF₁ and F₂ populations were constructed by parent Y05 and parent R08.Phenotypic observation of F₁ population:The flower color of F₁ generation was orange flower,which was roughly same as that of the parental R08,which is orange flower,speculating that orange color was the dominant character.In F₂generation,orange and yellow flowers were separated,there were 441 orange flowers and 166 yellow flowers among 607 plants,and the chi-square test for plants showed that the separation ratio of orange and yellow flowers was 3:1（χ²<3.84）,suggesting that orange flowers were controlled by a pair of dominant genes.2、Genes localization of orange flowerIn F₂population,the petals RNA mixing pool and leaves DNA mixing pool were constructed by yellow flower materials and orange flower materials.RNA pools were sequenced by 6G transcriptome and parental DNA and DNA pools were resequenced by30×resequencing.MMAPPR process mapped the orange flower genes to 17-23 Mb on chromosome A07,and QTL-seq mapping showed that the candidate interval of orange flower gene was 17-19 Mb on chromosome A07.Both methods mapped the candidate interval of orange flower genes on chromosome A07,and the candidate interval overlapped.71 In Del primers and 116 SSR primers were designed in the candidate interval,and 8 In Del markers and 6 SSR markers closely linked to orange flowers,these markers were all screened by polyacrylamide gel electrophoresis,respectively.3、Transcriptome analysisTranscriptome sequencing was performed on the mixed pools progeny RNA,and the sequencing results were analyzed as follows:Five differentially expressed genes were identified.The genes Bna A07g25840D,Bna A07g25800D,Bna A07g25870D and Bna A07g33370D were in candidate interval.GO enrichment and KEGG enrichment analysis of differential genes showed that gene Bna A07g33370D was enriched in the metabolic pathway of the spliceosome,involving the alternative splicing process.Counting the number of alternative splicing genes in the RNA progeny pool of yellow and orange flowers,193,228 and 191,842 alternative splicing genes in yellow flowers,194,429 and 196,485 alternative splicing genes in orange flowers.The number of splicing genes in orange flowers was about 3,000 more than that in yellow flowers.Thus,preliminarily speculating that gene Bna A07g33370D was the candidate gene for orange flower in Brassica napus L.Quantitative analysis of Bna A07g33370D gene by q RT-PCR showed that the expression level of Bna A07g33370D gene was the highest in the petals of Orange flower and much higher than that of yellow flower.Therefore,this study identified gene Bna A07g33370D as a candidate gene of orange flower trait.4、Candidate gene cloning and bioinformatics analysisIn this study,the candidate gene Bna A07g33370D was cloned.The CDS sequence was 1830bp from yellow flower materials and 1833bp from orange flower materials,and the sequence similarity was 97.32%.There were 49 nucleotide bases differences between the two sequences,among which 46 were caused by base substitution and 3 by base deletion.The two sequences encoded 610 and 611 amino acids,respectively.Ten amino acid differences were found by sequence alignment between the two materials,among which one was amino acid deletion and the other nine were amino acid substitution.The candidate gene Bna A07g33370D was analyzed by bioinformatics:The difference of 10amino acids resulted in small difference in physicochemical properties of proteins between the two materials,and subcellular localization showed that both proteins were located in the nucleus.They were non-secretory proteins without signal peptides and transmembrane domains.The hydrophobicity analysis results showed that the hydrophobicity curves of the two proteins were basically the same,and most of the curves are in the hydrophilic part,which are hydrophilic proteins.Analysis of secondary structure and tertiary structure of the two proteins:there were four secondary structures in both proteins,the proportion of each secondary structure to the total secondary structure is different,and the tertiary structures of the two proteins were basically the same.The results of domain prediction of the two proteins showed that there was an important domain"SKIP-SNW superfamily"in both coding proteins,which participated in the formation of spliceosome and then regulated the transcription process.