Development Of High-density SNP Breeding Chip And Its Application In Pol-CMS Thermosensitive Sterility Research In Brassica Napus

Rapeseed is one of the most widely planted oil crop in China.China imports a large number of canola to fill the domestic consumer market demand.Therefore,it’s particularly important to speed up the work of high-quality and high-yield rapeseed breeding.This research developed a high-density SNP breeding chip（Bnapus50K）for rapid and universal genotyping,and serving the breeding and genome research of B.napus.The pol cytoplasmic male sterile line of B.napus has been successfully applied to two-lines seed production based on its thermo-sensitive characteristics.Therefore,it is particularly important to clone thermosensitive genes for breeding and to analyze the molecular mechanism of thermo-sensitive occurrence.In this study,Brassica DNA chip were used to mapping thermo-sensitive genes.In addition,RNA-seq and Iso-seq were used to analyze the gene expression profile of pol thermo-sensitive sterile lines at different temperatures to reveal the mechanism of temperature-regulated fertility transition.The main findings are as follows:1.The design of Breeding Chip:Here,we integrated Brassica 60K DNA array genotyping data,re-sequencing data from 505 inbred rapeseed lines and the functional gene sequences,to develop an Illumina B.napus 50K SNP array,named Bnapus50K chip.This array with a more consistent genetic and physical distribution of probes.The 1,618functional probes related to important agronomic traits such as oil content,disease resistance,male sterility,and flowering time ect.144 cultivar lines,27 parent lines,and their corresponding 21 F₁ were used as test materials for the Bnapus50K detection,a cluster file containing 28,509 high-quality probes for allotetraploid rapeseed was constructed.2.Detection effect of the Bnapus50K array:The test result of the test sample shows that the high-quality probes have a good detection efficiency.The genetically modified probes can detect genetically modified components with a content greater than 3.3%.In this study,Bnapus50K chip was used to identify different cultivars with genetic relationship,and the results showed that the chip has practical value in cultivar identification.In addition,we combined bulked segregant analysis（BSA）and chip genotyping to locate the cloned gene Rfp,the results showed that the filted difference markers are mainly located near Rfp loci,indicating that the chip also have high utilization value in gene mapping.3.Application of the DNA chip in thermo-sensitive gene mapping:We investigated the fertility degree of the F₂ segregated population which constructed by pol thermo-sensitive sterile lines 596 and pol stable sterile lines 1318 in the field.Under persistent low-temperature conditions,we found that the F₂population showed a continuous phenotypic distribution from sterile to fertile,indicating that the thermo-sensitive recovery of pol CMS under low temperature is a quantitative trait.We combined BSA and DNA chip to genotype the extremely bulked pools from BC₁ segregating populations and their parental lines,the thermo-sensitive candidate gene was initially located on the A07chromosome.And the results were verified by resequencing analysis and polymorphic molecular marker scanning of the BC population.90 individual plants from the F₂segregated population were genotyped by Bnapus50K DNA array,a QTL was scanned on scaffold A08 chromosome,which explained 18.1%of phenotypic variation.Polymorphic markers were also used to screen the F₂ segregated population,and the candidate interval results were validated.Sum up,by using DNA chips,we discovered the loci that control thermo-sensitive sterility trait may located on the chromosomes of scaffold A07（1.092 Mb-3.379 Mb,7.048 Mb-10.588 Mb）and scaffold A08（12.900 Mb-14.192 Mb）.4.Phenotypic observation of pol thermo-sensitive sterile line:Under low temperature（16℃）,the anthers of Polima thermo-sensitive cytoplasmic male sterile line596 developed normally,and produced fertile pollen that can be stained with 1%acetocarmine staining solution;under high temperature（25℃）,596 had relatively short anthers with no viable pollen.Cytological observation of anther tissues at different temperatures by semi-thin section showed that the sterility period of the thermo-sensitive CMS line was the same as that of the stable CMS line,both of which were abnormal at the3-4 stages of anther development and the differentiation stage of spore cells.5.RNA-seq analysis:Flower buds of pol TCMS line and stable pol CMS line grown at 16°C（596-L and 1318,respectively）,and the pol TCMS line grown at 25°C（596-H）were obtained for RNA sequencing.1,038 upregulated Differentially expressed genes（DEGs）and 599 downregulated DEGs in 596-L compared with 596-H and 1318.GO enrichment analysis of DEGs between fertile and sterile samples showed that these genes were mainly involved in various metabolic processes and performed the function of ion binding.Transcripts of genes related to mitochondrial function were further analyzed,and it was found that there was an orf224/atp6 co-transcript containing 357 nt of the 3’end of the sterile gene orf224 in 596-L;genes related to non-phosphorylated respiratory pathways were up-regulated expression in 596-L,and the results of FCCP treatment the buds indicated that the balance of redox levels within mitochondria plays an important role in anthers development.In addition,some heat shock transcription factors related to ROS response were differentially expressed among different samples,and the results of NBT staining showed that ROS accumulation was the lowest in 596 at low temperature.6.Pac Bio Iso-seq:The transcripts of 596-L and 596-H were sequenced using the Pac Bio Iso-seq platform,serval new isoforms,fusion genes and lnc RNA were identified.The results of alternative splicing（AS）analysis of transcripts showed that the elevated temperature increased AS types and numbers in buds.GO and KEGG enrichment analysis were performed on the genes that specifically occurred AS events at different temperatures,and it was found that these genes were mainly involved in various metabolic processes at low temperature and involved in genetic information processing at high temperature.7.Identification of candidate genes:Comprehensive screening of gene DNA level variation,RNA level expression and protein annotation in the candidate interval of thermo-sensitive locus,five genes（Bna A07G0016300ZS,Bna A07G0027800ZS,Bna A07G0034100ZS,Bna A08G0072900ZS）were identified as possible candidate genes for fertility conversion of pol TCMS under low temperature.