Assembly Of Haplotype-resolved Genome By Single Pollen Cell And The Research Of Gene Regular In Self-incompatibility

An improved haplotype-resolved genomes is very important for the study of species in evolution,re-sequencing,and gene function.However,the haploid genome assembly is a huge problem for some highly heterozygous genome and a large number of repetitive sequences like Pyrus spp.In addition,self-incompatibility also restricts the subsequent agricultural production.In this study,we firstly phased 38304 Bacterial artificial chromosome(BAC)into 34 haplotype chromosomes by haplotype single pollen genome sequence and two haplotype-resolved pear genomes were assembled.Then,alleles in haplotype-resolved genomes and chimeric assembly in reference genome were identified.In addition,RNA-seq was used to identify differently expressed genes in self-pollinated and cross-pollinated styles,and the relationship among the S-RNase and plant hormone were constructed.The results are as follows:1.The pear pollen was cultured and the protoplasts were prepared by enzyme combined with the improved pollen medium formula.Then,the haplotype genome information of 12 pollen single cells was amplified by single cell amplification technology and sequenced.Then,using the "12 bit binary marker’,the relationship between artificial chromosome(BAC)and 12 haplotype pollen cells whole genome sequence was established,and BACs were separated into 34 haplotypes chromosome.After that,two haplotype-resolved genome in pear were independently assembled with 546 MB in haplotype-A and 536 MB in haplotype-B.Finally,the 12 pollen genomes with the largest number of genotype calls were characterized and indicated 264 MCO events with an average of 1.3 events per chromosome,and most of the meiotic recombination events occurred in the ends of chromosomes.2.Difference among haplotype-A,haplotype-B and reference genomes were compared.Using the BUSCOs software,it was found that the assembly quality of haplotype-A and haplotype-B was higher than that of the reference genome,and the lost BUSCOs was significantly reduced by 1.4%in haplotype genomes.A total of 33559 and 37805 protein encoding genes were annotated in haplotype A and haplotype B respectively,41904 genes in all.In addition,anchored genes in haplotype-resolved genomes increased by 3930 than reference genome.S17-RNase,an important gene related to self incompatibility,was finally anchored in the 968kb position of chromosome 17 in haplotype B,while F-box(SFB)was anchored in the 3.9-4.0M position of chromosome 17 in haplotype B.The physical distance between S-RNase genes and F-box(SFB)is about 3M and there is no meiotic recombination in this region in 12 pollens.In addition,a total of 3479 genes with mosaic assembly in reference genome,including 1034 genes cause the error of amino acid coding.Moreover,a total of 33904 allelic genes were identified in anchored haplotype-A(29465 allelic genes)and haplotype-B(28984 allelic genes).A total of 236 allelic genes were only exist in haplotype-A and 291 allelic genes were only exist in haplotype-B.At last,the expression level of alleles in fruit development was studied through the data of the transcriptomedata during fruit development.We found a total of 1926 differently expressed alleles and 2079 genes with monoallelic expression,including 95 genes related with fruit quality.3.Using transcriptome comparisions,differently expressed genes of unpollinated(UP),cross-pollinated(CP),and self-pollinated(SP)styles were identified after 48 h.A total of 1796 and 1890 genes were identified in unpollinated styles vs.cross-pollinated styles and and unpollinated styles vs.self-pollinated styles,respectively.KEGG analysis revealed that genes involved in the "plant hormone signal transduction pathway" and "plant-pathogen interaction pathway" were significantly enriched in DSI(UP vs.SP)compared to those in DSC(UP vs.CP).The expression level of S-glycoprotein ribonuclease(S-RNase)was dramatically reduced in cross pollinated(CP)styles.To better understand the relationship between the expression patterns of S-RNase and two major KEGG pathways,The content of 6 hormones in the unpollinated style,self-and cross-pollination style was measured.It was found that methyl jasmonate,abscisic acid,gibberellin,indoloacetic acid and zein nucleoside were affected by pollination,and the content of methyl jasmonate decreased significantly after pollination,and the content of the methyl jasmonate was relatively stable after self pollination.In addition,the expression f S-RNase in 6 styles of steroids was induced by methyl jasmonate and abscisic acid.Finally,we found the relationship between pollination,plant hormonal signal transduction and pathogen interaction.