Genome Assembly Of Spinach Female(XX) And Male(YY) And Structural And Evolutionary Analysis Of Sex-determining Region

Spinach（Spinacia oleracea L.）is an important leafy vegetable crop that belongs to the subfamily Quinoa,family Amaranthaceae.Generally,spinach（2n=12）is a dioecious species（i.e.,separate sexes）,while some genotypes have monoecious plants.Spinach has an XY sex-determination system,males are heterogametic（XY）,and females are homogametic（XX）.Sexual reproduction is the primary means of reproduction for the vast majority of plants and animals,in which sex chromosomes play a central role in sexual dimorphism.Sex chromosomes have evolved independently multiple times in divergent lineages of plants and animals.Homomorphic sex chromosomes usually represent the early stages of evolution.Spinach has homomorphic sex chromosomes and viable YY individuals,thus making it an ideal vegetable crop to investigate early evolution.However,the complete sex-determining（SD）region has not been reported yet due to the lack of a high-quality spinach reference genome.Especially,the complete Y chromosome hinders the further investigation of spinach sex chromosomes evolution and SD mechanism.In the present study,we de novo two genomes of both homozygous female（XX）and male（YY）genotypes in spinach,which allowed us to resolve X and Y haplotypes.Then,we unraveled the fine structure of a complex SD region in spinach.The structure was further confirmed using bioinformatics and experimental approaches.In addition,we also investigate the characteristics of the SD region,the evolutionary process,and the formation mechanism of the SD region in spinach.The main findings are as follows.1.A rare male（XY）seed-bearing individual from inbred line 10S15 was identified,whose progeny could produce XX,XY,and YY genotype plants with a 1:2:1 segregation ratio.Then,we sequenced and assembled one female（XX）and one male（YY）genome from the inbred line 10S15 using the ONT platform,named Sp＿XX＿v1 and Sp＿YY＿v1,respectively.The Sp＿XX＿v1 shared a length of 978 Mb with a contig N50 size of 34 Mb.The Sp＿YY＿v1 had a length of 926 Mb with a contig N50 of 41 Mb for male.And 359contigs were generated in Sp＿XX＿v1 and 88.85%of them were anchored to the chromosomes,while 117 contigs were assembled in Sp＿YY＿v1 and 95.72%of them were anchored to the chromosomes.We predicted 28,359 and 28,573 protein-coding genes in the female and male genomes,respectively.The BUSCO values,LTR Assembly Index（LAI）scores,Hi-C interactions heat map,and syntenic relationship of the two genomes indicated high-quality assemblies for both Sp＿XX＿v1 and Sp＿YY＿v1.2.Based on the two genomes,in combination with reported molecular markers associated with spinach sex,we detected a 24.10-Mb SD region in the Y chromosome,corresponding to a 13-Mb counterpart in the X chromosome.The SD region in the Y chromosome contained a 10-Mb male-specific region of the Y chromosome（MSY）and a large-scale inversion.Eighty samples including 48 males and 32 females of resequencing data were used for the genome-wide association study（GWAS）,and all of the significant SNPs associated with sex（α<0.05）were located in the SD region.In addition,304 Y-specific sequences were identified using the k-mer approach between the female and male pools,of which 93.40%were enriched in the MSY region.Also,1,360 sex-related sequences were identified,of which 70.90%were located in the SD region,a result that reinforces the accuracy of the SD region.To verify the structure of SD region,ONT long reads from female and male and female spinach published previously were used to confirm the inversion and the MSY region,respectively.3.The SD region of spinach has recombination suppression and accumulated abundant repetitive sequences,especially transposable elements（TEs）.The percentage of repetitive sequences in the SD region was 89.28%and 90.44%on the X and Y chromosomes,respectively,which was higher than that in the whole genome（73%）.The average gene density was 10 genes per Mb in the SD region of the Y chromosome,which was much lower than the average of 16 genes per Mb in the X counterpart.The gene density in the SD region was markedly lower than that in the genome-wide（28/Mb）.In addition,genome coverage in males and females did not exhibit divergence in the IV2 region between the X and Y chromosomes.However,within the IV2 region,males have a significantly higher SNP density than in the pseudoautosomal region（PAR）and males.The genetic differentiation(F_ST)and synonymous substitution rate（Ks）of the X/Y gene pairs showed that the gene pairs within the inversion exhibited markedly higher divergence than that of the PAR on the Y chromosome.These results suggested that recombination has been suppressed very recently,which shows significant divergence but without major Y chromosome decay.A total of 19differentially expressed genes（DEGs）were detected within the SD region,and 16 genes out of them were highly expressed in males while low or not expressed in females,indicating that male-biased expression of genes in the SD region.The expression levels of genes in the MSY were about twice as high in YY males as in XY males.However,the genes showed no significant difference in PAR,IV2 region,and autosomes,indicating a lack of dosage compensation in the Y chromosome.Divergence analysis revealed the presence of two stages perfectly corresponding to the MSY and the inversion region.Estimated divergence time showed the mean divergence time of 3.10 MYA for MSY and 2.56 MYA for inversion,thus indicating the first occurrence of the MSY,followed by the inversion event,which was further confirmed by the LTR-RTs insertion time in the MSY（3.15 MYA）and the inversion region（2.97 MYA）.