The Evolutionary Mechanism Of Sex Differentiation And Papain-like Cysteine Proteases Lineage-specific Gene Expansion In Papaya

Sex determination in papaya is controlled by a pair of recently evolved sex chromosomes.The female genotype is XX,male is XY,and hermaphrodite is XYh.A complete sequence assembly of the sex determining region is required to understand the evolutionary and molecular basis of sex determination in papaya.Papaya has a Y(h)Y(h)embryo-lethal phenotype,which is a typical stage of sex chromosome evolution.Thus,there is need to characterize the function of CpEMB506,a candidate gene likely responsible for the Y(h)Y(h)embryo lethal phenotype.During evolution,Y chromosome has degenerated faster than X chromosome and has experienced a faster rate of gene decay,resulting in an imbalance in the gene dosage between the male and female.The phenotypic differentiation of sex types is due to sex biased gene expression.Therfore,studying the dosage compensation and sex biased gene expression will improve our understanding of the mechanism of sex differentiation in papaya.In addition,the gene expression profiling of the sex organs at different developmen stages can help to decipher the gene network regulating the development of sex organs.The main discoveries are described as below:The sex determining regions(X and HSY)of papaya were reassembled using PacBio sequencing reads.Compared to the previous assembly,the new assembly improved significantly.The number of gaps dropped from 121 to 27 on the X pseudomolecule and from 107 to 13 on the HSY pseudomolecule.We also partially filled the largest gap on HSY and mapped the resulting sequence to the X chromosome at approximately 1.9 Mb downstream of knobl,which is present in the autosomal region shared by X and Y chromosomes.This assembly therefore helped delineate the left border(also called border A)between the autosomal and the sex determining regions of the papaya sex chromosomes.We discovered many previously unknown sex-specific repeats in the new assembly,and many regions rich in tandem repeats,including 5S rDNA,on both X and Y chromosomes.CpEMB506,a homolog of the key embryonic developmental gene AtEMB506in Arabidopsis,was identified in the sex determining region in papaya.In Arabidopsis,loss of AtEMB506 function causes embryo lethal phenotype.Since the CpEMB506 allele on HSY is a pseudogene,we predicted that CpEMB506 likely causes the YY embryo-lethal phenotype in papaya.CpEMB506 is highly expressed in embryos at 15-35 days post pollination,which is coincident with the time of YY embryo abortion.The CpEMB506 allele from papaya X chromosome can restore the 25%seed abortion phenotype in the emb506 heterozygous Arabidopsis mutant.To rescue the YY embryo lethal phenotype,we transformed CpEMB506 into papaya callus and obtained transgenic plants.Vasconcellea monoica,a close monoecious relative of papaya,does not have sex chromosomes.We therefore used it as a proxy to assess the ancestral gene expression level for the sex determining genes in papaya.We compared the gene expression between the male and the female leaves and flowers and found that some genes in the sex determining region show dosage compensation.Based on the sequence divergence of shared genes between the X and HSY,the two evolutionary strata,corresponding to the two inversions on the HSY were identified.The median expression level of Y alleles in the older evolutionary strata was lower than the ones in the younger strata and the collinear region,suggesting that genes on Y decreased their expression during sex chromosome evolution.Sex-biased genes were identified between male and female papaya tissues,including leaves,flowers,and different developmental stages of sex organs.Similar to animals,the reproductive organs in papaya showed more sex-biased gene expression than the vegetative organs.Although the total number of male-biased genes and female-biased genes were similar across most developmental stages,there were more male-biased genes with high magnitude of biased expression(>10 fold).The male-biased genes also had a higher Ka/Ks ratio,suggesting that these genes evolved faster than other genes.The transcriptomes of stamens at different developmental stages can be used to study stamen morphogenesis in papaya.Genes involved in stamen development and in hormone biosynthesis pathways showed similar expression between papaya and A rabidopsis,suggesting their conserved function.Using the WGCNA co-expression network analysis,we obtained a pollen-related gene cluster that was enriched in genes involved in pollen germination and pollen tube elongation.The network analysis also identified six core transcription factors that have multiple connections with other pollen expressed genes,suggesting their vital role in pollen development.Papain is a very important product from papaya.We studied the papain-like cysteine protease(PLPC)gene family in papaya.Thirty-three PLCPs were identified in papaya and grouped into 9 subfamilies.The numbers of PLCP genes vary among the 9 subfamilies and among plant species.PLCP subfamily ? has expanded in papaya via three consecutive tandem duplications,estimated to have occurred at approximately 48 MYA,34 MYA and 16 MYA.Most of the papaya PLCP genes from subfamily ? are expressed at high levels in the leaf and green fruit compared to the flower and the mature fruit tissues.