Molecular Mechanism Of Male Sex Determination In Papaya

Papaya(Carica Papaya L)is one of the world’s top ten tropical fruits.Papaya is a rare trioecious plant species with three sex forms:female,male and hermaphrodite.Sex determination in papaya is controlled by a pair of recently evolved sex chromosomes,the genotype XX controls female,genotype XY male,XYh is hermaphroditic plants.Yh and Y chromosomes have high similarity,but any combination of Y and Yh genotype(YY,YhYh,YYh)will cause seed abortion.The hermaphroditic variety ’SunUp’ genome was sequenced.Analysis of the Yh chromosome specific region(hermaphrodite-specific region of the Yh chromosome,HSY)reveals HSY that has about 8.1 million base pairs(mega-base pair,Mb)and its X counterpart has 3.5 Mb.The male-specific region of the Y chromosome(MSY)was also sequenced and annotated using the same sequencing method with Yh chromosome.The sequencing of the papaya sex-determinant region will serve as foundation for study of papaya sex determination.The objective of this thesis project is to explore the molecular mechanism of carpel suppression in male development using ethyl methanesulfonate(EMS)induced male to hermaphrodite sex reversal mutants.The main results of this research are as follows:Firstly,two male specific DNA markers were developed.1,887 indels(deletion and insertion)and 21,088 SNPs(single nucleotide polymorphisms)were detected by sequence comparison of the hermaphodite-specific region of Yh chromosome(HSY)and male-specific region of Y chromosome(MSY).A large male-specific retrotransposon insertion of about 8kb was located between gene CpXY(h)9 and PCpY(h)2.Besides,a deletion hotspot about 1.5 Mb was found.Three SNPs hotspots were located in inversion 1(0.2-0.4 Mb),inversion2(4.1-4.3 Mb)and collinear(7.4-7.6 Mb)region,respectively.Two of them were located in HSY genes close region.Two male-specific primers were designed according to the indels of sex determination region,and were name d PMSM(Papaya Male-specific Marker)1 and PMSM2,respectively.This means the three different sex characteristics in papaya could be quickly and accurately identified by the simple polymerase chain reaction(PCR).Secondly,six male to hermaphrodite sex reversal mutants were generated from 10,000 EMS treated papaya plants.The six male to hermaphrodite sex reversal mutants were identified using flower morphology observation in the field and male-specific marker primers to identification sex method,and were named MHM(Male to Hermaphrodite Mutant)1,MHM2,MHM3,MHM4,MHM5 and MHM6,respectively.Genome re-sequencing of six mutants and control revealed that the average variant rate isl/132 kb in genome-wide region,but the sex-determination region has lower average variant rate at 1/626 kb.A total of 818 non-synonymous point mutations were identified,corresponding to 747 encoded protein gene.A functional group reproductive growth were enriched using GO annotation.We found 30 encoding transcription factors using Arabidopsis transcription factors database.,including three transcription factors related to the regulation of plant hormone,ethylene response factor(ERF),auxin response factor(ARF)and brassinosteroid-related factor(bHLH).Thirdly,differentially expressed genes were identified betweem sex reversal mutant and wild type.The flower of less than 5 mm in size were collected from each mutant and wild type for RNA sequencing.1,770 differentially expressed genes(DEGs)compared with the wild type were shared in all mutants,including 939 up-regulated and 831 down-regulated.A total of 115 hormone-related differentially expressed genes were identified,mainly associated with plant signal transduction.The ethylene-related encode protein expression increased.110 differentially expressed transcription factors also were found,which ERF and NAC transcription factors upregulated.However,the development of carpel-related YABBY(CRC)was down-regulated transcription factors.KEGG annotation of differentially expressed genes,are enriched in plant hormone signal transduction pathways(ko04075).Fourthly ethylene response factor 4(ERF4)may play a role in sex differentiation.Sex reversal mutants were selected as materials by whole genome re-sequencing and RNA sequencing method of combining in this study,reveal that papaya carpel development in male plants is mainly related with plant hormone response and signal transduction,whereas the carpel initial growth were induced by gene CRC low expression.Nonsynonymous mutations ethylene response factor(ERF4)was upregulated in all mutants,indicating that the transcription factor may play an important role in the sex expression.In this study,the two papaya male-specific markers(PMSM1 and PMSM2)can be used to detect papaya plants sex types rapidly and accurately in combination with a positive control marker(71E)from autosome and the male/hermaphrodite specific marker(W11).The seedling plants sex type can be identified using present method,and selecting different sex type plants were transplant grown according to the actual situation.This greatly reduces the cost of production,avoid unnecessary waste of human and material resources,it has a certain practical significance for papaya production.The sex determination candidate genes were predicted by whole genome sequencing and transcriptome sequencing will be useful in the papaya sex determination transgenic and TILLING research.