Construction Of Genetic Linkage Maps And Analysis Of DNA Methytiaon-related Gene Expression In Hickory

Hickory(Carya cathayensis)is an important non-timber tree species in Zhejiang Province.Previous studies had shown that hickory and its relative pecan(C.illinoensis)were reciprocally crossable,their progenies exhibited heterosis and phenotypical similarity to their maternal parents.Recent studies have revealed the existence of imprinted quantitative trait loci(iQTLs)and apomixis in hickory,as well as poor polymorphism by traditional molecular markers such as RAPD,AFLP,and SSR.Therefore,it is difficult to carry out an in-depth study at the genetic levelin hickory.For this reason,this paper adopted MSAP(Methylation sensitive amplification polymorphism)markers to analyze hickory,pecan and progenies from their reciprocal crosses and a genetic map of hickory was constructed.On this basis,transcriptome sequencing was introduced to investigate the expression of methylation-related genes in hickory,pecan,and progenies from their reciprocal crosses,so as to understand the role of DNA methylation in the traits of hickory.The results would help reveal the genetic mechanism of hickory,and at the same time lay the foundation for the basic theoretical research on trees.The main results are as follows:1.With MSAP markers,analysis of methylation patterns for hickory,pecan and their progenies from reciprocal crosses demonstrates that 161476 loci in total were detected.There were 1828 loci with no changes in methylation pattern between parents and the progenies,5357 maternally inherited methylation loci,1220 paternally inherited methylation loci,4047 demethylated loci in the progeneis,4153 methylated loci different between parents and the progenies,12203 De novo methylated loci in the progeneis,and 132668 hypermethylated or mutated loci found in the progenies,out of which hypermethylated or mutated loci are those with no amplification products.The number of maternally inherited methylation loci is much higher than that of paternally inheritedones,indicating that maternal methylation plays a vital role in maintaining the expression of the maternal gene.The genetic locus resulting from demethylation accounted for 76.5%of the sum of nonmethylated loci,semi-methyalted loci and internal cytosine-methylated loci,which suggests that methylation has some effect on the discovery of genetic loci in hickory.2.A 15-linkage-group genetic map in hickory was constructed,which contained 7 RAPD markers,38 ISSR markers,23 SRAP markers,and 109 MSAP markers and covered a chromosomal length of 3580.2 cM with a mean interval of 20.2 cM.Four linkage groups were occupied only by MSAP markers.At the same time,a 14-linkage-group methylation locus-containing genetic map was constructed in which 337 markers were from MSAP.This map covered a chromosomal length of 7742.3cM with an average map distance of 19.4 cM,of which 7 linkage groups were almost covered by MSAP markers.The results indicate a high degree of methylation in the genome of hickory,which may play an important role in the expression of the growth-and development-associated genes in hickory.3.QTL detection showed that no significant QTL was found with genetic loci and phynotypic data of seedlings,but two loci associated with basal dimeter of seedlings and one locus associated with seedling hight were identified with both genetic and methylation loci and phynotypic data of seedlings.4.With unigenes from repeated transcriptome sequencing data at three temporal points of maternal and parental parents and their progenies rsulting from reciprocal crosses,PCA found that the progeny resulting from reciprocal crosses between hickory and pecan were close to their maternal parent,which confirms that the progeny from muciprocal crosses between hickory and pecan phynotypically resemble to their maternal parent at the transcriptome level.Meanwhile,25 groups of methylation-related genes were found in hickory leaves.ANOVA showed that they were relatively stable on different dates,but there were differences in expression among species and between organs or tissues.For the progeny from each of the reciprocal crosses,the expression level of methylation-related genes was not significantly different from that of their female parent,but there were significant differences between both parents,between the progenies from reciprocal crosses,and between the paternal parent and progenies of both crosses.A total of 22 groups of methylation-related genes were found during the seed development in hickory,the expression of which decreased to some extent along with the growth and development of the seed.Therefore,methylation plays a crucial role in the growth and development of hickory trees.5.SNP analysis showed that there was an average of 48197 SNPs in the leaves of hickory.The number of SNPs in hickory seeds was more than that in leaves,and most of them were synonymous but had the potential for application in the analysis of genetic diversity.SSR analysis showed that there were 31142 SSRs in the leaves of hickory,28574 SSRs in the pecan,26392 SSRs in the progeny of hickory xpecan,and 40852 SSRs loci in the progeny of pecan xhickory.The SSR number varies either among species or between tissues.