MSAP Marker-based Analysis Of Methylation In Hickory (Carya Cathayensis Sarg.)

Hickory(Carya cathayensis Sarg.)is an important non-timber species in Zhejiang Province.It has been found recently that there exist apomixis and iQTLs(imprinted quantitative trait loci)in hickory and polymorphism is low in natural populations in terms of cytobiology and molecular markers.iQTLs are related with DNA methylation,and DNA methylation is an inheritable biological phenomenon,which plays a vital role in the biological phenotype.Methylation-sensitive amplified polymorphism(MSAP)is a PCR-based method derived from AFLP(amplified fragment length polymorphism)to detect DNA methylation.Based on this,we try to use the MSAP marker to conduct a preliminary study on DNA methylation in hickory.The results are as follows:1.An MSAP protocol applicable to hickory was established and used to analyze hickory trees from natural forest,the result of which was then compared with data obtained with other molecular markers(AFLP,RAPD,ISSR,SRAP and SSR)used in hickory.It was found that MSAP markers had the highest level of polymorphism(93.57%).2.The total relative methylation and non-methylation levels in hickory were 25.994±2.415% and 33.381±3.657%,respectively,and the genomic relative non-methylation level was higher than the total relative methylation level(p<0.001).The relative semimethylation and internal cytosine methylation levels in the total methylation were 7.892±1.525% and 18.102±3.940%,respectively,and the relative internal cytosine methylation level was far higher than the relative semimethylation level(p< 0.001).3.Shannon diversity index(I)was used for analysis of genetic(NML)and epigenetic(MSL)diversities with samples from 3 natural populations and their corresponding half-sib populations.It showed that the epigenetic diversity index was far higher than the genetic diversity index(p<0.001)either at the parental or at the half-sib levels.The higher genetic and epigeneticdiversities of the parental populations,the higher ones of their half-sib populations.4.AMOVA showed that almost all of genetic variation existed within the population(98.76%),and only a small proportion(1.24%)existed among populations with no significant difference.76.7% of epigenetic variation existed within the population,and there was 23.3% of epigenetic variation existing among populations.The epigenetic differentiation coefficient(0.2331)was far higher than the genetic differentiation coefficient(0.01236)among populations.The epigenetic variation accounted for 17.4% and the genetic variation accounted for 11.31% among the half-sib populations;and the epigenetic variation accounted for 82.6%,and the genetic variation accounted for 88.69% within the population.The epigenetic differentiation coefficient(0.1739)was higher than the genetic differentiation coefficient(0.1134)among the populations.5.The MSAP protocol established in hickory was also suitable for pecan.The loci and the number of loci of pecan were different from those of hickory.The preliminary study showed that hickory has a methylation level and its epigenetic and genetic diversities were abundant.However methylation in hickory affects its the biological characteristics and their heritability,which need further study.