Genetic Characteristics Revealed By Msapanalysis In Hickory Based On Reciprocal Crosses

Hickory?Carya cathayensis?is an important non-timber species in Zhejiang Province.Many years'research has found that hickory and pecan?C.illinoensis?,another species belonging to the same genus as hickory,are reciprocally crossable?reciprocal crosses?,the progeny of which shows heterosis.Research in recent years has found there were imprinted quantitative trait loci?iQTL?and apomictic phenomena in hickory.Apomixis and heterosis are contradictory with each other,but both phenomena observed were all beneficial traits in production.In this paper,these 2 species and progenies from mutual crosses of these two species were analyzed with methylation sensitive amplification polymorphism?MSAP?markers which were suitable for both hickory and pecan,the results of which were compared with the result from hickory trees and their open-pollinated progenies,to reveal the genetic mechanisms behind hickory.The study could also lay a foundation for basic theoretical research on perennial tree species which are large in genomes and highly heterozygous.The main results are as follows:1.MSAP analysis of the progenies from reciprocal crosses indicated that the total number of loci,methylation-susceptible loci?MSL?,polymorphic MSL,no methylated loci?NML?and polymorphic NML of the progeny from pecan×hickory was more than that from hickory×pecan,but the Shannon diversity index?I?of MSL and NML of the progeny from pecan×hickory was slightly higher than that of the progeny from hickory×pecan;and the percentage of NML,hemimethylated loci and internal cytosine?C?methylated loci of the progeny from hickory×pecan was higher than that of the progeny from pecan×hickory.2.As compared with the result of analyzing the progeny of reciprocal crosses with RAPD,ISSR,and SRAP,MSAP molecular markers MSAP revealed much genetic loci and the percentage of polymorphic loci was nearly 100%,which was twice or triple as much as the 3 types of markers mentioned above.3.Wilcoxon Rank Sum test indicated that the progeny of hickory×pecan?W=251521?p<0.0001??differed significantly from that of pecan×hickory?W=436529?p<0.0001??in genetic diversity and epigenetic diversity.Analysis of molecular variance?AMOVA?showed that there was significant difference between progenies of reciprocal crosses in either MSL or NML?p<0.0001?and the variance within the progeny accounted for 80%;and principal coordinate analysis?PcoA?of MSL and NML indicated that differentiation of either NML or MSL between the progenies of reciprocal crosses was obviously different.Mantel test indicated that NML had significant effects on MSL in the progenies of reciprocal crosses?r=0.3647102,p=0.000999,p<0.01 for the progeny of hickory×pecan;and r=0.2648822,p=0.000999,p<0.01 for that of pecan×hickory?.Based on the genetic information acquired,clustering was performed with all the individuals from each of reciprocal crosses,which indicated at the molecular level that individuals from each of reciprocal crosses differed from one another and hybridization was feasible between hickory and pecan.4.Seedlings at different ages resulting from open-pollinated hickory and pecan trees as well as from reciprocal crosses between these two species differed in height?H?and basal diameter?D?significantly,and both pollination modes and ages had a significant interaction;and F₁ hybrid seedlings showed heterosis.Both H and D of seedlings from 4 pollination modes had no significant difference in the first two years except those of 2-year-old seedlings from open pollinated hickory.The H and D of4-year-old hybrids of hickory×pecan were 158.61 cm and 14.75 mm,respectively,while those of pecan×hickory were 128.54 cm and 14.93 mm,which were significantly greater than those of seedlings from open pollinated parents?101.69 cm and 10.82 mm in hickory as well as 87.65 cm and10.73 mm in pecan,respectively?.F₁ hybrids showed strong heterosis as compared with open pollinated ones.Likelihood ratio test?LRT?indicated that 204 out of the total 718 MSAP genetic markers were associated with seedling growth to a significant degree,the contribution of which to growth traits ranged from 17.98%to 97.9%.5.MSAP analysis of 29 pecan clones achieved a total of 1307 loci,the number of NML?56.24%?was higher than that of MSL?43.76%?,but variation in MSL was richer than that in NML.Internal C methylated loci accounted for 19.84%among 4 types of loci,and the rate of no methylated loci and hemimethylated loci was less than 10%,but the rate of no methylated loci was a little bit higher than that of hemimethylated loci.Wilcoxon Rank Sum test indicated that genetic and epigenetic differences for pecan clones was significant?W=150865.5?p<0.0001??.The I of genetic loci?0.20±0.08?was smaller than that of epigenetic loci?0.51±0.14?,but the CV of I for NML?40.9%?was higher than that for MSL?27.1%?.6.A comparison between two species,viz.hickory and pecan,based on MSAP analysis indicated that the percentage of NML in hickory was much higher than that in pecan.I indicated that the epigenetic diversity in pecan paralleled with that in hickory,but the genetic diversity in pecan was higher than that in hickory.Wilcoxon Rank Sum test indicated pecan?W=150865.5?p<0.0001??differed significantly from hickory?W=16656.5?p<0.0001??in either genetic or epigenetic diversity.AMOVA indicated that pecan and hickory had significant differences in both MSL and NML,most of which?67.28%for MSL and 77.48%for NML?was within the species.PcoA indicated that pecan differentiated significantly from hickory,and epigenetic diversity was very abundant.Mantel test indicated that NML had a significant effect on MSL in pecan but this was not the case in hickory.The values of genetic parameters?number of alleles?Na?,number of effective alleles?Ne?,Shannon diversity index?I?,expected heterozygosity?He?,unbiased expected heterozygosity?uHe??in hickory were very close to those in pecan,which also suggested that the reciprocal crosses between these 2species were feasible.In conclusion,analysis of progenies from reciprocal crosses between hickory and pecan indicated that methylation influenced the discovery of genetic loci;there was hybridization between these 2species;and methylation may play an important role in t apomixis of hickory.