Heterosis and Differential DNA Methylation in Soybean Hybrids and Their Parental Lines

Abstract

Heterosis is an important biological phenomenon and is widely applied to increase agricultural productivity. However, the underlying molecular mechanisms of heterosis are still unclear. Here we constructed three combinations of reciprocal hybrids of soybean, and subsequently used MethylRAD-seq to detect CCGG and CCWGG (W = A or T) methylation in the whole genome of these hybrids and their parents at the middle development period of contemporary seed. We were able to prove that changes in DNA methylation patterns occurred in immature hybrid seeds and the parental variation was to some degree responsible for differential expression between the reciprocal hybrids. Non-additive differential methylation sites (DMSs) were also identified in large numbers in hybrids. Interestingly, most of these DMSs were hyper-methylated and were more concentrated in gene regions than the natural distribution of the methylated sites. Further analysis of the non-additive DMSs located in gene regions exhibited their participation in various biological processes, especially those related to transcriptional regulation and hormonal function. These results revealed DNA methylation reprogramming pattern in the hybrid soybean, which is associated with phenotypic variation and heterosis initiation.

Keywords: DNA methylation; heterosis initiation and formation; reciprocally hybrid seeds; soybean.

Figure 1

DNA methylation landscapes of hybrids and their parental lines in hybrid contemporary seeds. (A,B) Relative average methylation levels in different genomic elements. (C) Distribution of methylation sites in soybean genome. (D) The significant difference of CCGG and CCWGG methylation levels. The number above black line of two sites is the p value determined by the paired Student’s t-test.

Figure 2

Association of DNA methylation in reciprocal hybrids with parental methylation status. (A,B) Average methylation levels at three combinations in developing seeds. Four groups are classified based on parental methylation differences in two cytosine contexts, including one parental line higher methylated than another parental line (e.g., P1 > P2 or P2 > P1); both parents are methylated with equal methylation levels (e.g., P1 = P2 > 0); cytosine contexts showing no methylation in two parents (e.g., P1 = P2 = 0). (C) Association of DNA methylation in reciprocal hybrids with parental-unequal methylated sites. HDS, reciprocal hybrids differentially methylated sites; PDS, parental differentially methylated sites; HDS&PDS and Ins, intersection ration of HDS and PDS; PSD, parental selective difference sites; PSD/Ins, PSD as a percentage of Ins; PSD/HDS, PSD as a percentage of HDS; F1221/F1331/F2332, the hybrid combination, e.g., F1221 is a combination using P1 and P2 as crossing parents, the same as figure below. (D) Parental selective difference sites in reciprocal hybrid’s unequal region. F12_F21/F13_F31/F23_F32, the methylated differential site of reciprocal hybrids, e.g., F12_F21 denotes those sites between F12 and F21.

Figure 3

Non-additively methylated sites in all hybrids. (A) The non-additive methylation sites number of all hybrids. (B) Association of non-additive methylation sites with parental methylation status. (C,D) Non-additive categories of methylated sites in all hybrids. These sites were classified when methylation change in all hybrids is identical. “Above” or “Below” means methylation level of one hybrid is greater than two parents or less than two parents of parental equivalent regions. In parental unequal regions, “HP” or “LP” means methylation level of one hybrid is similar to the highest or lowest methylation level, “<LP” or “>HP” means methylation level of one hybrid is lower than in lowest parent or higher than in highest parent. (E,F) The distribution ratio of non-additive sites in three reciprocal hybrids for two methylation contexts.

Figure 4

Non-additively methylated sites in reciprocal hybrid seeds. (A) The proportion of reciprocal non-additive methylated sites (re-DMSs) in whole genome and parent methylated region. The left pie chart shows the ratio of these sites in parent methylated region, red color denotes parent-equal regions and blue color symbolizes parent-unequal regions. The right chart displays the scale of these sites in genomic element. (B) Non-additive categories of re-DMSs in three group reciprocal hybrids. Re-DMSs were classified when methylation change in both reciprocal hybrids is identical (e.g., both “<LP” in F12 and F21). “Above” or “Below” means methylation level of reciprocal hybrids is greater than two parents or less than two parents of parental equivalent regions. In parental unequal regions, “HP” or “LP” means methylation level of reciprocal hybrids is similar to highest or lowest methylation level, “<LP” or “>HP” means methylation level of reciprocal hybrids is lower than in lowest parent or higher than in highest parent. (C,D) Statistics of re-DMSs in three reciprocal hybrids for two methylation contexts. (E) Enriched GO processes of non-additively methylated genes for three reciprocal hybrids. The color is bluer as p value is more insignificant, grey means insignificant enrichment.