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.

The soybean pod borer (Leguminivora glycinivorella) (SPB) is a major cause of soybean (Glycine max L.) yield losses in northeast Asia, thus it is desirable to elucidate the resistance mechanisms involved in soybean response to the SPB. However, few studies have mapped SPB-resistant quantitative trait loci (QTLs) and deciphered the response mechanism in soybean. Here, we selected two soybean varieties, JY93 (SPB-resistant) and K6 (SPB-sensitive), to construct F₂ and F_2:3 populations for QTL mapping and collected pod shells before and after SPB larvae chewed on the two parents to perform RNA-Seq, which can identify stable QTLs and explore the response mechanism of soybean to the SPB. The results show that four QTLs underlying SPB damage to seeds were detected on chromosomes 4, 9, 13, and 15. Among them, qESP-9-1 was scanned in all environments, hence it can be considered a stable QTL. All QTLs explained 0.79 to 6.09% of the phenotypic variation. Meanwhile, 2298 and 3509 DEGs were identified for JY93 and K6, respectively, after the SPB attack, and most of these genes were upregulated. Gene Ontology enrichment results indicated that the SPB-induced and differently expressed genes in both parents are involved in biological processes such as wound response, signal transduction, immune response, and phytohormone pathways. Interestingly, secondary metabolic processes such as flavonoid synthesis were only significantly enriched in the upregulated genes of JY93 after SPB chewing compared with K6. Finally, we identified 18 candidate genes related to soybean pod borer resistance through the integration of QTL mapping and RNA-Seq analysis. Seven of these genes had similar expression patterns to the mapping parents in four additional soybean germplasm after feeding by the SPB. These results provide additional knowledge of the early response and induced defense mechanisms against the SPB in soybean, which could help in breeding SPB-resistant soybean accessions.

Tiger nut (Cyperus esculentus L.) is recognized for its high oil and oleic acid content in underground tubers. However, the molecular mechanisms governing growth, development, and fatty acid accumulation in these tubers are not well understood. This study employed gas chromatography–mass spectrometry (GC–MS) and small RNA sequencing on tiger nut tubers across five developmental stages. The findings indicate that the critical period for accumulating dry matter and oils, particularly oleic acid, occurs between 35 and 75 days after tuber formation. A total of 183 microRNAs (miRNAs) were identified, comprising 31 known and 152 novel miRNAs. Approximately half of these miRNAs (such as ces-miR156b-3p and ces-miR166a-3p) exhibited differential expression during and around the key periods of metabolite synthesis. The predicted target genes of these miRNAs were significantly enriched in glycerate 3-phosphate metabolism and cell growth processes. Furthermore, 13 miRNA–mRNA interaction modules related to oil accumulation and tuber growth were identified, and these target genes’ expression levels showed significant differences during the tuber developmental stages. These findings advance the understanding of the molecular mechanisms underlying tuber development and oil accumulation in tiger nut.