Transcriptomics and proteomics-based analysis of heterosis on main economic traits of silkworm, Bombyx mori

Highlights

• The phenotypic traits of hybrid silkworms showed heterosis.

• Most of the genes and proteins in the silk gland of hybrid silkworms were lower than parents.

• Accelerated metabolism and protein synthesis in hybrid silkworms contributes to heterosis.

• Heterosis may be related to the decreased expression of intracellular transport proteins.

Abstract

The application of silkworm hybrids have promoted the innovation and development of agricultural technology, but the mechanism of heterosis in silkworm has not been explained clearly. In this study, the heterosis of silkworm in the aspects of body weight, silk gland and cocoon weight was investigated by means of silkworm hybridization and multi-omics approaches, including transcriptome and proteome. The results showed that heterosis of silkworm body weight, silk gland and cocoon weight was overdominant, but only part of genes and proteins were overdominant, and most of genes and proteins were non-additive. Combined analysis obtained six up-regulated genes and four down-regulated genes that were consistent both in transcriptome and proteome. Gene functional enrichment analysis indicated that most up-regulated genes and proteins were mostly related to metabolism, which led to accelerated metabolism and protein synthesis and contributing to improved heterosis. The up-regulation of 6-phosphate glucose dehydrogenase (G6PDH), phosphatidylethanolamine-binding protein (PEBP) and sHSP20.4, which are involved in metabolism, might be related to silk gland heterosis.

Significance

A combination of transcriptomic and proteomic analysis was used to understand the molecular mechanism of silkworm heterosis. We found that the phenotypic traits of silkworm are overdominant, while the analysis of transcriptome and proteome showed that only part of genes and proteins were overdominant, and most of genes and proteins were non-additive. Some of the genes had unique expression in F1, which was speculated that genes under heterozygous condition may result in rearrangement and cause metabolic changes in the hybrids. Those both up-regulated in transcriptomic and proteomic analysis were found to be involved in various metabolic processes, so as to accelerate metabolism and protein synthesis, thus exhibiting heterosis.

Introduction

Heterosis, or hybrid vigor refers to the biological phenomenon that hybrid F1 outperforms its purebred parents in disease resistance, yield, product quality and other traits [1]. Hybrid breeding based on heterosis has been applied in many crops and animals and improved their yield and quality, but the molecular mechanism of heterosis has not been accurately explained [2]. The re-exploration of maize heterosis a century ago has revolutionized crop breeding and production [3,4]. Many hypotheses on the genetic mechanism of heterosis have been proposed. Shull and East put forward the over dominant hypothesis [4,5]. Jones further added the dominant-linked gene hypothesis [6]. Sheridon proposed the epistasis hypothesis in 1981 [7]. Then came the gene network system hypothesis [6], active gene effect hypothesis [8] and genetic vibration synthesis theory [9]. These hypotheses have been supported in different studies [10,11]. Li et al. confirmed the epistasis hypothesis in rice [12]. The hypothesis of additive and non-additive expression mechanisms has also been confirmed in studies on arabidopsis thaliana, indica rice and japonica rice [13]. Molecular analysis of transcriptome, proteome and epigenome analysis of parents and hybrids have been applied to the study of heterosis [11,13], including the identification of gene expression changes [2] and the role of epigenetic processes in heterosis [14]. Studies have found that interactions between parental genomes can cause changes in the transcription process and even change the protein types of F1. Genome-wide changes in protein expression in hybrids had an impact on heterosis [[15], [16], [17]], which was also found in silkworm [18]. Additive and non-additive proteomic models were also found in various tissues of many species, such as leaf [15], embryo [16,17,19], seed root [20], cells [15] and mitochondria [21] of maize. The results of 2-D electrophoresis showed that the heterosis of additive and non-additive was obvious, and the same phenomenon was found in rice leaf [22], rice embryo [23], rice seedlings [24], sunflower [25] and wheat [17], while non-additive effect was found more advantageous in reports of lamb [26], chicken [27], cattle [28]. Schwartz et al. proposed single-molecule heterosis model [29], and presented evidence for a class of allelic interactions that resulted in increased enzyme activity of certain hybrids and thereby affecting heterosis.

Single-molecule interactions can also improve hybrid performance and tolerance [30]. Researchers have demonstrated that two coding proteins showed potential compensatory tendencies in vitro, forming amyloid-soluble β-oligomers [31]. Subsequent studies showed that the conversion rate of the mixed oligomers composed of two alleles into amyloid protein was much slower than that of the oligomers with two allele homologies [32]. This example of molecular heterosis observed in vitro provided the basis for maintaining diversity in the population and reinforced the concept of over dominance. Singer et al. provided a genetic explanation for the activity of single-gene hybrids in oil palm shelled by heterodimerization [33].

The silkworm is a good model for heterosis research. Its larval stage is only about 25 days, and the breeding environment is easy to control [34]. In 1919, more than 90% of the silkworms in Japan originated from crossbreeding between Japanese and Chinese silkworms, which reached 100% in 1928 [35], making silkworms a model of heterosis in agricultural production like the maize [36]. The breeding of silkworm hybrids has greatly improved the economic benefits of the silkworm industry, and there have been many reports on the study of silkworm heterosis. Strunnikov found that the level of heterosis is largely determined by the relationship between the effects of useful and harmful genes, the former belonged to the category of semidominant. Their favourable, joint well-coordinated effects increased in relation to the number of genes in a geometric. However, those of genes which control quantitative characters increased in relation to the number of genes in an arithmetic progression [37]. Singh et al. found that heterosis is invariably higher in single crosses compared to three-way and double crosses. Within a restricted range, the greater genetic distance was the greater heterosis [38]. Cai et al. found the more different it was between parents, the greater the esterase activity of heterosis, and the higher possibilities the heterosis [39]. Reddy et al. ‘s report confirmed this view [40]. Wang et al. studied the molecular mechanism related to silk production by using iTRAQ based proteomics and RNA sequencing based transcriptome technology, and found that the main factors of low silk production were the decrease of energy utilization rate, the decrease of protein translation rate and the increase of protein degradation rate [41].

So far, the theoretical basis of heterosis of silkworm is still relatively weak. Therefore, this study selected a low-yielding strain from China and a high-yielding strain from Japan for hybrid breeding. Body weight, cocoon shell weight and silk gland weight were investigated under strictly consistent feeding conditions, and heterosis was evaluated. The molecular mechanism of heterosis of silkworm was proposed on the basis of transcriptomic, proteomic and joint analysis on the silk gland of the fifth instar lava.