Homoeolog expression changes in different tissues of high hybrid
To observe the pattern of homoeolog expression in different tissues of high hybrid, a case by case gene comparison was performed for high hybrid (H) and its parents (maternal=A, paternal=B). The results are detailed in Table 1. Notably, a pattern of homoeolog expression was conserved in this hybrid for the greater number of genes in all tissues under investigation. Fewer homoeolog gene copies reflected novel biased pattern. For example, 36853 total number homoeolog gene pairs were identified in each tissue. We observed that 96% in the root, 97% in leaf, and 97% in flower buds had maintained parental expression patterns in hybrid. The total expression level of a homologous gene pair had no change or equal to that of both parents. An average of 1% homoeolog gene pairs showed no bias in each hybrid tissue. According to the results of novel bias expression, 2.27% homologous gene pairs had novel bias in the root. Only 2.1% homologous gene pairs in leaf exhibited novel bias expression. It was observed that 2.02% homologous gene pairs had novel bias in flower buds. Majority of novel biased homologous gene pairs exhibited maternal like expression patterns in root and leaf (Figure 1a-b). Wherein, parental like novel expression were observed in flower buds (Figure 1c). Further analysis indicated that 1.2% homoeolog gene pairs in the root, 1.06% in leaf, and 1.1% in flower buds demonstrated overall maternal parent biased expression. Whereas, overall homoeolog gene pairs with paternal parent biased expression was 1.1%, 1.04%, and 0.95% in the root, leaf, and flower buds, respectively. The overall statistics of homoeolog expression changes in different tissues of high hybrid seem to show a highly balanced biased expression of homoeolog gene pairs in each tissue. These results suggested homoeolog gene pairs that displayed fewer novel patterns of homoeolog expression may be responsible for the high level of heterosis in upland cotton.
Homoeolog expression changes in different tissues of low hybrid
The results of homoeolog expression analysis in different tissues of low hybrid (L) and its reciprocal parents (A= maternal, B= paternal) are represented in Table 2. Interestingly, the homoeolog expression patterns in hybrid were in the same direction for the majority of homoeolog gene pairs as observed in parents. For instance, 97.3% in the root, 97.2% in leaf, and 97.6% in flower buds out of all homoeolog gene pairs (36853) reflected equal parental expression patterns in this hybrid. These result predicted that expression biases pre-existing in parents was simply conserved in these tissues of hybrid. The group of homoeolog gene pairs with bias expression in parents and reverted to no bias expression in hybrid had an average of 1% in root and leaf. On the other hand, only 0.60% of homologous gene pairs in flower buds had this reversion. According to the results of novel bias expression in hybrid, 1.75, 1.78, and 1.76% homologous gene pairs adopted novel bias in the root, leaf, and flower buds, respectively. Majority of homologous gene pairs exhibited novel biased expression similar to maternal parents in root and leaf (Figure 2a-b). In contrast, flower buds had most of homologous gene pairs that had shown novel biased expression with higher paternal contribution in progeny (Figure 2c). Overall, a group of maternal or paternal biased homologous gene pairs had very similar trends in each tissue. There was 0.83% maternal and 0.84% paternal biased homologous gene expression in the root, whereas leaf had 0.98 and 0.81% maternal and paternal biased expression, respectively. Flower buds also showed a similar percentage of maternal and parental biased expression. Altogether, these results suggest that the majority of the homoeolog gene exhibited balanced or non-differential homoeolog expression patterns in low hybrid. Further, homologous genes with few novel patterns of homoeolog expression probably contribute to the low level of heterosis in upland cotton.
Distribution of novel expressed genes among different hybrids
This study further investigated the unique and common novel expressed genes among each tissue of hybrids and among different hybrids. In high hybrid, there were 374, 325, and 301 unique expressed novel genes in root, leaf, and flower buds, respectively (Figure 3). Moreover, a major portion of genes were overlapped among comparison of these tissues. For example, 94 genes were overlapped between comparison of root and leaf whereas 100 genes were overlapped between root and flower buds. The distribution results had shown that 298 novel genes were unique in root of low hybrid (Figure 4). The unique novel genes in leaf were 324 and 308 in flower buds. There were 57 overlapped novel genes between root and leaf while flower bud and leaf had 76 overlapped novel genes. The novel gene distribution between high and low hybrids revealed majority of novel genes were unique and less were overlapped among both hybrids (Figure S1). Only 122 genes were overlapped among hybrids wherein 877 genes were unique in high hybrid and 808 in low hybrids. These results anticipated that novel genes with unique expression most likely caused contrasting level of heterosis in hybrids.
Functional analysis of homoeolog genes exhibiting novel expression changes in hybrids
As to understand possible functions of homoeolog genes that had novel biased expression under hybridization in cotton, we performed GO and KEGG enrichment analysis in each hybrid. GO enrichment analysis (p-value < 0.05) for high hybrid showed a majority of novel biased expressed homoeolog genes had functional annotation related to catalytic activity, nucleus, metabolic process, transmembrane transport, binding of nucleotide, nucleic acid, protein, ATP, and RNA (Fig. 5a). Genes associated with binding of protein and ATP had the highest portion of novel biased expressed homoeolog genes in this hybrid. Most enriched GO terms for novel biased expressed homoeolog genes in low hybrid were involved in functional processes related to the nucleus, catalytic activity, binding of ATP, protein, and nucleotide (Fig. 5b). Interestingly, genes that performed the binding function of protein and ATP also had the majority of novel biased expressed homoeolog genes in this hybrid.
KEGG analysis revealed that the majority of novel biased expressed homoeolog genes in high hybrid were enriched in pathways such as biosynthesis of amino acids, mRNA surveillance pathway, Glycolysis / Gluconeogenesis, biosynthesis of antibiotics, metabolism of carbon, glutathione, cysteine/ methionine, biosynthesis of secondary metabolites, and microbial metabolism in diverse environments (Fig. 6a). Biosynthesis of secondary metabolites had more significant and gene enrichment as compared to other pathways. It was observed that a major portion of novel biased expressed homoeolog genes in low hybrid had enrichment in mRNA surveillance pathway, RNA degradation, Oocyte meiosis, metabolism of pyruvate, carbon, sugars, and biosynthesis of secondary metabolites (Fig. 6b). In a low hybrid, biosynthesis of secondary metabolites also had a maximum number of novel biased expressed homoeolog genes as compared to other pathways.