Analysis Of DNA Methylation And Differential Gene Expression Between Cotton Hybrids And Their Parents

Exploitation of heterosis is one of the major example of genetics that could increase the yield of crops, save the land, have made the significant contribution to the world. The efforts for exploring the mechanism of heterosis have lasted for nearly one centure. However, many aspects remain unknown. As the most important textile crop and the world's second most important oilseed crop, cotton plays a vital role in the economic, political, and social affairs of the world. Hybrid cotton has been widely used commercially and studied for many years in India and China. However, until now, there has been no report on the molecular aspects of cotton heterosis because of its allopolyploidy and relatively large genome size. In this paper, we study the heterosis of growth, yield-related traits, quality traits and root traits in different cotton crosses, then we investigated both the differences in cytosine methylation patterns among two cotton hybrids and their parental lines by using the methylation-sensitive amplified polymorphism (MSAP) method and the differential gene expression between the two hybrids and their parents by using the differential display technique, finally we analyzed the correlation between differential expression patterns and heterosis at full opening flower stage. This work was carried out for dissecting the molecular basis of heterosi on epigenetic regulation level and on gene differential expression level, respectively. The main results are as follows:1. One female parent crossed with two male parent, resulting in a highly heterotic hybrid, 345xCRI41 (Hybrid A), which shows high heterosis in growth, yield-related traits and root traits, and a low heterotic hybrid, 345×SGK321 (Hybrid B), which only shows heterosis in some root traits.2. To investigate the relation between DNA methylation and heterosis, we investigated the cytosine methylation level in cotton heterotic hybrid/nonheterotic hybrids and their parental lines at flowering stage by using the methylation-sensitive amplified polymorphism (MSAP) method. The results showed that about 20% of the 5'-CCGG sites in cotton genome at the flowering stage were methylated.3. According to the inheritance and alteration of cytosine methylation from parent to hybrid, the cytosine methylation patterns between hybrids and their parents were compared. Our results showed that extensive cytosine methylation alterations including hyper-and demethylation as well as the potential conversion of methylation types (from external cytosine to internal cytosine or vice visa) occurred in the hybrid compared with the parents; For the group showing a decreased level of methylation in the hybrid compared with the parents, the demethylation loci that occurred in cross 345/ CRI41 (Hybrid A) were greater than those in 345/SGK321 (Hybrid B). Another group showed an increased level of methylation in the hybrid, but these hyper-methylation loci account for only a small portion of the total polymorphism loci in both hybrids.4. On the basis of the whole analysis of methylation status at 5'-CCGG sites in the three parental lines and the two hybrids at the seedling stage and the flowering stage, we found that more loci were demethylated at the flowering stage than at the seedling stage.5. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. The loci that underwent methylation alterations in the hybrid compared with the parents were sequenced and found to be homologous to functionally characterized genes, including transcription regulator, signal recognition, leucine-rich repeat, calcium channel, PDR-like ABC transporter, decarboxylase, phosphatase, isomerase and ATPase.6. Using differential display technique, we analyzed the gene differential expression in leaves between cotton heterotic hybrid (345×CRI41)/non-heterotic hybrid(345×SGK321) and their parents at the seedling, squaring, flowering and boll-forming stages, respectively. The results indicated that there existed significant differences of gene expression between cotton hybrids and their parents during the four stages, whatever these hybrids were heterotic ones or non-heterotic ones. Both quantitative and qualitative differences were observed. The major differences of gene expression occurred in the qualitative level for each stage. The qualitative differences include: (â…°) bands observed in both parents but not in F₁ hybrid (BPnF₁); (â…±) bands occurring in either of the parents but not in F₁ hybrid (UPnF₁); (â…²) bands present only in F₁ hybrid but not in either of the parents(UF₁nP); (â…³) bands detected in either of the parents and F₁ hybrid(UPF₁).7. The amount of differential patterns was changeable for each stage and it was relatively higher at the flowering stage than those at other stages, which implies that the differential gene expression at this stage plays a crucial role in yield forming and heterosis performance of hybrid.. The amount of UF₁nP and UPF₁ patterns in highly heterotic hybrid was obviously higher than that in non-heterotic hybrid at the flowering stage. This indicated that higher percentage of these two patterns at this stage may be positively related to the observed heterosis at the later stage.8. The correlation between differential expression patterns and heterosis was estimated in a cotton diallel cross involving 24 hybrids and ten parents at full opening flower stage. The results showed that hybrid-specific expression pattern had a significant positive correlation with heterosis and silenced expression pattern was significantly negative correlation with heterosis at full opening flower stage.