Differentially Expressed Genes And Variations Of DNA Methylation Level In Cotton Hybrids Developed By CRI-12 And Its Pedigree Derived Lines

As a major source of fibers, important oil and albumen plants, cotton is considered to be a high value crop and plays an important role in the global economy. During the past decades, the hybrid cotton, especially pest-resistant hybrid cotton was greatly planted by the Yellow River Valley and the Yangtze River Valley, The extension and application of cotton hybrids have played a decisive role in cotton productionCRI-12, a Chinese elite Upland cotton variety with high yield, elite fiber quality and disease resistance, is characterized by its high heritability, combining ability and genetic stability. As the germ plasm of cotton breeding, it displays enormous appliance values of Foundation parental. At present, the study of hybrid cotton and CRI-12 is mostly focus on breeding, physiology and biochemistry. It is urgent to go deeply into the genetic mechanism study of CRI-12 as Foundation parent from the molecule level, with molecular biological methods increasingly improvement, it will offer theoretical evidence to study the important role of CRI-12.In this research, CRI-12 and its pedigree-derived lines were used to develop high heterosis cotton hybrids such as CRI-28, CRI-29, XZM2 and Jimian18, Leaves and root at seedling stage, leaves at budding stages and flowering stages were picked from these hybrids and their corresponding parents sampled for cDNA-AFLP and MSAP analysis, the gene differential expression patterns and DNA methylation variation level and pattern between hybrids cotton and their parents. The results were listed as the following:1,Temporal Spatial expression of heterosisThe roots and leaves at seedling stage of four hybrids and their corresponding parents were sampled for vegetative growth heterosis analysis. It was found various heterosis at roots and leaves, It suggested vegetative growth heterosis has tissue specification.Heterosis analysis was undergone through the dry weight measure of four hybrid and their corresponding parents at three-leaf stage, budding and flowering stages, It was found Jimian 18,CRI-29 and XZM 2 exhibited the positive over-mid-parent heterosis, and CRI-28 exhibited over-parent heterosis at three-leaf stage; CRI-29 and XZM 2 exhibited the positive over-mid-parent heterosis, Jimian 18 and CRI-28 exhibited over-parent heterosis at budding stage; XZM 2 exhibited the positive over-mid-parent heterosis, Jimian 18,CRI-28 and CRI-29 exhibited over-parent heterosis at flower stage. It suggested vegetative growth heterosis has timeliness.Yield of four hybrid and their corresponding parents were detected for yield heterosis analysis. It was found Jimian 18, CRI-28 and CRI-29 exhibited over-parent heterosis, XZM 2 exhibited the positive over-mid-parent heterosis. It suggested hybrid cotton by CRI-12 lines could exhibit various heterosis.Fiber quality traits have small heterosis of four hybrid cotton, but it is very lower relatively, only XZM 2 exhibited over-parent heterosis in fibre strength. It suggested hybrid cotton heterosis aren't distinctness in fiber quality traits.Integrated above results we presumed that heterosis had different temporal and spatial exhibition in various organ and various growth periods with various characterization, It mainly exhibited in vegetative growth and yield aspect.2,Gene differential expression and heterosisThe roots and leaves at seedling stage of four hybrids and their corresponding parents were sampled for cDNA-AFLP analysis. The gene differential expression patterns were detected between the hybrid and its parents and were described as follows:I. Up expression in hybrid showed the strong bands expressed only in hybrid but not in both parents; II. Dominant expression in single parent showed the strong bands expressed in one of the parents and hybrid but not in another parent, including the expression pattern in female parent and hybrid without in male parent and the expression pattern in male parent and hybrid without in female parent; III. Down expression in single parent showed the strong bands in one of the parents, including the expression pattern in male parent without in hybrid and female parent and the expression pattern in female parent without in hybrid and male parent; IV. Down expression in hybrids showed the strong bands in both parents but not in F1. Differential expression bands in leaves and roots accounted for 29.20%-46.09% and 15.65%-22.49% in total numbers of detected bands, respectively. The differential expression bands in leaves were more than those in roots, that explained some useful genes associated with heterosis maybe begin to express in seedling leaves.The gene differential expression patterns were detected between four hybrid and their parents at three-leaf stage, budding and flowering stages, The result as follows:The percentages of domain expression in single-parent were highest than other patterns, down expression genes in single-parent were higher than up expression in hybrids, down expression in hybrids were lowest of four patterns from three growth stage accumulative total number. The gene differential expression ratio of hybrid and parents was various in three growing stage, It suggested gene differential expression had timeliness.Further analysis of the gene differential expression ratio and vegetative growth heterosis and yield correlation revealed that the type of bands expressed only in F1 contributed to heterosis of vegetative growth at seedling and budding stage and it played an important role in yield heterosis occurrence at flowering stage; the type of bands expressed only in Female parent contributed to heterosis of vegetative growth at budding stage, but it possible decreased hybrid yield at flowering stage.Differentially expressed genes patterns in the four hybrids were detected, It revealed that many possible modes of gene action were involved in hybrids, with additivity,dominance,over-dominance,under-dominance and low-parent dominance. All possible modes of gene action coexisted supported the hypothesis of multiple molecular mechanisms contributed to heterosis. The up-regulated expression genes in hybrid were showed positive correlation with heterosis of vegetative growth and yield heterosis, over-dominance may play a vital role. The down-regulated expression pattern in female parent was harmful for yield heterosis and high yield in hybrids. It suggested that the inhibition of parental genes, especially female parental genes, was not beneficial to the occurrence of heterosis and formation of hybrid yield.Good hybrid cotton was breeding from good parents. CRI-12 was the high value parent of Jimian 18, CRI-29 and CRI-28 at the seedling and budding stage, high-yield of hybrid cotton based on dry weight accumulated heterosis of vegetative growth. Correlation analysis of differentially expression gene and yield heterosis revealed the type of bands expressed only in Female parent possible decreased yield of hybrid at flowering stage, which also provided confident evidence for CIR-12 played a predominant role in the expression of genes responsible for heterosis in CRI-28,CRI-29 and Jimian18 at molecular level.3,Variation of DNA methylation level and pattern in Hybrid CottonMS AP (methylation-sensitive Amplified fragment length polymorphism) was used in this study to detect the DNA methylation patterns in the 5'-CCGG sites of two cotton hybrid by CRI-12 and their parents.This study was used to understand the developmental stability and inheritance of cytosine methylation. It was found MSAP ratios in the two cotton hybrids were 12.41%-18.34% at seedling stage,18.35%-20.05% at budding, and 15.94%-17.07% at flowering stage, respectively. cytosine methylation profiles were variable during plant growth and development, DNA methylation experienced from increases to decrease progress throughout cotton development, which can partly explain differential gene expression in different growing stages.Compared the level of various DNA methylation,the Full methylation of internal cytosine (6.90%-11.47%) was highest than hemi-methylation of external cytosine and Full methylation of the internal Cs or external Cs. It suggested full methylation of internal cytosine was the dominant in two cotton hybrids. Full methylation of the internal Cs or external Cs at flowering stage distinct less than seedling stage and budding, that showed have demethylation occured at flowering stage, DNA methylation level of flowering stage decreased 2.77% than it at budding stage.Meanwhile, The MSAP profiles enable monitoring of inheritance or variation of parental methylation patterns in hybrid progenies. It was found that a great majority (from 96.61% to 98.86%, depending on crosses) of the methylation profiles in cotton inbred lines transmitted to the inter-strain hybrids; however, the otrer sites from 1.14% to 3.39% of the profiles in the hybrids exhibited variation from the expected parental additivity. Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies were variable among the cross-combinations, and during plant growth and development. In addition, sequencing of differentially methylated fragments and subsequent homology analysis of isolated bands that showed variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements, Such as leucine-rich repeat family protein, PDR-like ABC-transporter, putative oligopeptide transporter, GTP-binding protein, Similar to pathogenesis-related protein, DOMON domain-containing protein, putative adenosine phosphosulfate kinase, putative protein, RNA-directe DNA polymerase et al. The remaining 14 bands showed no homology to the database sequences.Compared DNA methylation level of hybrid and parent, the methylation average level of hybrid (17.08%) was lower than methylation average level of parents (17.29%) Combined heterosis of vegetative growth and yield of hybrid at seedling stage, budding and flowering stage, methylation level near mid-parent value may be contributed to over-parent heterosis occurrence. In general, the correlation between methylation level and heterosis of cotton was lowness, like rice.