Synthesis,Identification And Transcriptome Analysis Of Distant Hybrids In Gossypium

G.hirsutum and G.arboreum are important cultivated cotton species.G.sturtianum and G.thurberi are wild cotton species with many excellent characteristics.In this study,AD distant hybrid of G.arboreum(AA)and G.thurberi(DD),and allohexaploid hybrid(AADDCC)of G.hirsutum(AADD)and G.sturtianum(CC)were obtained by interspecific distant hybridization and chromosome doubling by colchicine.The hybrids(AD)was identified by morphological traits and SSR molecular markers.The hybrids(AADDCC)were identified by morphological traits,chloroplast number of stomatal guard cells,diameter of pollen grains,flow cytometry and meiotic observation of pollen mother cells,comparing with its parents and triploid hybrid.Finally young leaves of allohexaploid,triploid hybrid and its parents were used for RNA-seq to compare the expression differences between triploid and hexaploid,further exploring the changes of gene expression and the tendency of genome evolution following hybridization and polyploidization in new synthesized hexaploid.The main results are as follows:1.The hybrids(AD)between G.arboreum and G.thurberi showed mid-parents morphology characteristics,but its growth was blocked with short height and it can not show buds and flower.SSR identification showed that the genetic ratios of AD hybrid are 39.47%,42.11%,18.42%of its parents and new genetic recombinations respectively.The result proves that the obtained plants were true hybrids of G.arboreum and G.thurberi,and the hybrids(AD)got genetic variations beyond two parents.2.In this experiment,we obtained fertile allohexaploid hybrid(AADDCC)between G.hirsutum and G.sturtianum.23 plants of M1 were preliminary identified by flow cytometry and other ploidy tests.It showed that there were significant differences in the performance of leaf index,leaf area and chloroplast number of stomatal guard cells between triploid and hexaploid.Pollen grain diameter of its female parents,male parents,triploid and hexaploid were 100-120um,90-110m,20-100um,and 100-140um respectively.Flow cytometry tests showed that the value of its female parents,male parents,triploid and hexaploid were 140.44,98.81,122.89,and 239.88 respectively.Notably,there were significant difference among M1 populations in flow cytometry and pollen grain diameter.It proves that there were true allohexaploid plants and some aneuploid plants among M1 populations.The plants’ pollen grain diameter returned to normal with certain fertility,when DNA contents reached 79.56%of the normal allohexaploid among M1 populations.Finally,in the obtained two true allohexaploid plants,the meiosis behavior of pollen mother cells has become normal with the proportion of normal tetrads 94.5%,but the fertility of two plants was significantly different with 53 seed sets and 14 seed sets.In addition,in the allohexaploid hybrids,the traits of seed gland retarding development can be expressed normally.3.This study obtained 49746 Unigenes of G.sturtianum by De novo RNA-seq,annotated 40017 Unigenes,and detected 5401 SSR markers from the sequencing results.By analyzing the results of transcriptome sequencing,we found that the transcriptional level of the allohexaploid hybrid genome is not stable,and chromosome doubling can make the transcript expression level of the distant hybrids recover toward both parental original level.In addition,the expression levels of genes related to photosynthesis,plant hormone signal transduction,lipid metabolism,stress tolerance,and resistance to pests and diseases will change significantlly following with chromosome doubling of the distant hybrids between G.hirsutum and G.sturtianm.