Character Identification Of Allohexaploid Progenies Of Gossypium And Transcriptome Analysis Of Drought Stress

Cotton distant hybridization is an important way for germplasm innovation and new variety breeding.Wild species have many excellent characters that cultivated cotton does not have.Introducing the excellent characters of wild cotton into upland cotton and cultivating excellent varieties with stress resistance can effectively broaden the germplasm resources of cotton.In this study,the distant hybrids F1 of G.hirsutum and G.sturtianum were identified by morphology,cytogenetics and SSR molecular markers;then the F1 hybrid was induced by polyploid mutation to obtain a new heterohexaploid germplasm M1(AADDCC),and the seeds of M1 generation were planted.After the normal growth and development of cotton,the seeds of M2 generation were harvested after budding,flowering and bolling,and then the characters of offspring were identified and analyzed.In this study,the morphological and stomatal characters,pollen mother cell meiosis and pollen grains morphology of F1 and heterohexaploid M1 and M2 were compared and identified respectively.The authenticity of heterohexaploid germplasm was verified by comparison and identification,and the restoration of hybrid F1 fertility was further discussed.Finally,the hexaploid cotton M1 was used as the material which were treated under drought stress,and the normal treatment was used as control.After a period of time,the young leaves were selected as the material for transcriptome analysis,in order to compare and analyze the differentially expressed genes before and after drought stress and screen the drought resistance related genes.The main results were as follows:1.Morphological,cytogenetics and SSR molecular markers were used to identify the distant hybrids F1 of G.hirsutum and G.sturtianum synthesized in the early stage of laboratory.Morphological analysis and comparison of parents and hybrids were carried out from three aspects of plant,leaf and flower.The results have shown that the hybrid plants were higher than their parents,and the leaf was larger than their parents,showing significant heterosis;the leaf color was similar to the male parent,which was emerald green;the flower base spot of hybrid was similar to the male parent,which was dark red;the pollen mother cell meiosis of hybrid was observed in cytogenetics,and the results were as follows.The results have shown that there were many abnormal behaviors during meiosis in F1 hybrids.Among them,24.00% was diploid,10.40% was triad,40.60% was tetrad and 25.00% was polyd.In the tetrad,normal tetrad accounted for 72.91%,while the abnormal one accounted for 27.09%,so that a lot of abnormal pollen grains were formed,accounting for 24.00%.Normal pollen grains accounted for 76.00%,which was the main reason of hybrid sterility.Finally,the SSR molecular marker identification has shown that not only complementary bands of G.hirsutum and G.sturtianum appeared in F1 hybrids,but also specific bands were amplified.The proportion of genetic components was 7.69% for male parents,34.62%for female parents,23.07% for complementary bands of parents,and 34.62% for specific new bands.It not only indicated that gene recombination occurred in the process of hybridization,but also confirmed that the hybrid was a true hybrid of G.hirsutum and G.sturtianum at the molecular level,and provided valuable materials for cotton genetic breeding and germplasm innovation.2.Through the identification of morphological and cytological characters of hybrid F1 and heterohexaploid M1 and M2 between G.hirsutum and G.sturtianum,it was found that there were differences in leaf and hybrid of heterohexaploid.In addition,there were significant differences in cytology,leaf area,leaf shape index and chlorophyll content.Compared with F1 hybrid,the stomatal density of heterohexaploid M1 and M2 decreased,the length of guard cell and the number of chloroplast increased,which confirmed the authenticity of heterohexaploid.In morphology,there was no significant difference in leaf correlation between M1 and M2,which indicated that the physiological characters of hexaploid were gradually stable.The cytological analysis has shown that the proportion of normal tetrad was 72.91% in meiosis,87.00% in M1 and93.47% in M2.It has proven that meiosis has been normal,and the fertility of hybrid has been recovered and stabilized in M2 generation.3.Transcriptome sequencing was used to analyze the differential gene expression of heterohexaploid leaves before and after drought stress.The deep mining has shown that there were significant differences in gene expression.The DEGs analysis has shown that there were 2302 increased genes and 2681 decreased genes.The Go enrichment analysis has shown that the differentially expressed genes were mainly enriched in three aspects: molecular function,cell composition and biological process,and were mainly annotated into transcription regulation DNA template,regulation of cell macromolecular biosynthesis,redox process,oxidoreductase activity,monooxygenase activity,iron ion binding,auxin response and other pathways.Through the KEGG enrichment analysis,it was found that the functional annotation included plant hormone signal transduction,plant MAPK signal pathway,cysteine and methionine metabolism,starch and sucrose metabolism,which indicated that the normal growth,physiological regulation and drought resistance of cotton were related to these metabolic pathways.In order to further study the mechanism of drought resistance in cotton,the functional annotation and enrichment analysis of differential genes were carried out,and candidate genes related to drought resistance were screened.The results have shown that these genes were related to energy metabolism,nucleic acid metabolism,lipid metabolism,carbohydrate metabolism,signal transduction,hormone synthesis and photosynthesis and other metabolic processes.The preliminary results have shown that these pathways had a great correlation with cotton drought resistance.