Distant Hybridization Between Cultivated Cotton And G. Gossypioides,and The Allopolyploid Synthesis,identification And Transcriptome Analysis

G.hirsutum and G.arboreum are important cultivated cotton species in the world.However,due to long-term cultivation,their genetic diversity is limited.G.gossypioides is one of the earliest cotton species with D genome.It has many excellent characters that cultivated cotton lacks,such as cold resistance,drought resistance,high resistance to Fusarium wilt,rust resistance,no nectaries and so on.The introduction of excellent genes from G.gossypioides into cultivated cotton by distant hybridization not only expands the genetic diversity of cultivated cotton,but also makes cultivated cotton better meet the growing demand for cotton breeding.In this study,G.arboreum(AA)and G.hirsutum(AADD)were used as female parents respectively,and G.gossypioides(DD)as male parents.At the same time,a new artificial allohexaploid plant M0 was created by mutagenesis doubling of G.hirsutum × G.gossypioides true hybrids.Morphological identification and SSR molecular identification of the hybrid(AD)of G.arboreum and G.gossypioides,and the G.hirsutum × G.gossypioides hybrid(ADD)and allohexaploid M0(AADDDD)plants were Identified by morphological,cellular,physiological and biochemical indexes.Transcriptome analysis was carried out on G.hirsutum × G.gossypioides hybrid and allohexaploid M0.The expression differences of transcriptome between hybrid F1 and hexaploid M0 and their parents were compared and analyzed.To explore the change and direction of gene expression after genome doubling.The main results are as follows:1.Gossypium arboreum as a female parent had distant hybrid with Gossypium gossypioides as male parent.Artificial pollination was used to synthesize hybrid F1,and the authenticity of the F1 hybrids was verified,aiming to further doubling it into a new allotetraploid germplasm.Repeated pollination and gibberellin boll keeping measures were used to improve the boll setting rate of hybrid,and the F1 was identified by morphology and SSR molecular markers.The results showed that the F1 hybrids had typical post-zygotic reproductive isolation,and the hybrids died in the process of plant culture.Most leaf types of the F1 hybrid seedlings were amid parents and tended to resemble male parents as a whole.The results of SSR molecular markers demonstrated that the hybrid F1 not only amplified complementary bands of parents,but also new bands that parents did not have appeared.Statistical analysis revealed that the total genetic components of the hybrid F1 accounted for 45.91% of the female parents,40.98% of the male parents and 13.11% of new combination bands.The study not only proves the authenticity of hybrids at the molecular level,but also shows that AD genome interaction and genetic recombination occur along with the hybridization process.2.Using G.hirsutum as the female parent and G.gossypioides as the male parent,F1 hybrid was produced,and allohexaploid plants were obtained following with chromosome doubling with colchicine.The morphology(leaf phenotype),cytology(stomatal density,stomatal length,chloroplasts number in guard cell pairs,pollen grain diameter),physiological and biochemical indexes(SOD,POD,CAT,soluble protein and chlorophy II fluorescence parameters)of the hexaploid were identified.Through flow cytometry,three hexaploid plants were selected.The statistical results of hexaploid,triploid and its parents showed that there was no significant difference between the leaf shape index of hexaploid and G.hirsutum.It showed that the leaf shape of hexaploid was in favor of G.hirsutum as a whole,while the triploid distant hybrid was in favor of G.gossypioides.The leaf area and stomatal length of hexaploid increased,and the number of chloroplasts in guard cells also increased significantly.The phenotypic character of hexaploid flower is between its parents,and the diameter of pollen grain is larger than that of triploid.The normal pollen grain ratio of hexaploid was 73.25% and that of triploid was 51.13%,indicating that the fertility of hexaploid is recovering.The content of SOD,POD,CAT and soluble protein in hexaploid leaves was significantly higher than that in other generations.There was significant difference of maximum fluorescence yield Fm between hexaploid and that of other generations.It also showed significant difference in initial fluorescence F0,leaf variable fluorescence Fv,original light energy conversion efficiency(Fv/Fm),PSII potential photochemical efficiency(Fv/F0)and open PSII capture excitation energy efficiency(Fm/F0)between hexaploid and other generations.The combined result proves that the new germplasm of hexaploid between G.hirsutum and G.gossypioides were successfully synthesized by hybridization and chromosome doubling,and it was tested that allohexaploid has excellent performance in stress resistance and photosynthesis.3.Transcriptome sequencing analysis was carried out on G.hirsutum × G.gossypioides triploid hybrid F1,hexaploid M0 and their parents.The results are as follows: the percentage of Clean Reads and Clean Data is more than 92%,and the percentage of Q30 bases is between 93.42% and 93.92%,indicating that the sequencing result is accurate and the data is reliable.The intra-group correlation coefficient of each material sample group is greater than 0.83,indicating that the sample has good repeatability.Among the expressed genes,Wayne diagram showed that among all the expressed genes of parents and F1 and M0,there were 1742 genes specifically expressed in F1 and 1341 genes specifically expressed in M0.The whole gene expression of F1 was unstable after hybridization.although the expression of M0 gene was unstable after doubling,it showed a tendency to be close to the parents.Comparing the sum of differences between F1,M0 and their parents,the results showed that the genomic expression of hybrid F1 was more inclined to the male parent than that of the female parent,while the hexaploid M0 was more inclined to the female parent.The GO entries and KEGG pathways of differentially expressed genes between F1 and M0 were analyzed,and most of the significantly enriched pathways were about "photosynthesis" and "resistance".The differentially expressed genes between F1,M0 and their parents were mainly non-additive expression,while the non-additive genes in F1 were mainly down-regulated by super-parents,while those in M0 were mainly up-regulated by super-parents.