Pollen Fertility Analysis Of Wild Banana(Musa Itinerans)in Fujian

Banana(Musa spp.)is a perennial herb belonging to the genus Musa in the family Musaceae.The world’s main cultivated bananas are selected from the Cavendish(AAA)through nutritional propagation.Due to the close kinship between varietiesand,the lack of good resilient varieties has led to the problems of variety degradation and weak resistance.Cultivated bananas originated from intraspecific or interspecific crosses between diploid wild species(Musa acuminata)and(Musa balbisiana).Bananas have rich species resources,many of which exist in the wild and have developed many excellent characteristics during long-term natural selection,such as cold resistance,pest and disease resistance,etc.Therefore,the best gene pool for genetic improvement of bananas is the wild banana resources.The cultivated banana varieties are polyploid with low pollen fertility and most of the cultivated varieties are male sterile,which makes it difficult to study the crossbreeding of banana,so the first problem of crossbreeding is to screen the parents with excellent resistance and pollen fertility.In order to fully exploit the germplasm resources of Fujian wild banana(Musa itinerans),this paper firstly examined the vigor changes of Fujian wild banana pollen by TTC staining and in vitro germination,and identified,expressed and functionally verified the GDSL lipase gene family related to fertility,and further did transcriptome sequencing analysis with germinable pollen.The main results are as follows:1 Pollen viability detection and optimization of conditions for in vitro germinationThe study examined the pollen viability of Fujian wild banana at different periods by TTC and in vitro germination.The results showed that the pollen staining rate was more stable in sunny weather and gradually decreased during the ripening process until the anthers were fully mature and open,and then increased again.The pollen staining rate at different times of the day also showed a trend of increasing and then decreasing,reaching the highest staining rate at 9:00 am.The result of optimization of the isolated germination medium showed that pollen germination of wild bananain in Fujian required higher concentrations of boric acid compared to diploid bananas from other regions.The wild banana pollen under low-temperature stress showed germination disorders.The male buds were incubated at 24°C,28°C and 32°C for two days at constant temperature,then the pollen germination rate after incubation at 28°C was significantly higher than that at 24°C and 32°C.2 MaGDSL lipase gene family identification and expression analysisTo reveal the sequence characteristics of banana GDSL lipase gene family and its potential functions in the growth and development of banana.A series of bioinformatics approaches were used for genomewide identification of the banana GDSL lipase gene family,and the expression patterns of MaGDSLs in high(45°C)/low(4°C)-treated leaves,roots infested with the wilt fungus Foc TR4,and fruits at different ripening stages of natural/ethylene ripening were analyzed based on the transcriptome database,while real-time fluorescence quantitative PCR(q RT-PCR)to analyze the expression of some MaGDSL members in pollen.The banana A genome has 76 MaGDSL members distributed on11 chromosomes,which can be divided into 9 subfamilies with coding regions ranging from 1014 to 2193 bp in length,among which 5 members contain different numbers of transcripts;most of the GDSL members contain 5 exons and 4 introns.Three tandem duplicate pairsclusters of MaGDSLs on chromosomes 4 and 8.six tandem duplicate pairs on chromosomes 1,6,7,8,9 and 10,and 22 segmental duplicate pairs on all chromosomes except chromosome 11.The expressions of MaGDSLs in banana leaves and roots were highly variable,and individual members were regulated by high and low temperature stresses as well as by the wilt fungus.Especifically,MaGDSL1-1 was highly expressed in pollen.q PCR results analyzed that low temperature-stressed pollen grains underwent rapid permeability rearrangement and possible reconstruction of cell wall in order to counteract low-temperature stress,and their germination ability could be restored.The results suggest that MaGDSLs may play an important role in the growth and development of banana,while some members specifically respond to biotic and abiotic stresses and also participate in the regulation of pollen fertility.3 Phenotypic analysis of wild banana MaGDSL1-1 genetically transformed Arabidopsis thaliana in FujianTo understand the functional mechanism of GDSL lipase in regulating pollen fertility,MaGDSL1-1 was transduced into Arabidopsis.q PCR results were analyzed that MaGDSL1-1 was highly expressed in whole Arabidopsis plants.And compared with the wild type,the bud-drawing period of transgenic Arabidopsis was improved by 2 days.4 Transcriptome analysis of wild banana germination pollen in FujianThe transcriptome sequencing analysis was performed on wild plantain pollen that was subjected to loss of germination by low temperature stress and revitalized by constant temperature culture.GO enrichment showed that the differentially expressed genes were mainly enriched in carbohydrate kinase activity,m RNA modification,sugar phosphorylation,and other pathways.GO functional annotation screened 23 DEGs related to pollen growth and development,including pollen wall formation,pollen tube growth,pollen development,pollen recognition and acceptance,and pollen protein synthesis.Some members of MaGDSL lipase were differentially expressed in the pollen transcriptome,especially MaGDSL4-1,which was significantly up-regulated in normal germinating pollen compared with the control.In particular,MaGDSL4-1was significantly up-regulated in normal germinating pollen compared with the control,with an increase of about 22-fold,indicating that MaGDSL lipase has an important role in pollen development.