Gene Mining Of Seedless Grape Based On Transcriptome Sequencing

Grape(Vitis)is widely cultivated because of its outstanding economic and social value.Nowadays,seedless grape has gradually become the development trend of grape production and consumption in the world,and seedless grape is also the main goal of grape breeding.At present,the progress of seedless grape breeding is slow,and the molecular mechanism is not clear enough,which causes some difficulties for seedless grape breeding.The research of molecular biology provides a powerful tool for the study of seedless mechanism of grape.The exploring of seedless grape related genes is of great significance for accelerating seedless grape breeding.In this experiment,we used grape cultivar ‘Victoria’ and its seedless budding plants transformation as materials to observe the differences of flower organs and fruit characters,determine the pollen germination rate,and observe the pollen cytology and morphology.Through the screening and analysis of transcriptome data,the genes related to anuclear are explored.The main results are as follows:1.Through the observation of seedless budding of ‘Victoria’ and the phenotype of flower organs and mature fruit of ‘Victoria’,as well as the investigation of fruit and leaf characters of two plants,it is found that the anther of ‘Victoria’ seedless budding plants is withered,the flower cap of the flower ear of ‘Victoria’ seedless budding plants at full flowering stage do not fall off,and the mature fruit is seedless,which is caused by pollen abortion The seedless fruit also leads to the change of fruit volume and weight.2.The pollen germination rate of ‘Victoria’ and its seedless budding is determined.It is found that the pollen of ‘Victoria’ could germinate normally after 4 hours in the same environment,and the germination rate is 86.6%,while the pollen germination rate of ‘Victoria’seedless budding is 0.It can be concluded that the pollen germination rate of ‘Victoria’ seedless budding plants is far lower than that of ‘Victoria’.It can be confirmed that the pollen of ‘Victoria’seedless budding plants has abnormal development,which leads to abnormal pollen germination.3.The results showed that the tapetum of seedless pollen is degraded in advance,which could not provide enough nutrients for microspore,and then affected the development of pollen.Scanning electron microscopy(SEM)was used to observe the mature pollen of ‘Victoria’ and‘Victoria’ seedless budding plants.It is found that the germinating groove of mature pollen of‘Victoria’ seedless budding plants is narrower and shallower than that of ‘Victoria’,which also affected the fertility of pollen.4.After sorting out and analyzing the transcriptome data,729961224 raw reads are generated by the transcriptome sequencing in this study,and 713847698 clean reads are obtained after filtering out the low-quality fragments.The comparison efficiency of each sample read with the reference genome is between 89.22% and 94.05%,and the number of unique comparison positions is more than 87.23%.There are 1455 differential genes in VBFL vs VFL,including 725 up-regulated genes and 730 down regulated genes.There are 6416 differential genes in VBFR vs VFR,including 2866 up-regulated genes and 3500 down regulated genes.The main KEGG metabolic pathways of these DEGs are flavonoid biosynthesis,circadian rhythm plant,carbon fixation of photosynthetic organisms,glycerophospholipids metabolism and so on.Through analysis and screening,flavonoid biosynthesis pathway and phenylpropane biosynthesis pathway are identified as the key pathways related to pollen development.5.A total of 24 candidate genes are screened in flavonoid biosynthesis pathway and phenylpropane biosynthesis pathway,and 12 of them were verified by q RT-PCR(VIT＿01s0010g03720,VIT＿03s0180g00260,VIT＿15s0048g02450,VIT＿16s0039g01360,VIT＿16s0200g00840,VIT＿16s0039g01240,VIT＿18s0001g01140,VIT＿16s0100g00840,VIT＿16s0039g01280,VIT＿16s0100g00920,VIT＿16s0100g01040 and VIT＿09s0002g04460).The results shows that these genes were consistent with the trend of transcriptome data.6.We clone 4 genes(VIT＿16s0100g00920,VIT＿03s0180g00260,VIT＿15s0048g02450and VIT＿01s0010g03720)and analyze them.It turns out that there is no difference in CDS sequence between VIT＿15s0048g02450(COMT)and VIT＿01s0010g03720(4CL)gene.Among the remaining two genes,There are 54 base differences in VIT＿03s0180g00260(CAD),all of which occurred at homozygous loci.Heterozygosity occurred in ’Victoria’.VIT＿18s0072g00920(STS)has the least base difference in sequence,only 7 base changes,which lead to the changes of three amino acids.