Dissection Of Genetic Variation Underlying Grape Fruit Shape Based On Transcriptome And Association Analysis

Grape is one of the most important fruit plants in the world,that is commercially processed into raisins,juice,and wine.China stands seconds with a total area of grapevine cultivation and ranks first in terms of table grape production.The grape fruit characteristics,such as size,shape,and color,determine the commodity and economic value of the fruit,and it is also one of the important target breeding attributes.With the improvement of living standards,consumers are satisfied with the traditional supply of fruits and turn to pursue new,strange,and special high-quality products.Therefore,it is very important to cultivate excellent varieties with beautiful appearance,high nutrition,and high efficiency.The size and shape of different varieties of grapes vary greatly,which is very important for the final selection of fruit with ideal characters.The genetic basis of fruit size and shape variation has been an area of interest to geneticists and breeders for decades.Breeders and growers face continuous challenge:to combine high quality（production）with high quality（appearance,taste and consumer perception,and processing quality of processing industry）.At present,there are few studies on the molecular mechanism of grape seed shape formation.It will be helpful to explore the genetic basis and gene location of fruit shape.Because of the important role of grape berry shape,this project was researched to understand the genetic control of grape berry shape using the grapevine germplasm resources from Dalai.The main research methods and results are as follows.Analyzing the berry shapes of grape germplasms can provide a good basis for understanding the berry shape characteristics and breeding parent selection.In this study,820 grape germplasms were selected（286 through field surveys,534 from ’Chinese Ampelography’ and ’Chinese Grape Varieties’）.The type of different berry shapes and varieties of grape,the distribution of the fruit weight and its correlation with fruit diameter,and the number of different berry shapes were studied.The results showed that the particles of obovoid and narrow lipoid fruits were larger,while the round grape fruits were smaller.The Vitis vinifera L.has broader lipsoid and ovoid shapes,and there are more obovoid varieties of Vitis vinifera × V.labrusca.Table grapes are more elongated than wine,raisin and juice grapes.Black grapes are mainly round and nearly round,but white and red grapes have a distinct berry shape.From the breeding perspective,the berry shape of the grape is mainly round,and then became mainly round,nearly round and broad lipsoid,and more and more fruit forms appear one after another.Round,broad lipsoid and obovoid grape horizontal,longitudinal can fit well with fruit weights.There may be differences in the berry shape of the same grape variety,with broad lipsoid changes being prevalent.To find the gene locus to control the shape of grape fruit,the whole genome association analysis,and the whole phenotype group association analysis were carried out on 360 Grape Germplasm.The genome DNA of Grape Germplasm was sequenced,the average sequencing depth was 8x,the average coverage was 91.99%,4,241,729 SNPs were obtained.Further,172,778 SNPs were obtained according to the standards of completeness greater than 0.95 and the frequency of minor genotype greater than 0.05.GWAS identified 592 significant quantitative trait loci（QTLs）and 3,306 preliminary candidate genes for 7 fruit-related traits and 4 main components.The phenotype group association study（GPWAS）determined 1,231 preliminary candidate genes.By combining GWAS and GPWAS analysis,many promising candidate genes,such as VIT₀2s0025g02690（protein IQ domain 1）,VIT₀2s0025g02350（MADS box transcription factor 31）,VIT₀6s0004g05240（ethylene receptor 2）,VIT₁4s0108g00830（myb related protein 306）,VIT₁4s0083g01110（brassinolide-6-oxidase）,VIT₀9s0012g08270（EIN3 binding F-box protein 1）are preferred for further research.This is the first time that GWAS and GPWAS combined analyses have been performed on grape.Many SNP loci and candidate genes found in this study are consistent with previous QTL studies.These findings reveal the genetic basis of the related traits of grape berries and provide a reference for the molecular breeding of grape.Based on cell observation,transcriptional analysis,and correlation analysis,the physiological and biochemical changes of the fruit in grape were studied during the formation and maturation.In this study,the differences in the number,cell size,and gene expression patterns of round,oval,and long ellipsoid fruit varieties were revealed.The results showed that the shape of fruit was affected by the double effects of cell division and cell elongation.To determine the key molecular events controlling the formation of grape fruit,a transcriptional expression profile of fruit development was created based on the fruit samples extracted from three grape genotypes per week.The main changes of the transcription group involved the cell division stage,the transformation stage of fruit development cell division to cell expansion and cell expansion stage.It revealed three important transcription processes,and the key factors of fruit shape difference represented by the cell wall,microtubule,and hormone.The core regulatory network of transcripts was determined by co-expression analysis,and 277 core regulatory genes were identified by association analysis.The plant-specific IQ67-domain（IQD）protein family members are downstream targets of calcium sensors,known to regulate plant growth and lateral organ polarity,and basal defense response against environmental cues.No systematic study of the IQD gene family has been performed on the grapevine.The public availability of the grapevine genome enables us to perform identification,phylogeny,chromosomal orientation,and gene structure analysis of the IQD genes in the grapevine.We identified 49 VvIQD genes（VvIQDlVvIQD49）and further classified them into eight subgroups based on phylogenetic relationships.The 49 VvIQD genes were assigned to 19 different chromosomal positions.The collinear relationship between grapevine and Arabidopsis IQDs（VvIQD and AtIQD）,and within grapevine VvIQDs,was highly conserved.In addition,most of the duplicated gene pairs showed Ka/Ks ratio less than 1.00,indicating purifying selection within these gene pairs,implying functional discrepancy after duplication.Transcription profiling of VvIQD genes sheds light on their specific role in grapevine tissue and organ development.The qRT-PCR validation of the 49 VvIQD genes in grape berry tissue from cultivars with distinct berry shape during developmental phases suggested candidate genes involved in the shape of grape berries.The subcellular prediction of VvIQD22,VvIQD23,VvIQD38,and VvIQD49 genes validated their localization in the nucleus and plasma membrane.The VvIQD49 protein interaction with VvCaM2 was also verified by bimolecular fluorescence complementation analysis in the plasma membrane.Our findings will be valuable for the functional genomic studies for desirable shape development of grape berries.