| Grape(Vitis vinifera L.)is one of the most widely grown fruit crops.Grape berries are commercially grown for fresh fruits,juices and raisins,but are mainly used to ferment into wine.Berry development is a complex process that involves profound physiological and metabolic changes.At the fruit ripening stage,further metabolic changes make the fruit edible and attractive,which promotes seed dispersal,including changes in peel color,cell swelling and influx of water,softening of berries,and accumulation of sugar in the pulp.The loss of organic acids and the synthesis of tannic acid and volatile aromas,but the subsequent cracking will seriously affect the yield and quality of grapes.In order to further understand the causes of grape berry-cracking,anatomical observations of different degrees of fruit cracking were conducted,and the genes related to grape berry-cracking were excavated through genome-wide association analysis,and the mechanism of spraying calcium fertilizer to reduce the occurrence of berry-cracking was analyzed through transcriptome and metabolome,in order to provide a reference for better understanding of grape berry-cracking mechanism.1.During fruit ripening,fruit cracking occurs in most horticultural crops,which seriously affect their economic value.The cracking of grape berries,which are one of the most important fruits in China,can also cause serious economic losses.The first step in studying the mechanism underlying berry cracking is to screen for crack-resistant grape varieties.In the present study,the ripening berries were soaked in distilled water,and the berry-cracking performance of 291 grape varieties,was evaluated using two indices,the berry-cracking rate and the berry-cracking index.The types of berry cracking in the field were also investigated.Initially,104 extremely crack-resistant varieties and 44 varieties that were extremely susceptible to cracking were selected.It was found that the berry-cracking rate and the berry-cracking index of Vitis vinifera L.were higher than those of V.vinifera ×V.labrusca.There were four main types of berry cracking types:longitudinal cracking,ring cracking,longitudinal cracking+ring cracking and cuticle cracking.Among these,ring cracking,longitudinal cracking+ring cracking and cuticular cracking occurred only in V.vinifera L.,and longitudinal cracking occurred mainly in V.vinifera L.The occurrence and type of berry cracking in grapes is related to the grape variety.The varieties with different berry-cracking properties can be further studied to elucidate the mechanism underlying grape berry cracking.2.In order to study the relationship between berry-cracking and phenotypic characteristics and anatomical structure of grape germplasm resources,291 and 53 germplasm resources were selected for phenotypic characteristics and anatomical observation,respectively.Berry cracking was measured using the distilled water immersion method and the severity of cracking was evaluated by two indices,berry cracking rate and berry cracking index.The results showed that with a thin pericarp and crisp flesh texture were prone to berry cracking.The berry cracking rate and berry cracking index were higher in seedless varietie than in varieties with seeds.Correlation analysis showed that the berry cracking rate and berry cracking index of grapes were positively correlated with berry water increment,bunch length,bunch width,bunch size,bunch weight,and berry shape index,while berry cracking indices were negatively correlated with seed number.The severity of berry cracking was found to be closely related to berry types,berry shape index and seed number.3.Grape berry cracking refers to the phenomenon of peel cracking that occurs during berry growth and development.It is related to be berry structure and the characteristics of the berry tissue.To study the relationship between berry-cracking and berry anatomical structure,53 grape germplasm resources were selected.The ripening berry was soaked and cracked in vitro,after which the berry-cracking rate was measured and paraffin sections were prepared to analyze the structural parameters of different berry tissues.Berry cracking was found to be influenced by the shape of cuticle,the arrangement of epidermal cells and,subepidermal cells,and the thickness of cell layers.With regard to cuticle shape,grapes with local suberization had the highest cracking rate(58.33%),and those with a flat cuticle shape had the lowest rate(30.67%).Grape berries with a compact epidermal cell arrangement had the highest berry cracking rate(57.00%).As for subepidermal cell arrangement,the highest berry-cracking rates were observed in varieties with a looser arrangement(83.33%);and the lowest rates were found in varieties with a more compact subepidermal cell arrangement(32.33%).Correlation analysis between the grape berry-cracking rate and cell layer thickness showed that berry-cracking rate was negatively correlated with the thickness of the cuticle,epidermis,and subepidermis.Moreover,the cell shape and size parameters in the different cell layers may also affect the occurrence of berry cracking.Using paraffin sections to analyze cell structure characteristics,we can predict the cracking of grape berries,which would then provide theoretical support for further screening of germplasm resources to identify cracking-resistant berries.4.Grape berries often crack preferentially in the near and far berry stalk during soaking in the field and in vitro soaking,respectively,which may be related to the vascular bundle moisture absorption and cell anatomy.In order to study the relationship between vascular bundle water uptake and cell anatomical structure and different cracking parts of berries,49 germplasm resources were selected.Among them,18 were prone to cracking near the proximal end,31 were resistant to cracking near the proximal end in the field;24 of them were prone to cracking at the distal of the berry,and 25 were resistant to cracking at the distal of the berry in vitro soaking.In vitro soaking experiment was carried out on the ripe berries to measure the difference of berry-cracking degrees among different varieties.In vitro staining was used to trace the water absorption of vascular bundles,and paraffin sections were prepared to observe and analyze the structural parameters of different tissues.The results showed that the cracking rate and water uptake of the berries prone to cracking were significantly higher than that of the berry resistant to cracking.Compared with the berry resistant to cracking,the berries are prone to cracking is characterized by thinner cuticle,epidermis and sub-epidermis.In addition,other shape parameters and size parameters of cells in each cell layer may also cause cracking.After immersing the pedicel in the dye and dyeing,it was found that all the materials were limited to near the berry brush after dyeing.After immersing the whole fruit in dyeing,it was found that the dye accumulated in the distal stalk of the berries which were prone to cracking,and even cracked.However,the accumulation of dyes on the surface of those whose were resistant to cracking is less,only sporadic distribution.Repression of water transport and differences in anatomical structure of cells are responsible for the cracking of the near the proximal end in the field;preferential water absorption and differences in the anatomical structure of the cell are the causes of cracking of the near the proximal end in the soaked condition in vitro.Through the vascular bundle water transport tracing and analysis of the differences in cell structure characteristics of different varieties,preliminary explanations can be made for the reasons of prone to cracking near the proximal end in the field and prone to cracking at the distal of the berry in vitro soaking,providing theoretical support for further research on the mechanism of berry-cracking.5.Fruit cracking is a common phenomenon during the growth and development of horticultural crops that seriously affects fruit yield and quality.To better understand the genetic basis of grape berry-cracking,we conducted a genome-wide association study of ripe grape berries.In vitro fuchsin staining was used to trace the water uptake of the vascular bundles in the berries,and the berries were treated with aquaporin promoters and inhibitors to verify if aquaporin is involved in the occurrence of grape berry-cracking.Twelve single nucleotide polymorphism(SNP)loci that were significantly associated with grape berry-cracking were detected,and these loci were distributed on three chromosomes,namely,6,13,and 18.These significantly-correlated SNP loci were associated with 63 unique candidate berry-cracking genes.We further screened six candidate genes mainly related to fruit water metabolism,plant hormones,and cell wall metabolism.The gene expression patterns indicated we found that the six candidate genes in the susceptible berry-cracking variety were more highly expressed than the resistant berry-cracking variety near the proximal end and equatorial part of the berry.In addition,in vitro tracing of the fuchsin solution indicated that water accumulated near the proximal end of the berry.This preliminarily verified that aquaporin genes may be involved in the occurrence of grape berry-cracking.The high expression of the candidate berry-cracking genes screened by genome-wide association analysis near the proximal end of the berry together with water accumulation may jointly promote the occurrence of berry-cracking.6.Spraying calcium fertilizers has been adopted to prevent berry cracking.However,studies on the mechanism of reducing berry cracking are limited.To understand the molecular mechanism of spraying calcium fertilizers to reduce the occurrence of berry cracking,‘90-1'was used as the study material,and transcriptome sequencing and metabolome were conducted to identify the differentially expressed genes and differential metabolites.Different metabolites enriched secondary metabolic pathways during color change and maturity stages,including the flavonoid biosynthesis pathways;the mature metabolites enriched the functional category of galactose metabolism and starch and sucrose metabolism pathways.Enrichment analysis using KEGG pathways showed that most of the differentially expressed genes enriched the functional category of flavonoid biosynthesis.Secondary metabolites have higher antioxidant capacity,and thus spraying calcium fertilizers may improve the antioxidant capacity of berries by regulating genes that are related to the flavonoid metabolism pathway,thus reducing the occurrence of grape berry cracking. |
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