The accumulation of anthocyanins and monoterpenes during grape berry color change at both metabolic and transcriptional levels were analyzed, to explore the spatio-temporal synthesis mechanisms of anthocyanins and monoterpenes, and provide a theoretical basis for the regulation of anthocyanin and monoterpene synthesis in table grape.

The grape berries of Ruiduhongyu were used as materials, sampling was started at 5 days before the veraison and continued until 40 days after the initial color change. The contents of total soluble solids and titratable acid in the berry samples were determined by conventional methods; The changes in monoterpene components and content in berries were determined with headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPEME-GC-MS); The content of total flavonoids and total anthocyanins were detected using a spectrophotometer; The expression changes of key genes involved in monoterpenes and anthocyanins synthesis were analyzed by transcriptome sequencing and real-time fluorescence quantitative PCR.

With the advancement of the coloration process, the main components of 25 free and bound monoterpenes in the Ruiduhongyu berries fluctuated. Most of the free monoterpenes began to be synthesized in large quantities from the 20th day of coloration and reached the highest level at the 35-40 th day of coloration. The content of bound monoterpenes reached the highest level at 30 days after coloration. The content of bound monoterpenes was higher than that of free monoterpenes. Flavonoids were synthesized and accumulated in large quantities before the fruit coloration; the synthesis of anthocyanins was initiated along with the fruit coloration and reached the highest level at 20 days after coloration, followed by a slight decrease. Based on transcriptome sequencing, a total of 5 836 differentially expressed genes were identified, and the number of differentially expressed genes varied significantly among different developmental stages. The differentially expressed genes were enriched in the pathways of phenylalanine synthesis, flavonoid synthesis, and monoterpene synthesis. Among them, 14 differentially expressed genes were related to the monoterpene synthesis pathway, and 11 were related to the anthocyanin synthesis pathway. The expression patterns of these genes were consistent with the accumulation of monoterpenes and anthocyanins. Further correlation analysis screened out 24 transcription factors that were significantly correlated with the expression of multiple genes involved in the monoterpene and anthocyanin synthesis pathways.

The synthesis of anthocyanins in red Muscat type grape berries initiates earlier than that of the aroma compound monoterpenes, and the synthesis of the two types of compounds is regulated in a spatio-temporal pattern. The accumulation of monoterpenes and anthocyanins is closely related to the expression of multiple key enzyme genes in their synthesis pathways, and their synthesis is regulated at the transcriptional level of various genes.

Grape berry size is one of important factors affecting grape appearance and the final productivity. It is a complex quantitative trait regulated by multiple genes. Mining the key genetic regulatory loci and the underlying genes for berry size related traits would help to improve grape yield.

In this study, 150 diverse grapevine varieties were selected as materials. The berry weight, seed number per berry, and seed weight were measured in 2019 and 2020, respectively. Based on high-density genotype data obtained by resequencing, the genome-wide association studies (GWAS) were carried out to detect significantly associated SNPs and to predict important candidate genes.

The three measured traits exhibited extensive phenotypic variation with 39.55%-68.89% of phenotypic variation coefficients; the phenotypic distribution of the observed three traits in the population showed continuous quantitative genetic characteristics; a significant positive correlation between each trait were observed in two years; based on the phenotypic data collected in two years, a total of 150 significant SNPs were detected for berry weight. In 2019, 99 SNPs were detected, each of which contributed the phenotypic variation from 14.48% to 25.59%; in 2020, 73 SNPs were detected, explaining 16.08%-26.83% of phenotypic variation; among these SNPs, 24 were detected repeatedly in both two years, mainly located on chromosome 1, 5, 11 and 16. Compared with the trait of berry weight, less SNPs significantly associated with the seed number were detected. A significant SNP was detected in 2019, and the phenotypic explanation value was 24.29%; in 2020, 17 significant SNPs were detected, which all located on chromosome 18; 1 and 2 SNPs located on chromosome 18 significantly associated with seed weight were detected in 2019 and 2020, respectively, accounting for 23.59%-48.29% of phenotypic variation. Within the genomic region of SNPs detected repeatedly for two years, 11 candidate genes related to berry weight were screened out based on the functional annotation, including ethylene signal pathway genes (VIT_05s0049g00490, VIT_05s0049g00500, VIT_05s0049g00510 and VIT_16s0100g00400), gibberellin signal pathway genes (VIT_11s0016g04630 and VIT_16s0022g02310), auxin responsive protein gene (VIT_11s0016g05640) and some important transcription factor genes (VIT_05s0049g00460, VIT_11s0016g05660 and VIT_16s0022g02330). A candidate gene VIT_18s0041g01880 (encoding a MADS box protein VviAGL11) associated with seed content was identified on chromosome 18, and different SNP genotypes on this gene significantly affected the grape berry seed number and weight.

A total of 150 SNPs significantly associated with berry weight were detected in two years, mainly located on chromosomes 1, 5, 11 and 16; A total of 19 significant SNPs associated with seed content were detected, mainly located on chromosome 18. Based on the results of gene annotation and genotype analysis, 11 candidate genes that might be involved in the regulation of grape berry weight including VIT_11s0016g04630 and VIT_16s0022g02310 were selected; the candidate gene VIT_18s0041g01880 was determined significantly correlated with seed content.

The eQTL mapping for monoterpene biosynthesis related gene expression traits were performed and the candidate genes were mined to deeply understand the regulation mechanism of monoterpene synthesis, so as to lay a foundation for the cultivation of new Muscat grape varieties and germplasm improvement.

The F₁ population generated by crossing Moldova and Ruiduxiangyu were used as materials in this study, and the grape berry samples were collected at verasion and ripening stage respectively. The phenotypic data of expression traits were obtained by detecting the expression levels of seven monoterpene synthesis pathway genes (VvDXS1, VvDXS3, VvDXR, VvHDR, VvLiner, VvTerp, and VvGermD) by using real-time quantitative qPCR technique. eQTL mapping of monoterpene gene expression traits were performed with the mapQTL6.0 software based on the interval mapping method. The associated markers of eQTL were mapped to the genomic region, and the genes within eQTLs were annotated and analyzed via the databases of Ensembl Plants and NCBI. The expression profiles of candidate genes in the samples of parents at different developmental stages were detected by grape whole genome microarray.

The expression levels of seven monoterpene biosynthesis related genes in F₁ population showed a continuous quantitative genetic distribution. A significant correlation between the expression of monoterpene genes was observed. At verasion, 13 eQTLs for the seven expression traits were mapped on chromosome 1, 6, 14, 16, 17, 10 and 12, respectively, and the phenotypic explanation value ranged from 12.2% to 23.5%. Among them, eQTLs (qDXS1-v14, qHDR-v14-1 and qTerp-v14) on chromosome 14 covered the same genetic interval of 57.582-76.979 cM, and qLiner-v10, qTerp-v10 and qGermD-v10 were co-located on chromosome 10. At the mature stage, 16 eQTLs were detected, mainly located on chromosome 1, 6, 12, 8, 13 and 19. qDXS1-m6-2, qDXR-m6-2, qLiner-m6 and qGermD-m6 were co-located in the genetic interval 139.212-143.161 cM of chromosome 6. In addition, a total of 18 eQTLs on chromosomes 1, 3, 7, 10, 12, 15 and 19 were detected for the change ratio of each gene expression between maturity and verasion, respectively. qDXS1-r12-1, qDXR-r12-1, qHDR-r12, qLiner-r12 and qGermD-r12 covered the same genetic interval of 6.330-6.967cM on chromosome 12. The eQTL region for multiple expression traits co-located were further annotated, 90 transcription factor genes were screened, and 11 candidate genes were finally identified by expression profile and correlation analysis. Among them, four candidate genes of VIT_06s0009g01380, VIT_14s0006g02290, VIT_12s0028g01170 and VIT_15s0046g00290 were predicted to participate in the regulation of hormone signaling pathway, one candidate gene VIT_12s0028g01110 encodes a phytochrome interacting factor related to light response, and some other genes encode Myb, WRKY transcription factors or unknown functional proteins.

A total of 37 eQTLs linked to monoterpene synthesis gene expression traits were detected at two different development stages, which mainly located on chromosome 6, 12 and 14. Based on the results of gene annotation and expression profile analysis, 11 candidate genes including VIT_14s0006g02290 and VIT_06s0009g01380 were identified, and these candidate genes were highly correlated with the expression of multiple monoterpene genes.