Analysis of anthocyanin components and contents in 301 grape germplasm resources

【Objective】The anthocyanin composition and content of grapes in different populations were studied for screening out superior germplasms, and promoting the innovative utilization of germplasm resources.【Methods】The components and contents of anthocyanins were determined by the high performance liquid chromatography-mass spectrometry (HPLC-MS) in the peels of 301 grape cultivars at ripening stage. The experimental materials comprised 133 accessions of Vitis vinifera, 126 accessions of V. vinifera-V. labrusca, and 42 accessions of wild grapes. The experimental materials were collected from the National Grape Germplasm Resources Repository of Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences. In the experiment, 1.0 m×2.5 m plant spacing, singlestem and double-arm tree system, as well as very short and slightly pruning method were applied. At the fruit ripening stage, the berries that are completely ripe and colored were taken. After the berry was peeled, the peels were frozen with liquid nitrogen and put in an ultra-low temperature refrigerator at -80 ℃ for storage. The extraction method for anthocyanins was as follows: 0.5 g of frozen berry skin powder was dissolved in 8 mL of methanol solution containing 2% formic acid. The mixture was then subjected to ultrasonic oscillation for 10 minutes, followed by shaking at 28 ℃ and 220 r·min-1 for 30 minutes. Subsequently, it was centrifuged at 4 ℃ and 10 000 r min-1 for 10 minutes, and the supernatant was collected. The remaining precipitate in the centrifuge tube was extracted three more times by repeat-ing the above steps. The supernatants from all four extractions were combined. The solvent was removed by rotary evaporation at 40 ℃, and the residue was finally reconstituted to a final volume of 10 mL with an aqueous solution containing 2% formic acid and 10% methanol. The extract was wrapped in aluminum foil and stored in an ultra-low temperature freezer at -80 ℃ for subsequent analysis. For anthocyanin identification, five standard compounds were used: Cyanidin-3-O-glucoside, Delphinidin-3-O- glucoside, Malvidin-3-O- glucoside, Petunidin-3-O- glucoside and Peonidin-3-O- glucoside. Quantification of anthocyanins was performed by constructing a standard curve using Mv-3-O-G at five concentration levels (0-500 mg·L-1 ) with three replicates. The anthocyanin content was determined using Mv- 3- O- G as an external standard, and the contents of other anthocyanins were calculated as equivalents of Mv-3-O-G.【Results】Substantial differences were observed in the anthocyanin composition and concentration among grape accessions with different berry colors. In green-colored accessions, 1-12 types of anthocyanins were detected, while black-colored accessions contained 16-31. The remaining accessions exhibited a range of 3-28 types of anthocyanins. The total anthocyanin content progressively increased with the deepening of berry color, showing significant differences both in total content and composition among the various color groups. Green grape accessions contained only trace amounts of anthocyanins. In red grape accessions, peonidins were the most abundant, reaching 60.09 μg · g-1 and accounting for 56.57% of the total anthocyanin content. In both purple and black accessions, malvidins were predominant, with average contents of 966.77 μg·g-1 and 1 990.53 μg·g-1 , constituting 79.27% and 73.54% of the total anthocyanin content, respectively. The content and relative proportion of malvidins increased with the intensity of berry skin color. Malvidins represented the category with the most pronounced content difference between purple and red accessions, averaging 966.77 μg·g-1 in purple accessions compared to only 19.61 μg · g- 1 in red accessions—a difference of approximately 50- fold. Wild grape accessions exhibited the highest total anthocyanin content, reaching 3 311.76 μg · g- 1 , which was substantially greater than that of V. vinifera-V. labrusca (589.64 μg · g-1 ) and V. vinifera (502.85 μg · g-1 ). Among wild grapes, the accession Cinerea had the highest anthocyanin content at 26 430.02 μg · g- 1 , followed by the V. amurensis accession Changbai No. 9 with 10 062.38 μg · g- 1 , both possessing blueblack berry skin. Within cultivated varieties, the three accessions with the highest anthocyanin contents were the V. vinifera-V. labrusca‘gf.67-198-3’(5 532.54 μg · g- 1 ), the V. vinifera‘Khyndogny ’ (6 642.13 μg·g-1 ), and the V. vinifera-V. labrusca‘Shanxi No. 80’(6 870.49 μg·g-1 ), all of which were purple-black berry skin. Malvidins were the predominant anthocyanins in all three groups, accounting for 59.7%, 73.1%, and 87.2% of the total anthocyanins in V. vinifera-V. labrusca, V. vinifera, and wild grapes, respectively. The relative proportions of peonidins were similar in V. vinifera, and V. vinifera-V. labrusca (13.1% and 14.8%, respectively), as were those of delphinidins in V. vinifera and wild grapes (4.0% and 4.1%, respectively). Cyanidins were present at relatively low levels across all groups (0.7%- 6.3% ). Furthermore, pelargonidins were not detected in any wild grape accessions in this study, and were found only in low amounts in a subset of V. vinifera and V. vinifera-V. labrusca grape accessions. Anthocyanin acyl modifications in grape berry skins exhibited diversity, with a total of six types detected. Coumaroylation was the predominant form of acylation, while acetylation and caffeoylation were less frequent and were even absent in some germplasms. The level of coumaroylation in wild grapes (28.54%) was lower than that in V. vinifera (44.97%) and V. vinifera-V. labrusca (42.15%). Additionally, feruloylation, succinylation, and glycosyl- acylated anthocyanins were identified, though these three types were present in only trace amounts, each accounting for less than 1% of the total. The overall degree of acylation showed no significant difference between V. vinifera and V. vinifera- V. labrusca (56.62% and 56.75%, respectively), both of which were considerably higher than that observed in wild grapes (34.18% ).【Conclusion】Differences in anthocyanin composition and content were observed among germplasms of different varieties and fruit colors. Malvidins were the most abundant across all tested grape accessions and also showed the greatest content variation between red-fruited and purplefruited accessions. The total degree of anthocyanin modification was higher in V. vinifera-V. labrusca and V. vinifera than in wild grapes. V. vinifera showed the highest level of coumaroylation, while V. vinifera-V. labrusca exhibited the highest degree of acetylation.