Establishment and application of a Multiple nucleotide polymorphism molecular identification system for grape cultivars

Grapes (Vitis L.) are one of the most important and popular fruits in China. However, efficient identification of grapes is important in the protection of domestic grapes and breeders' rights. Here, we developed a new method called multiple nucleotide polymorphisms (MNP), which combines the advantages of multiple PCR amplification and high-throughput sequencing to improve the efficiency of grape variety identification. We identified 582 universal MNP markers and screened them based on the whole genome sequencing data of 31 representative cultivars. Subsequently, MNP libraries of 60 common cultivars were constructed using multiple PCR amplification and sequencing. The pairing comparison of 60 grape materials revealed a high identification rate (>99 %) of MNP markers. Furthermore, MNP accurately distinguishes diploid and polyploid, which can assist in molecular identification of grapes. Overall, MNP molecular identification technology provides a new way for grape variety identification. Compared with SNP, MNP reduces the cost of identification because it does not need to make expensive gene chips. In addition, MNP can perform the amplification of thousands of primers in a single tube and uses sequencing instead of polyacrylamide gel electrophoresis, which greatly saves experimental consumables and labor compared with SSR. Collectively, the developed technique has the advantages of low cost and high efficiency, which is more competitive than other identification methods at present. The developed technology can assist in resource research and promote plant breeding.