Mining, screening, and validation of cold-resistance molecular markers in Vitis vinifera L.

To address one of the main challenges restricting the development of global viticulture, it is imperative to breed high-quality and cold-resistant grape varieties. In this study, intraspecific cross-breeding was carried out between two cold resistance and four high-quality varieties for eight populations, including six reciprocal crosses and two crossing combinations. The cold-resistance of six hybrid parents and 481 hybrid F₁ progeny was determined by differential thermal analysis, and the cold-resistance grade and frequency distribution were analyzed from the 14 crossing combinations. Bioinformatics and statistical methods were used to mine and design SSR marker primers for 40 cold-resistance related genes. Agarose gel electrophoresis and cold-resistance measurement were used to screen molecular markers of the tested materials. The results showed that varying cold resistance for different populations, with a continuous distribution of cold resistance of intraspecific hybrid progenies of different crossing combinations. The screening and verification of molecular markers revealed that OMT2.1-211, SIRT276, UBC317-800, S238-854, and S241-717 can be used as standard markers to determine cold-resistance of intraspecific hybrid progenies of Ecolly or Meili and Marselan, and P5CS173, P5CS240, and SIRT276 could be used to rapidly screen intraspecific hybrid progenies of Ecolly or Meili and Cabernet Sauvignon. Intraspecific cross-breeding of V. vinifera can be applied to breed suitable intermediate materials or new cold-resistant materials. This work also provides a reference method for the development of SSR molecular markers to target specific genes of interest.