Features of the primary structure of the Ph-3 gene, revealed by development of a new gene-based marker of late blight resistance in tomato

Late blight caused by the oomycete Phytophthora infestans (Mont.) de Bary is one of the most harmful diseases of tomatoes. Late blight control remains challenging due to the high genetic variability and complex racial composition of P. infestans . Therefore, the most promising method of combating late blight is the breeding of resistant varieties of tomato. When creating resistant varieties, the introgression of resistance genes from wild-growing related species is widely used. In particular, several late blight resistance genes identified in the wild tomato species Solanum pimpinellifolium have been introgressed into tomato cultivars. Among these genes, the Ph-3 gene is considered to be the strongest late blight resistance gene, as it provides resistance to a variety of P. infestans isolates. Therefore, considerable efforts of scientific groups around the world are directed to the study of this gene in order to include it in breeding programs and introduce it into new commercial varieties and lines of tomato. To date, DNA markers associated with this gene are known. However, homologues of this gene were found in the tomato genome, which do not have functional activity. Analysis of the multiple alignment of the nucleotide sequences of the Ph-3 gene and its homologues showed that the primers used in the known markers for amplification of this gene are in the conservative regions of these sequences, and it is impossible to specifically amplify the Ph-3 gene with them. Therefore, the aim of this work was to design a new highly specific marker of the Ph-3 gene and compare it with already known markers by analyzing the collection of tomato varieties of the Federal Scientific Center for Vegetable Growing for the presence of known and new markers and assessing the linkage of these markers with resistance to late blight disease in the studied varieties. To this end specific primers were designed (5'-AATATTGAAAATAGCTGCACTGA-3'/5'-CGAGATTTGGAGGGAATGTAA-3') that discern the Ph-3 gene from its homologues and amplify a 412 bp gene fragment (the Ph3-412 marker). Using these primers, 24 tomato ( Solanum lycopersicum L.) varieties bred at the Federal Scientific and Technical Center and tested for late blight field resistance (Federal Scientific and Technical Center, Moscow Province, 2021) were analyzed. Also, these varieties were analyzed with known marker NC-LB-9-6678. To determine the nucleotide sequence of the new marker, we cloned the amplified product obtained from the studied varieties into pAL-TA vector and sequenced the resulting clones. In addition, we cloned and sequenced 601 and 907 bp fragments obtained with a known marker. We compared the nucleotide sequences of all three fragments with the sequences of the prototype gene and its known homologues. As a result, we confirmed that the fragment amplified using primers designed by us belongs to the Ph-3 gene, while the 601 bp fragment obtained with the known primers corresponds to the SlRGA4 homologue, and the 907 bp fragment obtained with the same primers is homologous to the Ph-3 gene but it contains an insertion of the LTR retrotransposon of the Ty1- copy family with a size of 306 bp. Thus, the gene containing such insertion is most likely inactive. We also showed that in all analyzed varieties, in which the Ph-3 gene was found, this gene contains the abovementioned insertion. The presence of such insertion can lead to a loss of functional activity; this must be taken into account when marking the Ph-3 gene. For the breeding programs it is necessary to identify plants in which the Ph-3 gene does not have this retrotransposon insertion.