In the present paper, the potato cultivars bred at the Leningrad Research Institute for Agriculture “Belogorka”, were taken as an example for demonstrating the results of elaboration of methodological approaches that are currently developed at the N.I. Vavilov Institute of Plant Genetic Resources (VIR) for the preparing of nomenclatural standards and their genotyping. In 2018, joint research of VIR scientists and breeders from the Leningrad Research Institute for Agriculture “Belogorka” began in the field of preparing nomenclatural standards for potato cultivars bred at this institute. Nomenclatural standards were prepared according to the ‘International Code of Nomenclature for Cultivated Plants’. Plant material for herbarium specimens was collected in the experimental field of the “Belogorka” Institute in 2018 by cultivar authors and handed over to the VIR Herbarium of cultivated plants, their wild relatives and weeds (WIR). The plant material included stems with inflorescences and later - tubers of 21 cultivars which were bred at the “Belogorka” Institute. Two precultivars undergoing State variety testing and three breeding clones were also included in this study. Just before herbarium preparation, the obtained plant material was photographed, plant morphological characters described, and the results compared with the description given in such official documents as the “Cultivar Questionnaireˮ and “Description of selection achievementˮ. The nomenclatural standards of 21 cultivars registered in the VIR Herbarium Database and transferred for conservation to the VIR herbarium, are published in this paper. Before herbarium preparation, the plant material was sampled for DNA extraction and subsequent genotyping and molecular screening. The genetic passports include information about the polymorphism of 10 chromosome-specific microsatellite loci, as well as the data on the presence/absence of diagnostic fragments of 12 markers of the 11R-genes conferring resistance to diseases and pests, and for some cultivars – the information about their cytoplasm type. These genetic passports are valuable not only because different types of DNA markers were used in their preparing (SSR, SCAR and CAPS markers of theRgenes; markers specific to different loci of the nuclear and organelle genomes), but first of all because of the material itself, as the DNA samples were isolated from the plants with the assigned status of nomenclatural standard for each particular cultivar. Based on the genetic passports data, trueness to type of the “Belogorka” cultivar samples obtained from various sources was verified.

The N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) is developing new approaches to documentation of national cultivars, taking into account recommendations of the International Code of Nomenclature for Cultivated Plants in parallel with methods of genetic certification. The nomenclatural standard of a particular cultivar represented by a herbarium specimen can be used as a reference for verifying authenticity and uniformity of cultivar specimens obtained from various sources. The verification requires fast and reliable methods for cultivar genotyping. This paper presents protocols for modified methods of DNA extraction, PCR-analysis and SSR-genotyping, which allow potato cultivars identification without the use of expensive reagent kits. A set of ten chromosome-specific microsatellite markers was used to study polymorphisms in 66 modern Russian potato cultivars, as well as in 11 pre-cultivars and breeding clones, represented by nomenclatural standards and voucher specimens, respectively. This subset of 77 specimens has demonstrated a high level of polymorphism in ten studied microsatellite loci. The SSR analysis identified 73 alleles; 7.3 alleles per locus were observed on average, the number of which varied from 3 (STG0025 locus) to 11 (locus StI046). The PIC values varied from 0.544 (STG0025 locus) to 0.836 (StI046 locus). The alleles, unique for this subset, were found at six studied loci. The high level of polymorphism at the SSR loci made it possible to unambiguously identify almost every cultivar, with the exception of the expected coincidence of microsatellite profiles of two cultivars, which are somaclonal variants. Using an optimized set of eight microsatellite markers, the genetic relationships of modern Russian potato cultivars were studied.

Генетическое разнообразие сортов картофеля российской селекции и стран ближнего зарубежья по типам цитоплазм

The main objectives in potato breeding are increasing yield abilities and improving resistance to numerous pathogens and pests. Among them, the late blight caused by the Phytophthora infestans oomycete is one of the most destructive potato diseases both in Russia and worldwide. Wild relatives of cultivated potato are traditionally used in breeding as the source of valuable R genes conferring resistance to pathogens. Of particular interest are Mexican wild species because Mexico is the centre of origin and diversity of P. infestans and at the same time, it is the centre of potato species diversity. Mexican wild potato species S. bulbocastanum and S. stoloniferum are an important source of the R genes conferring broad-spec trum resistance against various isolates of P. infestans (Rpi-blb1, Rpi-blb2, Rpi-sto1). Recently these genes have been transferred into cultivated potato gene pool using the cisgene approach. At the same time there is a high probability of finding geno types with the Rpi-sto1 gene (functional homologues of Rpi-blb1) among conventionally bred varieties because for about 40 years S. stoloniferum has been used in breeding as a source of the Rysto and Ry-fsto genes of the extreme resistance to the most important viral pathogen PVY. In this study 188 potato varieties bred in Russia and in near-abroad countries were screened for the presence of six gene-specific markers of the RB/Rpi-blb1 = Rpi-sto1 and Rpi-blb2 genes conferring broad-spectrum resistance against P. infestans, and for the markers linked to the Rysto and Ry-fsto genes conferring extreme resistance to PVY. In addition, a marker for detecting male sterile mitochondrial DNA type gamma derived from S. stoloniferum was used. The genotypes selected through the molecular markers were divided into four groups: (A) 13 PVY resistant varieties carrying diagnostic markers of the Rysto, Ry-fsto genes and having sterile mt-type gamma; (B) four varieties possessing mt-type gamma and not having the markers of the R genes introgressed from S. stoloniferum; (C) eight genotypes carrying five gene-specific markers for the RB/Rpi-blb1/= Rpi-sto1; (D) the rest 166 (86.9 %) varieties not possessing any of the diagnostic markers associated with the S. stoloniferum genetic material. The sequences of the Rpi-sto1- and BLB1 F/R-amplicons were identical in all the genotypes of group ‘C’ and showed respective 99 % and 100 % similarity to the corresponding fragments of the Rpi-sto1 and Rpi-blb1 genes from the GenBank database. Among the genotypes of group ‘C’ various mt-types were detected, and some of them were male fertile.

Изучено генетическое разнообразие российских сортов картофеля и сортов селекции стран ближнего зарубежья из коллекции ВИР, которые создавались в 1931–2015 гг. На основании результатов анализа полиморфизма 14 монолокусных ядерных микросателлитов (SSR) проведено генотипирование 113 селекционных сортов. Дополнительно изучено распространение у сортов восьми маркеров трех R-генов, вовлеченных в контроль устойчивости растений к двум карантинным объектам – золотистой картофельной нематоде Globodera rostochiensis и возбудителю рака картофеля Synchytrium endobioticum, которые локально встречаются в отдельных регионах Российской Федерации. У всех изученных сортов, устойчивых к патотипу 1 S. endobioticum, выявлен диагностический компонент маркера Nl-25 гена Sen1, у восприимчивых сортов фрагмент Nl-251400 не обнаружен. Полученные результаты продемонстрировали различную эффективность маркеров генов Н1 и Gro1-4, детерминирующих устойчивость к патотипу Ro1 G. rostochiensis; наиболее перспективно использовать несколько маркеров для детекции нематодоустойчивых сортов. Результаты молекулярного скрининга с шестью маркерами генов Н1 и Gro1-4 позволили детектировать у сортов шесть гаплотипов. Пять гаплотипов включали сорта с различными сочетаниями изученных маркеров, большинство (87,9 %) из этих сортов были устойчивы или слабо поражаемы нематодой. Наиболее многочисленный гаплотип H1/0 включал 76 сортов, у которых не было выявлено ни одного маркера генов Н1 и Gro1-4; 96,1 % из них были восприимчивыми к G. rostochiensis. Обсуждается связь между родословными сортов, временем их создания, составом гаплотипов и устойчивостью селекционных сортов к золотистой картофельной нематоде и возбудителю рака картофеля.

In order to preserve the genetic diversity of cultivars, it is recommended to develop documentation approaches through the registration of cultivar nomenclatural standards in accordance with the International Code of Nomenclature for Cultivated Plants. The N.I Vavilov Institute of Plant Genetic Resources keeps developing this approach along with the DNA marker technologies: SSR genotyping and molecular screening with DNA markers of genes controlling valuable traits. In 2018, VIR, together with the leading national centre for potato breeding – the A.G. Lorkh All-Russian Scientific-Research Institute of Potato Farming (now the A.G. Lorkh Russian Potato Research Center), initiated the preparation of nomenclatural standards for potato cultivars bred both at the A.G. Lorkh institute and in co-authorship with other breeding institutions. This paper presents 30 nomenclatural standards which are maintained in the Nomenclature standard fund of the Herbarium of cultivated plants and their wild relatives and weeds (VIR Herbarium, WIR). Also, the paper presents genetic passports of the mentioned 30 cultivars, developed using DNA samples extracted from the plant material of nomenclatural standards. Genetic passports include information about the polymorphism of the 8 chromosome-specific microsatellites and data on the results of molecular screening with 15 markers of 11 genes conferring resistance to the most dangerous pathogens and pests: potato viruses Y and X, late blight and potato cyst nematodes. A comparison of SSR-profiles of nomenclatural standards with those of 66 cultivar samples obtained from various sources made it possible to verify authenticity and homogeneity of the latter.