1. Selection of potato breeding clones resistant to potato nematode Globodera rostochiensis Ro1 using artificial inoculation and adaption of molecular markers assisted selection in State Priekuļi Plant Breeding Institute. G. Ūsele 1, I. Mežaka 1, I. Skrabule 1 and N. Rostoks 2 1 State Priekuļi Plant Breeding Institute 2 Faculty of Biology, University of Latvia, 4 Kronvalda Blvd., Riga, Latvia E-mail: guna.usele@gmail.com Introduction/Problem Potato cyst nematodes (PCN) are the major problem in worldwide potato production especially in cool-temperate areas. Potato cyst nematodes are sedentary endo-parasites, requiring the roots of potato to complete their lifecycle. During PCN invasion, occupation and subsequent emergence into the rhizosphere, the roots are irreparably damaged, reducing their ability to take up water and minerals. In infested fields, mature females and cysts may be found on potato roots within eight to eleven weeks after crop emergence. Cysts are visible to the naked eye and can be seen as minute white or yellow globes on the root surface (Fig.1. and 2. ) G. rostochiensis females progress from white to a golden yellow colour before darkening into the cyst, the golden yellow phase lasting 4-6 weeks. Breeding of resistant cultivars is the most desirable and effective method of control of pest. Potato breeding material has been screened using artificial inoculation in State Priekuli Plant Breeding Institute since year 2000. Adaption of molecular markers for detection of nematode resistance genes has been initiated since 2010. Marker assisted selection (MAS) could fasten breeding work and save material and labor expenses. Methodology Inoculation Third year generation tubers have been planted in pots with soil infected with nematode. Inoculation were done in three replications for each breeding line. The pathotype Ro1 of Globodera rostochiensis was used for inoculation, as it is the only confirmed patotype presented in Latvia. Inoculation was carried out in May and after 8-11weeks infection degree was determined and susceptible to nematode clones was discarded. Genotypes were designated resistant when no cysts were, and susceptible when the test of resistance showed more than three cysts in every replication. If one or two of replications from one til three cycts were found, cultivar was counted as partialy resistant. Resistant standarts were the cutivars Brasla and Lenora, susceptible standart were the cultivar Mutagenagrie. First steps of implementation of MAS Marker assisted selection (MAS) has been initiated at 2010 to test nematode pathotype Ro 1 resistance. The first steps of MAS implementation is to verify results of molecular markers by comparing them with results obtained by artificial inoculations. If MAS proves to be reliable, application of MAS could help fasten selection towards nematode resistance testing and to save costs and labor because hybrids with no resistance would be discarded at early generations. Markers Ro1 (indicator of gene H1 that confers resistance to G. rostochiensis pathotypes Ro 1, Ro 4 from S. tuberosum spp. andigena) and Gro1 (indicator of the presence of resistance to G. rostochiensis pathotypes Ro 1, Ro 2, Ro 3, Ro 4 and Ro 5 from S. spegazzinii), published by Biryukova (2008), are being used to detect resistance genes in breeding clones and simultaneously the same clones were artificially inoculated with nematode pathotype Ro 1 to verify the results obtained with molecular markers. Table 1. Potao breeding sheme 2010, Priekuli, Latvia Conclusions Even if both of parents were resistant to nematode, no more than 74% of progenies were resistant therefore testing is especially significant because even choice of resistant parents does not guarantee resistant progenies. To test on variety resistance to PCN In 92% of samples data obtained from databases was consistent with data obtained by molecular markers. High level of compatibility of data assures that markers Ro1 and Gro1 published by Biryukova (2008), can be applied to wide set of germplasm. Results: Amount of resistant breeding clones in tested material varied from 68% to 74% depending on parents (Fig.3.- Fig.5.). Molecular markers usually are developed in biparental mapping populations and often are applicable only to test resistance in hybrids of the certain cross. To test whether markers would be applicable in wider set of germplasm, data on variety resistance to PCN pathotype Ro 1 was acquired from public database www.europotato.org and compared to resistance status acquired by molecular markers. Two known genes are known for resistance to PCN pathotype Ro 1 – Gro1 and H1. Therefore testing results were considered consistent if 1)presence of resistance allele of either gene or both genes was detected by molecular markers and inoculations tests revealed that variety is resistant or 2)succeptible allele of both genes was detected and inoculation tests revealed that variety is resistant. In 92% of samples data obtained from databases was consistent with data obtained by molecular markers. In 7% of cases susceptible allele was detected by molecular markers in varieties in which resistance was reported in databases. In 1% of cases resistant allele was detected by molecular marker in varieties in which were reported as susceptible. Inconsistency of results could be explained by several reasons: 1) resistance could be maintained by other gene than tested one; 2) there has been a crossover between marker and resistance gene; 3) there could be phenotyping of genotyping errors. Acknowledgements Implementation of molecular markers has been funded by EU Structural Funds (project: “Development, improvement and implementation of environmentally friendly and sustainable crop breeding technologies”, 2009/0218/1DP/1.1.1.2.0/09/APIA/VIAA/099 ),. Acknowledgements Implementation of molecular markers has been funded by EU Structural Funds (project: “Development, improvement and implementation of environmentally friendly and sustainable crop breeding technologies”, 2009/0218/1DP/1.1.1.2.0/09/APIA/VIAA/099 ),. YearBreeding materialNumber of genotypes 1.Hybridization -crosses150 2.Seedlings22000 3.1st year field clonal genetarion18000 4.2nd year field clonal generation1640 5.3rd year field clonal generation (inoculation and paralel testing with markers) 240 6.Primary assesment of yield, quality and diseases and pest resistance 150 7. – 9.Assesment of yield, quality and diseases and pest resistance 130 10. – 12.Official DUS and VSU tests for including in Plant Variety Cataloque 1 Results of inoculation from year 2005 til year 2010, depending on resistance status of parents Fig.1. Potato roots infected with Globodera rostochiensis cysts ten weeks after inoculation Fig.5. Both parents resistant References Biryukova V.A., Zhuravlev A.A., Abrosimova S.B., Kostina L.I., Khromova L.M., Shmyglya I.V., Morozova N.N., Kirsanova S.N., 2008, Use of molecular markers of potato Golden nematode resistance genes H1 and GRO1. Russian Agricultural Sciences 34: 365-368 Fig.4. Both of parents susceptible Fig.3. One of parents resistant Fig.2. Potato plant ten weeks after inoculation