Combination Breeding and Marker-Assisted Selection to Develop Late Blight Resistant Potato Cultivars

(1) Background: Although resistance to pathogens and pests has been researched in many potato cultivars and breeding lines with DNA markers, there is scarce evidence as to the efficiency of the marker-assisted selection (MAS) for these traits when applied at the early stages of breeding. A goal of this study was to estimate the potential of affordable DNA markers to track resistance genes that are effective against the pathogen Phytophthora infestans (Rpi genes), as a practical breeding tool on a progeny of 68 clones derived from a cross between the cultivar Sudarynya and the hybrid 13/11-09. (2) Methods: this population was studied for four years to elucidate the distribution of late blight (LB) resistance and other agronomical desirable or simple to phenotype traits such as tuber and flower pigmentation, yield capacity and structure. LB resistance was phenotypically evaluated following natural and artificial infection and the presence/absence of nine Rpi genes was assessed with 11 sequence-characterized amplified region (SCAR) markers. To validate this analysis, the profile of Rpi genes in the 13/11-09 parent was established using diagnostic resistance gene enrichment sequencing (dRenSeq) as a gold standard. (3) Results: at the early stages of a breeding program, when screening the segregation of F1 offspring, MAS can halve the workload and selected SCAR markers for Rpi genes provide useful tools

Diversity of Late Blight Resistance Genes in the VIR Potato Collection

Late blight (LB) caused by the oomycete Phytophthora infestans (Mont.) de Bary is the greatest threat to potato production worldwide. Current potato breeding for LB resistance heavily depends on the introduction of new genes for resistance to P. infestans (Rpi genes). Such genes have been discovered in highly diverse wild, primitive, and cultivated species of tuber-bearing potatoes (Solanum L. section Petota Dumort.) and introgressed into the elite potato cultivars by hybridization and transgenic complementation. Unfortunately, even the most resistant potato varieties have been overcome by LB due to the arrival of new pathogen strains and their rapid evolution. Therefore, novel sources for germplasm enhancement comprising the broad-spectrum Rpi genes are in high demand with breeders who aim to provide durable LB resistance. The Genbank of the N.I. Vavilov Institute of Plant Genetic Resources (VIR) in St. Petersburg harbors one of the world’s largest collections of potato and potato relatives. In this study, LB resistance was evaluated in a core selection representing 20 species of seven Petota series according to the Hawkes (1990) classification: Bulbocastana (Rydb.) Hawkes, Demissa Buk., Longipedicellata Buk., Maglia Bitt., Pinnatisecta (Rydb.) Hawkes, Tuberosa (Rydb.) Hawkes (wild and cultivated species), and Yungasensa Corr. LB resistance was assessed in 96 accessions representing 18 species in the laboratory test with detached leaves using a highly virulent and aggressive isolate of P. infestans. The Petota species notably differed in their LB resistance: S. bulbocastanum Dun., S. demissum Lindl., S. cardiophyllum Lindl., and S. berthaultii Hawkes stood out at a high frequency of resistant accessions (7–9 points on a 9-point scale). Well-established specific SCAR markers of ten Rpi genes—Rpi-R1, Rpi-R2/Rpi-blb3, Rpi-R3a, Rpi-R3b, Rpi-R8, Rpi-blb1/Rpi-sto1, Rpi-blb2, and Rpi-vnt1—were used to mine 117 accessions representing 20 species from seven Petota series. In particular, our evidence confirmed the diverse Rpi gene location in two American continents. The structural homologs of the Rpi-R2, Rpi-R3a, Rpi-R3b, and Rpi-R8 genes were found in the North American species other than S. demissum, the species that was the original source of these genes for early potato breeding, and in some cases, in the South American Tuberosa species. The Rpi-blb1/Rpi-sto1 orthologs from S. bulbocastanum and S. stoloniferum Schlechtd et Bché were restricted to genome B in the Mesoamerican series Bulbocastana, Pinnatisecta, and Longipedicellata. The structural homologs of the Rpi-vnt1 gene that were initially identified in the South American species S. venturii Hawkes and Hjert. were reported, for the first time, in the North American series of Petota species

Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR.

Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter