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.

(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.

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.

Clones of potato hybrids comprising genetic material from up to seven wild Solanum species were studied with phytopathological methods and DNA markers for Solanum genomes A, B and D and for late blight resistance genes R1, R2/Rpi-blb3, R3a, R3b and RB/Rpi-blb1. Late blight resistance of these clones was obviously associated with the presence of DNA markers for the R genes and significantly related to the number of these markers discerned in particular interspecific hybrids.

A novel plant hairpin-like defense polypeptide named EcAMP3 was isolated from latent barnyard grass (Echinochloa crusgalli L.) seeds. The native peptide and its recombinant analogue were characterized. EcAMP3 displays antifungal and antibacterial activity in vitro. The gene family encoding EcAMPs precursor protein was also characterized; the genes and pseudogenes of this family show 97-100% homology. Every member of EcAMPs precursor family contains seven identical cysteine motifs: C1XXXC2(11-13)C3XXXC4. One of those motifs corresponds to the isolated peptide. EcAMP3 is the first member of the plant hairpin-like peptide family that inhibits the growth of phytopathogenic bacteria. Obtained results can explain the nature of the complex resistance of barnyard grass to a variety of pathogenic microorganisms.

The panel of wild Solanum species and potato hybrid clones was studied by coupled phytopathological and SCAR-marker analyses. Late blight (LB) resistance was assessed in individual plants under the field conditions and in the laboratory, with the detached-leaf test. The novel lines derived from previously unexplored sources of LB resistance among the Bolivian diploid species S. alandiae manifested a successful field resistance in the trials through two epidemic years. SCAR markers were developed using the R1, R3a and RB gene sequences. By screening wild Solanum species and clones of potato hybrids after natural and artificial infestation by P. infestans, we established that the presence of SCAR markers R1-1205 was significantly related to LB resistance. This evidence suggests the practicability of employing this marker as a breeding tool for early prediction of LB resistance in a wide range of Solanum germplasms.

Day length controls development in many plants. In Arabidopsis thaliana, the CONSTANS (CO) gene has been firmly established as a key component in the photoperiodic pathway of floral transition; less is known about CONSTANS-LIKE1 (COL1) orthologues of this gene in Arabidopsis and several other species. The CONSTANS protein comprises two B-box-type zinc fingers, CCT domain, and a variable middle region (MR) which corresponds to exon 2 in the COL1 genes of Solanum species. Solanum COL1 proteins are over 85% identical within the genus and about 50% similar to Arabidopsis CO. Comparative COL1 analysis in several cultivated and wild Solanum species discerned two gene variants, which differed in the structures of exon 2 and introns 1 and 2. In exon 2, two variants were primarily discerned by the numbers of AAC/AAT and CAA/CAG repeats coding for polyasparagine and polyglutamine tracts in MR; therefore two variants were dubbed short and long COL1 genes (sCOL1 and lCOL1). However, intron 1 in lCOL1 was shorter than in sCOL1 due to three indels, whereas intron 2 in available COL1 sequences was represented by three different variants. The temporal profiles of sCOL1 and lCOL1 expression in tuberosum potato dramatically differed under short and long day, and the level of sCOL1 expression exceeded that of lSOL1 by an order of magnitude. Both sCOL1 and lCOL1 were found in each Solanum genome under study and in each individual plant, and the ratio of their copy numbers was not related to plant ploidy and photoperiodic response. Evidently the evolution of two COL1 genes preceded Solanum speciation, and the day-length response of diverse Solanum genotypes does not stem from the primary COL1 structure.

Although the CONSTANS gene and its CONSTANS-LIKE1 ( COL1 ) orthologs are known to control the photoperiod-dependent floral transition in many plant species, the role of these genes in Solanum development has not been sufficiently elucidated. Previously we characterized two forms of CONSTANS-LIKE1 genes, sCOL1 and lCOL1 , in potato ( Solanum tuberosum ssp. tuberosum ). To prove that these genes were functional, we followed their expression in potato cv. Early Rose with the real-time PCR technique. Both sCOL1 and lCOL1 displayed characteristic day-night patterns of expression under long-day and short-day conditions. The profiles and amplitudes of expression dramatically differed in two genes, with the maximum sCOL1 expression exceeding that of lCOL1 by an order of magnitude.

To develop breeder-friendly sequence characterized amplified region (SCAR) markers characteristic of particular Solanum species and workable as tools for introgression breeding, we compared several polymorphic regions of Solanum genomes, such as repetitive sequences identical in the genomes of wild Solanum species and derived potato cultivars, the characteristic fragments of the genes for potato late blight resistance (R genes) and the hot spot of variation in intron 2 of the developmental gene FLORICAULA/LEAFY. Multiple alignments of these sequences became the basis for designing the species-specific PCR primers. The specificity of SCAR markers amplified with these primers was verified against the accessions of several Solanum species. The markers reliably discerned the germplasm of wild Solanum species in potato cultivars and were feasible for monitoring the transfer of such germplasm into potato cultivars. The presence of SCAR markers derived from the R genes was associated with the foliage and tuber resistance of potato cultivars to late blight; it follows that such markers are potential predictors of these phenotypic traits in new potato crosses.

The putative homologs of CONSTANS and FLORICAULA/LEAFY are primary candidate genes for the control of flowering and tuberization in potato. CONSTANS homologs were cloned from potato (Solanum tuberosum subsp. tuberosum) 'Bastoneza' and 'Early Rose' (GenBank accession numbers DQ472734 and DQ499754) and S. demissum (DQ499755). Their amino acid sequences greatly resembled CONSTANS from S. tuberosum subsp. andigenum (S. Prat, pers. commun.) and CONSTANS 1 and CONSTANS 3 from S. lycopersicum (AAS67377 and AAS67379). Long-day tuberosum plants notably differed from short-day andigenum and demissum in the variable middle region of the CONSTANS protein, especially by the length of polyasparagine and polyglutamine motifs. Partial homologs of the FLORICAULA/LEAFY protein from potato (ABB69053, ABB72204, ABB76758-ABB76760) and demissum accessions (ABB69052, ABB72202, and ABB72203) shared 94% identity with tomato FALSIFLORA (AAF66101). FLORICAULA/LEAFY intron 2 (FLint2) revealed extensive inter- and intraspecific polymorphism. The search for putative cis-regulatory elements in FLint2 alleles returned SQUAMOSA/APETALA1 footprints whose distribution seems to differ in andigenum, demissum and tuberosum accessions.

The method of polymerase chain reaction was used to amplify a fragment of the LZ-NBS-LRR receptor kinase gene R1; the gene was transferred into potato (Solanum tuberosum) from its wild-growing relative S. demissum and confers the race-specific recognition of the pathogen Phytophthora infestans. To verify this method as a test for the presence of the late blight resistance gene R1, the amplified genome fragment was cloned from the potato hybrid comprising the germplasm of S. demissum. The primary structure of this fragment, which corresponded to the receptor domain of kinase, did not practically differ from the matching sequence in S. demissum. In addition, the method was verified by scoring the set of plant differentials, wherein the presence of R1 was established with race-specific Phytophthora isolates. By screening 70 potato cultivars, we established a significant relationship between the presence of the gene R1 fragment and the phenotypic characters of late blight resistance and late maturity. This evidence supports the idea that R1 was introgressed from short-day S. demissum into potato plants together with some gene(s) conferring late transition to flowering.

The MADS-box APETALA1 genes control plant transition to flowering and the floral morphogenesis proper. The experimental evidence of APETALA1 overexpression presumes that this class of genes can also directly affect time to flowering. We therefore cloned and compared homologs of APETALA1 class genes from potato ( Solanum tuberosum cultivars adapted to long day conditions) and its wild relative Solanum demissum , a short-day subtropical species. The homologs isolated from these plants belong to the subclass FRUITFULL . The inconsiderable variations in the primary structure of these homologs cannot explain the diverse photoperiodic reactions of particular Solanum genotypes.

A 2-kb genome fragment cloned from potato cv. Bastoneza closely resembles CONSTANS, the arabidopsis gene for the photoperiodic control of flowering, and its homologs in tomato, cottonwood, and barley. The potato homolog is immediately related to tomato CONSTANS 1; their exons and introns were 94 and 82% identical. Within the most variable middle region of CONSTANS protein and its homologs, asparagine- and glutamine-rich motifs are of particular interest as they are probably involved in transcription regulation.