Molecular screening of the pear collection maintained at the Maikop Experiment Station of VIR for scab resistance gene markers

Pear scab caused by Venturia nashicola is one of the most important diseases in pears in China. Better understanding of the relationships between soluble sugar and phenol contents in the leaves and pear scab will assist in developing resistant cultivars for management of this disease. However, such relationships were poorly understood. In this study, we determined soluble sugar and total phenol contents in the leaves of 29 pear cultivars with varying levels of scab resistance in May, June, August and September of 2012 and related their levels to pear scab resistance. Results of this study demonstrated that: 1) the changes in soluble sugar and total phenol contents in the leaves of interspecific pears appeared to be relatively stable in May, June and August. In September, however, significant changes in their levels were observed. The levels of soluble sugar content in the leaves of Pyrus bretschnrideri and P. communis were significantly lower. Total phenol content in the P. communis leaves reached the highest. 2) Differences in soluble sugar and total phenol contents in the leaves of pear cultivars with varying levels of scab resistance were insignificant in May, June, August, and September. Total phenol content in pear cultivars that were immune to scab was slightly higher than that in other resistant cultivars in May, June, August, and September. 3) Soluble sugar and total phenol contents in the leaves of different interspecific and scab-resistant cultivars had no significant correlations with scab incidence in May, June, August and September. 4) There was a negatively correlation (p=0.039, r=-0.386) between soluble sugar content in the leaves of different cultivars and scab incidence in August.

Scab caused by Venturia nashicola is one of the most serious diseases of Asian pears, including Japanese pear (Pyrus pyrifolia var. culta) and Chinese pears (P. bretschneideri and P. ussuriensis). Breeding scab-resistant pear cultivars is essential to minimize fungicide use and development of fungicide resistance. A survey of pathogenic specialization in V. nashicola is needed to ensure durable scab resistance in cultivated pears. V. nashicola race 1, 2, and 3 isolates, each differing in pathogenicity to Japanese pear cultivar Kousui and Asian pear strain Mamenashi 12, have been reported in Japan. In this study, isolates collected from scabbed pears in China and Taiwan were classified as V. nashicola based on conidial size and mating ability. However, various isolates had pathogenicity distinct from races 1, 2, and 3 according to tests on seven differential host genotypes of pear cultivars from Japan (Kousui and strain Mamenashi 12), China (Jingbaili, Yali, Linyuli, and Nanguoli), and Taiwan (Hengshanli). These new races were designated as races 4 to 7. Progenies characteristic of race 3 isolates were produced using a cross between race 1 and race 2 isolates, suggesting the possible role of sexual recombination in the emergence of novel races. Japanese pear cultivar Kinchaku and Chinese P. sinkiangensis 'Xiangli' (a Korla fragrant pear grown in China) did not show visible symptoms after inoculation with any of the seven races. Broad scab resistance in Kinchaku and Xiangli makes them a promising genetic resource for resistance breeding programs.

Genetic mapping of the pear scab resistance gene Vnlf using a pseudo-BC3 population derived from Japanese pear cultivars and European pear ‘La France’

Scab is one of the major fungal diseases infecting pear trees, causing the greatest economic losses. Identifying and pyramiding scab resistance factors should help in breeding new resistant pear cultivars. We have identified and mapped two new pear resistance loci against the fungal pathogen Venturia pirina. The first locus, mapped both as a major gene and as a QTL, is located on linkage group (LG) 01 of the hybrid P3480, deriving from the European pear cultivar ‘Wilder.’ It colocalizes with the Vnk resistance gene found in the Asian pear cultivar ‘Kinchaku’ against Venturia nashicola. A second locus, mapped as a QTL, is located on LG04 of the interspecific cultivar ‘Euras.’ In a small ‘Euras’ × P3480 progeny, seven seedlings carrying the resistant alleles at both loci have been selected using SSR markers.

Simple sequence repeat (SSR) markers were used to assess genetic diversity and relationship of Pyrus L. cultivars native mainly to East Asia. A total of 168 putative alleles were generated from six primer-pairs (BGA35, KU10, BGT23b, NH004a, NH011b and NH015a). All the SSR markers showed a high level of genetic polymorphism with a mean of 28 putative alleles per locus and the heterozygosity of 0.63. The Dice’s similarity coefficient between cultivars ranged from 0.02 to 0.98 and Occidental pears generally had low affinities to Asian pears. Ten major groups were generated from all the accessions by UPGMA clusters analysis. Chinese sand pears consisted of four groups with Chinese white pears and Japanese pears, of which Chinese sand pears occurred in all four groups, presenting a large genetic diversity, Chinese white pears were included in three groups, and Japanese pears only fell into one group. In the dendrogram, Chinese sand pears and Chinese white pears did not form discrete group, even subgroups. Some Japanese pear cultivars had high affinities to Chinese sand pear cultivars. These findings supports the authors’ previous viewpoints of Chinese white pears as a variety or an ecotype of Chinese sand pears (P. pyrifolia var. sinensis (Lindley) Y. Teng et K. Tanabe) and the progenitor of Japanese pears coming from China. Cultivars of P. ussuriensis Maxim. were clustered together with one clone of P. hondoensis Nakai et Kikuchi, a relative species of P. ussuriensis. Cultivars of P. communis L. and other Occidental species formed three independent groups and were distant from most Asian pears, except for P. betulaefolia Bge.

Identification and genetic mapping of novel resistance gene, Rvn4, for pear scab in Chinese pear

SummaryScab, caused by Venturia nashicola, is one of the most damaging diseases in pear. The development of varieties resistant to scab has long been an aim of Asian pear breeding programmes. The application of molecular markers to select scab-resistant plants will increase the efficiency of breeding programmes. To identify DNA markers linked to the scab-resistance gene named Rvn2 in PS2-93-3-98, the progeny of a cross between PS2-93-3-98 and ‘Yali’ pear was studied. As scab-resistance clearly segregated 1:1 in the F1 progeny, Rvn2 is a single dominant gene. Bulked segregant analysis and amplified fragment length polymorphism (AFLP) analysis were then applied to identify markers associated with Rvn2. Three AFLP markers linked to Rvn2 were identified. Mapping of the markers indicated that the three selected markers, E-AGT/M-CCA234, E-ATT/M-CCG328, and E-GGT/M-TCT217, were located 4.9, 3.2, and 0.8 cM from the Rvn2 locus, respectively. Rvn2 was found to be located in a different linkage group from previously identified scab-resistance gene loci. For marker-assisted selection of scab resistance, two of the AFLP markers (E-AGT/M-CCA234 and E-GGT/M-TCT217) were converted into cleaved amplified polymorphic sequence (CAPS) markers. These CAPS markers, designated PSC217-Xho I and PSC234-Hae III could distinguish between resistant and susceptible individuals.These CAPS markers therefore have the potential to increase the efficiency of selection for scab-resistance in pear breeding programmes.

Asian pear scab is a fungal disease caused by Venturia nashicola. The identification of genes conferring scab resistance could facilitate the breeding of disease-resistant cultivars. Therefore, the present study aimed to identify a scab-resistance gene using an interspecific hybrid population ((Pyrus pyrifolia × P. communis) × P. pyrifolia). Artificial inoculation of V. nashicola was carried out for two years. The segregation ratio (1:1) of resistant to susceptible individuals indicated that resistance to V. nashicola was inherited from P. communis and controlled by a single dominant gene. Based on two years phenotypic data with the Kruskal–Wallis test and interval mapping, 12 common markers were significantly associated with scab resistance. A novel scab resistance gene, Rvn3, was mapped in linkage group 6 of the interspecific hybrid pear, and co-linearity between Rvn3 and one of the apple scab resistance genes, Rvi14, was confirmed. Notably, an insertion in pseudo-chromosome 6 of the interspecific hybrid cultivar showed homology with apple scab resistance genes. Hence, the newly discovered Rvn3 was considered an ortholog of the apple scab resistance gene. Since the mapping population used in the present study is a pseudo-BC1 population, pyramiding of multiple resistance genes to pseudo-BC1 could facilitate the breeding of pear cultivars with durable resistance.

Monogenic scab resistance and columnar growth habit are important breeding traits of apple. The use of molecular markers very accurately determines the presence of necessary genes in the genome early during ontogenesis and reduces the time of selection. The purpose of this study was molecular genetic testing of initial forms of apple and hybrid seedlings of apple to identify carriers of the target alleles of genes for monogenic scab resistance (Rvi6) and columnar growth habit (Co) and the clarification of the pattern of inheritance of the Co and Rvi6 genes in hybrid progeny populations. This paper presents the results of molecular genetic analysis of varieties Valuta, Uspenskoe, Belarusskoe sladkoe and seedlings of the Valuta×Uspenskoe and Valuta×Belarusskoe sladkoe hybrid families for genes controlling columnar growth habit (Co) and scab resistance (Rvi6). The presence of the dominant allele of the Co gene was diagnosed with primers 29f1 and JWI1r flanking the 5’-end of the insert at the Co locus controlling columnar genotypes. Allelic status of the Rvi6 gene was determined with the AL07-SCAR marker mapping at about 0.2 cM from the Rvi6 locus. The correlation frequency of inheritance of the allelic states of these genes has been determined. Valuta×Uspenskoe crosse yielded 48.1 % columnar genotypes and 77.8 % scab-immune genotypes; Valuta×Belarusskoe sladkoe crosses, 46.8 % and 68.0 %, respectively. The observed segregation conforms to the expected Mendelian ratios: 1:1 for columnar habit and 3:1 for scab resistance. The joint inheritance of columnar growth habit and monogenic resistance to scab has been analyzed. The hybrid seedlings that had the dominant Co allele together with the Rvi6 gene in the homozygous dominant state (Rvi6 Rvi6) in their genome have been identified, which can significantly intensify the selection process and run it into 100 % of hybrid seedlings with monogenic scab resistance and up to 50 % of genotypes with columnar growth habit.

Differences in scab-resistance of Japanese, Chinese, and European pear cultivars were ex-amined by spraying with a conidial suspension (2 × 105 conidial•m1-1) of pear scab, Ventur-ia nashicola.The degree of scab resistance in Pyrus species or cultivars could be classified into four types: Highly Resistant (HR), no macroscopic evidence of infection on any leaves; Resistant (R), necrotic lesions on a few leaves but with no sporulation; Susceptible (S), sparsely sporulat-ing lesions on a few leaves; and Highly Susceptible (HS), abundance of sporulating lesions on several leaves.Differences in scab-resistance were observed among Pyrus species. All European pear cul-tivars (P. communis) were evaluated as HR; most Japanese pear cultivars and selected clones (P. pyrifolia) were rated as HS or S, although 'Kinchaku' and 'Okusankichi' were designat-ed as being HR and R, respectively. Pear cultivars classified as P. ussuriensis or P. pyrifo-lia which originated in China were evaluated as either HR or R. Cultivars of P. bretschneideri exhibited an entire range from HR to HS.

Background. The harmful organisms affecting potato cause great economic damage in all areas where the crop is grown. One of the most economically important pathogens is the potato late blight (agent Phytophthora infestans Mont. (de Bary)). Also, Potato Virus Y (PVY) and potato cyst nematode (PCN) of the Ro1 pathotype, the only one that occurs in the Russian Federation, cause significant yield losses. Materials and methods. Phytopathological and molecular screening was carried out on original interspecific potato hybrids obtained in 11 crossing combinations, seven of which were studied for the first time. Hybrids resulted from crosses that involved Mexican and South American potato species as sources of late blight and PVY resistance. Field observations of late blight resistance were conducted during three seasons under high infection pressure. A part of plant material was screened in laboratory tests. PCR tests employed 9 DNA markers of genes associated with resistance to Ph. infestans, PVY and PCN of Ro1 pathotype used in our previous study. Results. In field evaluation, hybrid clones mostly expressed high, and a part of them moderate late blight resistance. Of the 6 markers of the used Rpi genes, at least two were detected in most clones, while some had 3 or 4 markers. Some clones were found to have markers of PCN resistance genes and of several genes for resistance to PVY. The cytoplasm type was determined for all plant material. Conclutions. Within a small but pedigree-diverse set of interspeсific hybrids, the markers of resistance genes to all three pests were found. A part of clones obtained by using the sources of PVY and late blight resistance which we had identified, were found to contain two or more resistance genes to these pathogens. According to the results of molecular tests, the clones were found to carry several markers of late blight and PVY resistance genes. Original interspecific potato hybrids with determined late blight resistance levels, cytoplasm type and availability of markers of genes for resistance to three pests can be used in further hybridization.

Background. Unique apple cultivars have been created at the Sverdlovsk Horticultural Breeding Station and adapted to growing in the harsh natural and climatic conditions of the Middle Urals. Sharp temperature changes and a significant amount of precipitation in summer contribute to the development of fungal and bacterial diseases. Therefore, the main direction in breeding is the creation of forms with a complex of genes for resistance to various types of diseases. To search for sources of valuable features, the use of the molecular marker method remains relevant, which shortens the analysis time and allows for selection directly on the basis of gene presence instead of the external manifestation of а trait. The aim of our work was to search for scab and fire blight resistance genes in apple cultivars bred by the Sverdlovsk Breeding Station of Horticulture using DNA markers. The study used the VfC marker of the Rvi6 scab resistance gene, AE10-375 and GE-8019 QTL FBF7 markers of resistance to fire blight of apple trees. The results of molecular identification of the scab resistance gene were compared with those of evaluating the field resistance in the cultivars studied during the epiphytotic years. 21 apple cultivars were analyzed. Results. In the course of the research, the Rvi6 gene was identified in three apple cultivars – ‘Pervouralskaya’ ‘Aksyonaʼ and ʻBlagaya Vestʼ. An assessment of field resistance to scab showed that a number of cultivars were not affected. It was established that all varieties with a fragment indicating the presence of the Rvi6 gene were not affected by scab during epiphytotic years. The cultivars ʻTavatuyʼ, ʻRozochkaʼ, ʻDanilaʼ, ʻVEM Rozovyjʼ, and ‘Rodnikovayaʼ also showed no signs of the disease. The pedigree analysis showed that scab resistance donors carrying the Rvi5 gene were used to create these cultivars.In the analyzed collection, markers AЕ10-375 and GE-8019 QTL FBF7 of resistance to bacterial fire blight of apple trees were noted in almost the same number of cultivars (AЕ10-375 was identified in 12 accessions, and GE-8019 in 10). However, the presence of resistance is evidenced by the presence of two markers in one genotype. The five identified accessions are ʻIset Belaya’, ‘Pervouralskaya’, ʻAksyonaʼ, ʻSerebryanoye Kopyttseʼ, and ʻBlagaya Vest’. Conclusions. The performed research made it possible to establish the sources of valuable characters in apple cultivars bred in the Urals. The identified genotypes are promising for further use in breeding and industrial horticulture.

Potato virus Y (PVY) are among the most harmful viral pathogens that reduce the yield and quality of potatoes. The number of modern varieties resistant to a wide range of PVY strains is very limited, therefore, the selection of potatoes in this direction does not become irrelevant. Molecular markers of the Ry genes are universal tools for identifying new sources of resistance among existing biodiversity of potato genotypes. Since potato varieties and hybrids containing Rysto tend to exhibit cytoplasmic male sterility associated with mitochondrial DNA, the definition of cytoplasmic types is important. In the article, molecular markers of the Ry genes YES3-3A, YES3-3B, RYSC3, Ry186 were used for screening foreign and Russian varieties and hybrids potatoes from the collections of Lorch Potato Research Institute and N.I.Vavilov Institute of Plant Genetic Resources. Molecular screening and analysis of рedigree revealed that russian varieties and hybrids of potatoes characterized by extreme resistance to PVY were obtained on the basis of foreign varieties Alwara, Arosa, Bison, Bobr, Roko, as well as backcrosses of the Hungarian selection donors of the Rysto gene linked to cytoplasmic male sterility, and forms 128/6 a donor of the Ryadg gene, derived from S. stoloniferum. The marker RYSC3 coupled to Ryadg was found in interspecies hybrids of N.I.Vavilov Institute of Plant Genetic Resources 8-1-2004, 8-3-2004, 8-5-2004, 135-5-2005, 135-3-2005, having the same origin with the participation of S. okadae species K-20921 Hawkes et Hjerting and S. chacoense K-19759 Bitt. The marker Ry186 of the gene Rychc is rare. It is present in 5% of the potato genotypes. Molecular screening revealed samples of potatoes with markers of the Ry genes. They are of particular interest for further breeding. Data on the presence of Ry markers of genes in potato varieties and hybrids, serve as valuable information in the selection of initial forms for hybridization.

Swiss and more generally European apple (Malus × domestica) production is hampered by several diseases, the most destructive being fire blight, caused by Erwinia amylovora. On the other hand, there are apple scab, caused by Venturia inaequalis and powdery mildew, caused by Podosphaera leucotricha, which represent the major phytosanitary problems. Classical breeding has produced many scab and mildew resistant cultivars and efforts to breed also fire blight resistant cultivars are currently undertaken. Marker assisted selection (MAS) increases efficiency by allowing early non-destructive screening of seedlings and identifying genotypes showing pyramids of resistance genes. If the development of markers for MAS was the primary goal of genetic analysis in the 1990s, identification and cloning of resistance genes is now the goal. The first and until now the sole resistance gene which has been isolated and transformed into a susceptible apple cultivar is the gene HcrVf2 (Rvi6), responsible for the Vf scab resistance present in most classically bred scab resistant cultivars. Much effort is currently spent in the identification and positional cloning of other apple genes conferring resistance to apple scab and fire blight. In our labs, we identified the putative scab resistance gene Rvi15 and two fire blight resistance genes namely from ‘Evereste’ and Malus × robusta 5. The functionality of these candidate genes is currently under scrutiny by complementation experiments. However, the final goal is the creation of a product, e.g., an improved apple cultivar that is resistant to scab and fire blight. The ideal product would have advantages to the environment and producer, and should raise as little concern as possible with consumers. To accomplish this ‘ideal product’, we opted for the cisgenic approach by introducing the scab resistance gene HcrVf2 with its own regulatory sequences into the highly susceptible apple cultivar, ‘Gala’, through Agrobacterium transformation. All marker genes were eliminated after transformation. Similarly, we are currently introducing into both the readily developed cisgenic ‘Gala’ and in the untransformed ‘Gala’ the putative Malus own fire blight resistance gene candidates, aiming at both proof of functionality of the identified candidates and possibly at rapid development of a fire blight and scab resistant cisgenic apple.

A gene encoding a leucine-rich repeat receptor-like protein kinase (LRPK) was identified from Japanese pear (Pyrus pyrifolia var. culta) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The Japanese pear LRPKp protein was composed of a leucine-rich extracellular ligand-binding domain, a transmembrane-spanning segment, and a cytoplasmic kinase domain; it had the highest sequence homology with apple LRPKm and Arabidopsis receptor-like protein kinase (RLK). Southern blot analysis revealed that the LRPKp belongs to a multigenic family, and a polymorphism in restriction fragment length was observed among Asian and European pear cultivars. The LRPKp transcripts were present at a high level in leaves and at a low level in flowers, while no expression was detected in fruits. The expression pattern of LRPKp was also studied by real-time RT-PCR in the scab susceptible cv. Kousui and resistant cvs. Kinchaku and Flemish Beauty after challenge inoculation with Venturia nashicola, the causal agent of Japanese pear scab. Transcripts of LRPKp were transiently enhanced in both the susceptible Kousui and to a great extent in the non-host-resistant Flemish Beauty. However, in resistant Kinchaku, expression of this gene was only weakly enhanced. Moreover, treatment with a resistance inducer, acibenzolar-S-methyl (ASM), which significantly reduces scab severity on potted trees of Kousui, was also able to induce and to potentiate the expression of this gene after inoculation. This suggests that the LRPKp gene is possibly involved in ASM-induced resistance.