Identification of barley accessions from the VIR collection carrying the <i>mlo11(cnv2)</i> powdery mildew resistance allele

Background. The search for barley (Hordeum vulgare L.) genotypes that carry effective genes for resistance to powdery mildew agent Blumeria graminis f. sp. hordei is a present-day issue for Russian plant breeding. The mlo11 allele that confers long-term protection of barley against the pathogen is rarely found among the varieties, approved for cultivation in the territory of Russia. There is no information on the occurrence among Russian varieties of another effective allele, mlo11 (cnv2), therefore, the search for its source is a current necessity. Materials and methods. Seven barley accessions from Ethiopia and 7 accessions from Japan have been tested for resistance to the northwestern population of the powdery mildew agent in the field and in laboratory conditions. To identify of the Mlo gene alleles, nucleotide sequences of the Stowaway-MITE (Miniature Inverted-repeat Transposable Elements) and the adjacent promoter fragments were determined. Results. Phytopathological tests in the field and greenhouse conditions, as well as molecular markers were used to study 14 barley accessions from Ethiopia and Japan. According to the preliminary tests, plants were resistant to powdery mildew. The highly effective allele of powdery mildew resistance mlo11 (cnv2) was for the first time identified in four barley accessions from Ethiopia, k-20087, k-20523, k-20524 and k-28126. Under field conditions, adult plants were resistant, and in the greenhouse they were moderately damaged by powdery mildew (1-2 points). The disease symptoms were similar to those described for the sample Eth295, a carrier of the mlo11(cnv2) allele variant: single pustules and the absence of necrotic spots on the leaves. The fragments of Stowaway-MITE and adjacent Mlo 5' promoter sequences were amplified in all 14 accessions. The amplicons were cloned and sequenced. The unique marker SNPs within the MITE and Mlo 5’ promoter sequences, i.e. the substitutions of cytosine by thymine in positions 262 and 452, were found only in k-20087, k-20523, k-20524 and k-28126. These accessions belong to different botanical varieties and differ from each other in a number of morphological features, i.e. they are not duplicates. Conclusions. The genotypes selected as a result of the study can serve as a source of the mlo11(cnv2) allele in breeding powdery mildew-resistant barley varieties

POLYMORPHIC VARIANTS OF SUNFLOWER <i> RFL-PPR </i> GENES AS MOLECULAR GENETIC MARKERS

A sample of 131 sunflower lines from VIR genetic collection which differed for the ability to suppression of the CMS phenotype was studied using molecular markers specific for the CMS PET1 associated mitochondrial locus orfH522 and Rf1 nuclear gene, and also for CAPS-markers developed on the bases of single nucleotide polymorphism of RFL-PPR genes homologs. With the use of hybridological analysis a tight linkage of HRG01 and HRG02 marker fragments with the Rf1 gene controlling pollen fertility restoration trait was confirmed. The allelic variants of the RFL-PPR genes sequences were associated with the allelic state of the Rf1 locus. However, the CAPS marker elaborated for the QHL12D20 fragment showed independent inheritance from pollen fertility restoration trait in the F2 of the hybrid VIR116xVIR740

Diagnostic value of <i>Rf1</i> gene molecular markers in sunflower

Background. Modern production of sunflower seeds is currently based on the cultivation of high-yielding heterotic F1 hybrids from crossbreeding of lines with cytoplasmic male sterility (CMS) of PET1-type and fertility restorer lines. The paternal parent serves as a donor of the nuclear Rf1 gene functional allele, which is responsible for pollen fertility restoration in F1 plants. The detection of carriers of the Rf1 locus recessive and dominant alleles using diagnostic molecular markers accelerates breeding of female and male parental lines for creating hybrids.Materials and methods. The material for the study included 75 lines of various origins from the VIR sunflower genetic collection as well as hybrids from crosses of VIR 116A sterile line with fertile lines differing in the type of cytoplasm (fertile or sterile) and the presence of molecular markers, most of which were linked to the Rf1 locus. For marker validation, two different approaches were used: either by analyzing associations between the ability of a line to restore pollen fertility and the presence of molecular markers in its genotype, or by estimating recombination frequency between the Rf1 locus and marker loci in four segregating hybrid populations.Results. According to the obtained results, no markers demonstrated 100% efficiency in the analysis of the sample of genotypes. The ORS511 marker was most frequently observed among the lines presumably carrying the dominant allele Rf1. Pollen fertility of F1 hybrids from interline crossings was 89-99%. The segregation for fertility/sterility in F2 fitted the theoretical ratio of 3:1 expected in case of the monogenic control of the trait. The markers HRG01, HRG02 and ORS511 were linked to the fertility restoration trait, with recombination rates between Rf1 locus and markers varying in different cross combinations. The analysis of VIR 116А × VIR 740 and VIR 116А × RIL 130 hybrids showed that among the marker loci studied, the ORS511 was closest to the Rf1 locus Rf1 (recombination frequency of 2.2 and 3.3%, respectively). The recombination rate between the Rf1 and ORS511 loci equaled 7.5% in the cross VIR 116А × VIR 210 and 8.9% in VIR 116 × VIR 195.Conclusion. The markers ORS511, HRG01 and HRG02 are the most efficient for the identification of alleles of the Rf1 gene and for the marker assisted selection in hybrid populations produced involving sunflower lines from the VIR collection

The trait-specific collection of large-seeded sunflower at VIR: ba source for breeding cultivars and hybrids

Background. In recent years, an increase has been observed in the share of sunflower seeds in the confectionery and food industries. There are 30 cultivars and only 3 hybrids for confectionery use among 839 zoned sunflower cultivars and hybrids listed in the State Register for Selection Achievements Admitted for Usage in 2023. Thus, the development of source material for breeding large-seeded cultivars and hybrids remains a relevant task.Materials and methods. The studied material included 90 accessions selected for their large seed size from VIR’s sunflower collection. The following characters were assessed: 1000 seed weight, days from sprouting to maturation, plant height, head diameter, cypsela color and size, and resistance to downy mildew. DNA markers were used to identify accessions with the Rf1 gene and CMS.Results. Accessions k-2818 (Primorsky Territory), k-3633, k-3748, k-3782 (China), and k-3578 (Ukraine) were the best in 1000 seed weight. Russian cvs. ‘Alekseyevsky Krupnoplodny 2’ (k-3552), ‘Gyar-Gyar’ (k-1589), ‘SPK’ (k-3426), and ‘Lakomka’ (k-3526) confirmed their large seed size. The sterile type of cytoplasm was identified in 12 accessions. Fourteen accessions with fertile cytoplasm had diagnostic markers of the nuclear Rf1 gene.Conclusion. The large-seeded cultivars developed at VNIIMK, ‘SPK’, ‘Lakomka’, and k-3782 from China were the best not only in their large seed size but also in downy mildew resistance. As a result of long-term studies, a trait-specific collection was established for the large seed size trait. It included 90 accessions that can be used in the development of confectionery cultivars. Large-seeded sunflower accessions carrying the Rf1 gene were identified. Some of them became the ancestors of donor lines for both pollen fertility restoration genes and large seed size

Alternaria leaf blight of clusterbean

The annual  legume  crop clusterbean (Cyamopsis tetragonoloba  (L.) Taub.) is a promising  crop for cultivation  in the south of Russia. In 2018, phytosanitary examinations of clusterbean fields (13 collection accessions) were conducted in five VIR branches (Krasnodar Territory, Dagestan,  Astrakhan and Volgograd Regions). At all points,  several types of leaf spots were observed on clusterbean leaves and symptoms of plant damage by fungi of the genus  Alternaria Nees prevailed.  Using microbiological  methods and  Next-Generation Sequencing (NGS) of the  nuclear  ribosomal internal  transcribed spacer  two (ITS2), the  species  composition of micromycetes was identified.  It was found  that the  micromycetes Alternaria spp. in different  ecological  and  geographical conditions of the  south  of Russia cause two main types of leaf spots: the typical (beige and brown round spots, usually accompanied by concentric zonality) and brown  spot (small brown  bulging  merging spots). Overwhelmingly  the damage to leaf tissues is caused  by the fungus  A. tenuissima (Nees &amp; T. Nees : Fr.) Wiltshire. A quite  numerous accompanying mycoflora (first of all, fungi of the genus  Fusarium Link) was also detected. A. cyamopsidis Rangaswami &amp; A.V. Rao, one of the most  harmful guar pathogens in the countries  where the main acreage is located  (India, Pakistan, USA), was not found on clusterbean fields in Russia. The accessions  of clusterbean differ in degree of damage by A. tenuissima that causes Alternaria leaf blight. Differential interaction of parasite  and plant  host was revealed.  Therefore, to prevent epiphytotics, varieties protected by non-identical resistance genes  should  be grown.  At all VIR branches, accessions  k-52568 (Argentina) and k-52569 (Pakistan) were weakly damaged by Alternaria leaf blight, and some  accessions  were resistant only in the environmental conditions of one or two experimental stations. The accessions  studied were heterogeneous  in pathogen resistance,  which  allows selecting  disease-resistant lines from most  of the  collection  accessions.  Thus, in various VIR branches, plants  without symptoms of disease  were selected from accessions  k-52571, k-52573 and k-52580, and seeds were collected to create new donors of disease resistance

Structural variability of sunflower gene for methionine-rich albumin SFA8

Background. The 2S albumins of sunflower and other oilseed plants possess a high nutritional quality, the defense activity against fungi diseases casual gents and also valuable functional properties. The major component of albumin fraction, the SFA8 protein consists of 103 amino acid residues among which methionine constitutes 15 Mole %. In the cultivated sunflower gene pool the SFA8 structural gene is represented by the two alleles the products of which have different isoelectric points and differ by the electrophoretic mobility, however molecular mechanisms of the polymorphism are still unknown. Results. The amplified sequences of the SFA8 gene from seven Helianthus annuus L. accessions and three accessions of wild Helianthus L. species from VIR collection were sequences. The intron of 258-303 bp length depending on the genotype was firstly found in the central part of the gene. The length of the first exon constitutes 99 bp, the second exon is of 210 bp length. The nucleotide and translated amino acid sequences are polymorphic among different genotypes. The line VIR 130 in which the two expressing SFA8 proteins, the normal polypeptide with isoelectric point (pI) approximately 6.0 (normal SFA8) and its allelic variant with pI 6.5 (variant SFA8) have been earlier revealed possesses two types of the SFA8 encoding sequence. In one sequence the substitution 108С—G is present that results in the substitution of the polar uncharged amino acid serine for the positively charged arginine and respectively in alteration of the protein charge and isoelectric point. The intron sequence is also polymorphic and characterized by the presence of indels of approximately 45 bp. The intron sequences of all accessions contain dinucleotides GT at the 5΄ end and AG at the 3΄ end which are characteristic for consensus sequences of splicing sites in the U2-type introns. The variants of the secondary structure of the SFA8 intron sequences of H. argophyllus Torr. &amp; A. Gray and all the analyzed H. annuus genotypes are similar and differ from those of H. petiolaris Nutt. and H. giganteus L. Conclusions. The data on the SFA8 gene sequence polymorphism are important understanding the molecular mechanisms of genotypic differences in biochemical and functional properties of the protein, and he revealed differences in the intron secondary structure can be important for understanding expression patterns of the protein

LOCAL BARLEY ACCESSIONS FROM ETHIOPIA RESISTANT TO POWDERY MILDEW

The mlo11 allele associated with the loss of function of the Mlo locus and controlling mildew resistance in the majority of modern barley cultivars was identified in Ethiopian barley landraces. Twenty-seven accessions from Ethiopia were studied during laboratory experiments which resulted in identifying k-5448 as an accession resistant to powdery mildew. Three accessions (k-8555, k-8682 k-17554) were heterogeneous by the studied trait. With the help of molecular markers we showed that the resistant components of the selected forms are protected by the mlo11 gene

Powdery mildew resistance of barley accessions from Dagestan

Powdery mildew caused by the parasitic fungus Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Marchal is one of the most common diseases of barley. Growing resistant varieties can significantly minimize harmful effects of the pathogen. The specificity in the interaction between the fungus and its host plant requires a continuous search for new donors of the resistance trait. The powdery mildew resistance of 264 barley accessions from Dagestan and genetic control of the trait in resistant forms were studied under field and laboratory conditions. Forty-seven barley lines carrying previously identified powdery mildew resistance genes were also examined. During three years, the experimental material was evaluated under severe infection pressure at the Dagestan Experiment Station of VIR (North Caucasus, Derbent). Juvenile resistance against the Northwest (St. Petersburg, Pushkin) pathogen population was evaluated in a climatic chamber. The genetic control of B. graminis resistance in the selected accessions was studied with the application of hybridological and molecular analyses. The level of genetic diversity of Dagestan barley for effective resistance to powdery mildew is very low. Only two accessions, VIR-23787 and VIR-28212, are resistant against B. graminis at both seedling and adult plant stages. The high-level resistance of breeding line VIR-28212 originating from barley landrace VIR-17554 (Ep-80 Abyssinien) from Ethiopia is controlled by the recessive gene mlo11. Accession VIR-17554 is heterogeneous for the studied trait, with the powdery mildew resistant genotypes belonging to two varieties, dupliatrum (an awnless phenotype) and nigrinudum (an awned phenotype). In accession VIR-23787, a recessive resistance gene distinct from the mlo11 allele was identified. This accession is supposed to be protected by a new, effective pathogen resistance gene

MOLECULAR MARKING OF SUNFLOWER LINES WITH DIFFERENT ABILITY TO SUPPRESSION OF THE CYTOPLASMIC MALE STERILITY PHENOTYPE

Ninety five lines of sunflower genetic collection differing by their ability to suppress the CMS phenotype were molecularly marked with the use of 7 primer pairs. Using the STS marker orfH522, a sterile (PET1) cytoplasmon was identified in 79 lines, which confirmed indirectly the presence of fertility restoration genes in their genotypes. The majority of these lines also have a complex of molecular markers linked to the Rf1 gene. The HRG01, HRG02 and STS115 markers showed the best diagnostic value in revealing the Rf1 gene in the examined material. The data on allelic variation of the microsatellite loci ORS224, ORS511 and ORS799 were obtained for the first time

ALLELIC DIVERSITY OF THE <i> Ppd </i> AND <i> VRN </i> GENES INVOLVED IN CONTROL OF THE DURATION OF SHOOTING-EARING STAGE IN DAGESTANIAN BARLEY ACCESSIONS

The period between shooting and earing stages was investigated in a group of 265 barley accessions from Dagestan. The accessions k-15008 and k-15013 were characterized by the highest development rate in South Dagestan. In Northwest Russia the early accession k-15027 was revealed. In 207 barley accessions the dominant and recessive alleles of the Ppd and VRN genes involved in control of the period between shooting and earing stages were identified with the use of allele-specific molecular markers. Twenty two groups with different allelic combinations of the loci were revealed. The most numerous groups possessed the Ppd-H1Ppd-H2vrn-H1 Vrn-H2vrnH3 and ppd-H1Ppd-H2vrn-H1Vrn-H2vrnH3 allelic combinations and included 59 and 91 accessions correspondingly