Gene-specific Molecular Markers Research Articles

Screening of Apple Cultivars for Scab Resistance in Kazakhstan

Scab, caused by Venturia inaequalis, is the most destructive fungal disease of apple worldwide. Apple scab incidence was studied in apple orchards in the south and southeast of Kazakhstan, including the Almaty, Zhambyl, and Turkestan regions, during 2022 and 2023. Disease incidence was higher in the Zhambyl region than in the Turkestan and Almaty regions in both years. The field evaluation suggested that 19 genotypes showed resistance to apple scab. Molecular screening was carried out using eight gene-specific molecular markers (AM19, CH05e03, OPL19, Hi07f02, AL07, K08, HB09, and CH02f06). The results of the molecular screening revealed that in 38 of the 45 studied cultivars, which included 11 Kazakh cultivars and 34 foreign cultivars, the Rvi (Rvi2, Rvi4, Rvi5, Rvi6, Rvi8, Rvi9, Rvi11, Rvi14, and Rvi15) resistance genes were amplified. Resistance genes such as Rvi2, Rvi4, Rvi6, and Rvi9 are still useful for breeding, but their use is recommended only in extended pyramids of multiple resistance genes. Several cultivars will be strong candidates for further breeding programs against apple scab and for the pyramiding of scab resistance genes in new cultivars.

Development of novel specific molecular markers for the Sw-5b gene to assist with tomato spotted wilt virus-resistant tomato breeding

Tomato spotted wilt virus (TSWV) is a plant pathogen that causes devastating tomato yield losses worldwide. The Sw-5b gene is one of the most effective resistance genes for TSWV control in tomato plants, and has been widely used in resistance breeding. Molecular markers are specific DNA sequences with known locations on the chromosome; they are indispensable tools in marker-assisted selection, which detects the presence of target genes to expedite breeding. We developed gene-specific molecular markers for Sw-5b to facilitate the accurate distinction of resistance (Sw-5bR) and susceptibility (Sw-5bS) alleles of Sw-5b. Using these markers, we successfully detected Sw-5b and determined its genotype (homozygous Sw-5bR, heterozygous Sw-5bR/S, or homozygous Sw-5bS) in six tomato varieties. Then we successfully applied these markers to 46 commercial tomato cultivars to detect and determine the genotype of Sw-5b. The results revealed a striking absence of the Sw-5bR gene and high TSWV susceptibility among most of the analyzed commercial cultivars. With the assistance of the novel Sw-5b-specific molecular markers, we generated a TSWV-resistant and homozygous Sw-5bR Micro-Tom tomato line, demonstrating the practical application of these markers in plant breeding. In summary, we developed novel gene-specific molecular markers for Sw-5b, and applied them to distinguish Sw-5b alleles for TSWV resistance or susceptibility. This marker set provides a valuable tool for breeding TSWV-resistant tomato varieties.

Evaluation of the Allelic Variations in Vernalisation (VRN1) and Photoperiod (PPD1) Genes and Genetic Diversity in a Spanish Spelt Wheat Collection.

Allelic variation within genes controlling the vernalisation requirement (VRN1) and photoperiod response (PPD1) determines the adaptation of wheat to different environmental growing conditions as well as influences other traits related to grain yield. This study aimed to screen a Spanish spelt wheat collection using gene-specific molecular markers for VRN-A1, VRN-B1, VRN-D1, and PPD-D1 loci and to phenotype for heading date (HD) in both field and greenhouse experiments under a long photoperiod and without vernalisation. Fifty-five spelt genotypes (91.7%) exhibited a spring growth habit, and all of them carried at least one dominant VRN1 allele, whereas five (8.3%) genotypes had a winter growth habit, and they carried the triple recessive allele combination. The Vrn-D1s was the most frequent allele in the studied set of spelt accessions, and it was found in combination with both the dominant Vrn-A1b and/or Vrn-B1a alleles in 88.3% of the spelt accessions tested. All spelt accessions carried the photoperiod-sensitive Ppd-D1b allele, which may explain the late heading of spelt germplasm compared to the commercial spring bread wheat Setenil used as a control. The least significant difference test showed significant differences between allelic combinations, the earliest accessions being those carrying two or three dominant alleles, followed by the one-gene combinations. In addition, the genetic diversity was evaluated through capillary electrophoresis using 15 wheat simple sequence repeat (SSR) markers. Most markers had high levels of polymorphism, producing 95 different alleles which ranged between 53 and 279 bp in size. Based on the polymorphic information content values obtained (from 0.51 to 0.97), 12 out of the 15 SSRs were catalogued as informative markers (values > 0.5). According to the dendrogram generated, the spelt accessions clustered as a separate group from the commercial bread wheat Setenil. Knowledge of VRN1 and PPD1 alleles, heading time, and genetic variability using SSR markers is valuable for spelt wheat breeding programs.

Screening of Indian wild apple accessions with scab and powdery mildew disease resistant gene specific molecular markers

Investigations on resistance to pathogens within local wild populations is the most efficient way for developing resistant cultivars in breeding programmes. In this study, Indian wild crab apple (Malus baccata Borkh var Himalaica) accessions maintained at two field gene banks in Himachal Pradesh were screened to find out the resistant sources. Individual trees were tested for the presence/absence of Vf, Vrl and Vbj scab resistant and Pl1 and Pl2 mildew resistant genes with eight and one SCAR markers respectively tightly linked to these genes. The potted plants were evaluated after the inoculation of pathogen. All the accessions showed diversity in relation to resistant gene to both the pathogens. All the SCAR markers K08, T06, Al07, Am19, ACS07, ACS09, Z13 and S22 were present in eight crab apple accessions. PLBJ SCAR marker linked with major genes Pl1 and Pl2 was detected in six accessions. Molecular marker assessment results almost corresponded to the data received in pot assay experiments thus were in good agreement. The study confirmed that these accessions carry scab and mildew resistant genes which could play important role in selection of desirable genotypes for apple breeding as well as use as suitable rootstock.

Effects of Starch Synthesis-Related Genes Polymorphism on Quality of Glutinous Rice.

Glutinous rice (Oryza sativa L.) quality includes thermal properties, retrogradation and pasting viscosity properties, and so on, which have little or no amylose. However, the genetic network regulation of different quality indices has not been systematically studied. The aim was to investigate the relationship between starch synthesis-related genes (SSRGs) and the physicochemical properties of glutinous rice by targeted-gene association analysis (TGAS). The genotypes of 17 SSRGs were analyzed using 46 gene-specific molecular markers in 63 glutinous rice accessions. TGAS and gene interactions analysis indicated that soluble starch synthase (SS) IIa, SSI, starch branching enzyme (BE) IIa, and pullulanase (PUL) had significant genetic effects on glutinous rice quality. SSI and SSIIa were the major genes that regulated thermal properties and retrogradation properties (RP). PUL was central in the regulation of gel consistency (GC), and it participated in the regulation of pasting viscosity parameters (PVP) except for the pasting time and the pasting temperature. BEIIb, ISA1, SSIVb, BEIIa, SSIVa, and their interactions with SSIIa regulated gelatinization temperature (GT) and PVP. The starch properties of glutinous rice are mainly controlled by SSIIa, SSI, PUL, and their interactions, but SSIIa is central among them. These findings indicate that starch properties in glutinous rice have a complex genetic system. It provides crucial information for promoting glutinous rice quality.

Morphological and Molecular Screening of Soybean Genotypes against Yellow Mosaic Virus Disease

Background: The growth and productivity of soybean are adversely affected by an array of biotic factors. Viruses are one of them as they cause great loss to the yield of soybean in India. The present study was conducted with an objective to identify yellow mosaic virus (YMV) resistant genotypes among the selected set of 53 soybean genotypes. Methods: The field screening was performed to identify YMV resistant genotypes. The field data was compared with molecular data recorded on the basis of gene specific SSR molecular markers. Result: During field study, 11 genotypes were found to be highly resistant, 26 resistant, 6 moderately resistant, 4 moderately susceptible, 3 susceptible, while three genotypes namely: JS335, JS 97-52 and RVS 2001-4 were found to be highly susceptible. In molecular analysis three genotypes viz.,: JS 20-29, JS 20-69 and JS 20-98 were found to be resistant against YMV. Among the polymorphic SSR markers the highest genetic diversity (0.4785) was observed with Satt554 while lowest genetic diversity (0.037) was observed with Satt308. Similarly polymorphism information content (PIC) was highest (0.364) in Satt554 and lowest (0.0363) in Satt308 among all polymorphic markers used for screening against YMV. The resistant genotypes identified in this study may be used as donor of resistance gene against YMV to develop improved genotypes which would stand as barrier against spread of the disease to newer areas and thus it can boost production and productivity of soybean in the country.

Development and validation of high-throughput and low-cost STARP assays for genes underpinning economically important traits in wheat.

We developed and validated 56 gene-specific semi-thermal asymmetric reverse PCR (STARP) markers for 46 genes of important wheat quality, biotic and abiotic stress resistance, grain yield, and adaptation-related traits for marker-assisted selection in wheat breeding. Development of high-throughput, low-cost, gene-specific molecular markers is important for marker-assisted selection in wheat breeding. In this study, we developed 56 gene-specific semi-thermal asymmetric reverse PCR (STARP) markers for wheat quality, tolerance to biotic and abiotic stresses, grain yield, and adaptation-related traits. The STARP assays were validated by (1) comparison of the assays with corresponding diagnostic STS/CAPS markers on 40 diverse wheat cultivars and (2) characterization of allelic effects based on the phenotypic and genotypic data of three segregating populations and 305 diverse wheat accessions from China and 13 other countries. The STARP assays showed the advantages of high-throughput, accuracy, flexibility, simple assay design, low operational costs, and platform compatibility. The state-of-the-art assays of this study provide a robust and reliable molecular marker toolkit for wheat breeding programs.

Ethylene and expansin biosynthesis related genes polymorphism in local apple (Malus domestica Borkh.) cultivars from VIR Collection of plant genetic resources

At Pushkin and Pavlovsk Laboratories of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) a diverse collec tion of local apple cultivars is maintained. Some of the cultivars are widely used in breeding programs for their ecological plasticity, increased adaptation to abiotic stress and disease resistance, still there have been no large-scale studies of these local cultivars for fruit storage ability. Fruit softening during storage is an important problem for apple production. Retention of desirable firmness after prolonged storage is one of the key requirements for new apple cultivars. Expansin and ethy lene biosynthesis related genes are known to be involved in control of fruit softening in apple, and gene specific molecular markers have been reported. In this study the polymorphism and allelic configuration of ethylene and expansin biosynthesis related genes Md-ACS1, Md-ACO1 and Md-Exp7 involved in control of fruit softening in 87 local apple cultivars from VIR Collection of Plant Genetic Resources were analyzed. PCR markers Md-ACS1, Md-ACO1 and SSR-marker Md-Exp7 were used in the study. The allele frequencies in the collection generally coincided with the data from previous studies. Md-ACS1 allele 2 associated with reduced ethylene production was found only in three local cultivars, while all the studied local cultivars were heterozygous for the Md-ACO1 locus, as well as most modern Russian apple cultivars. Half of the studied local cultivars were heterozygous for Md-Exp7 (198 : 202). Thirteen local cultivars with rare Md-Exp7 alleles (206, 210 and 212) were detected. No association was found between the Md-Exp7 genotype and the cultivars’ maturation time. The obtained results can be used for additional evaluation of the cultivars’ potential for breeding.

Virulence structure of Magnaporthe oryzae populations from Fujian Province, China

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. The aim of the present study was to elucidate the virulence structure of M. oryzae populations in Fujian Province, China, over the period 2006–2015. For this purpose, 456 M. oryzae isolates collected from diverse cultivars from eight rice cropping regions in Fujian were screened for the presence of 11 known avirulence genes: Avr-Pik, Avr-Pita1, Avr-Pita2, Avr-Pita3, PWL2, Avr1-CO39, ACE1, Avr-Piz-t, Avr-Pia, Avr-Pii and Avr-Pikm, with gene-specific molecular markers. The results showed that Avr-Pik and Avr-Pita3 occurred at the highest frequency (94.5% and 91.7%, respectively), while Avr1-CO39 and Avr-Pii were not detected. Further, the remaining avirulence genes occurred at frequencies ranging from 5.9% to 89.0%. Temporal population dynamics revealed that Avr-Pik was uniformly distributed in all 10 years, at frequencies of more than 87.5%. Spatial distribution analysis showed avirulence genes were present at different frequencies among the geographic regions. In addition, 24 rice monogenic lines of IRRI-Japan with known blast resistance genes were inoculated to assess the virulence of 60 isolates. The results revealed that the resistance gene Pik showed the broadest resistance spectrum to the isolates tested and therefore would be the most useful in rice blast resistance breeding. The resistance genes Pi-z5, Pi-1(1), Pi-kp, Pi-9(t), Pi-ta(1) and Pi-kh also were effective and would therefore also be of value to resistance breeding programmes. The present study provides information that should be useful for the development and deployment of rice blast resistant cultivars in Fujian Province.

Assessment of some genetic attributes in wheat (Triticum aestivum L.) using gene-specific molecular markers

Twenty-four wheat genotypes were characterized for the presence of three stress related genetic attributes using gene-specific molecular markers. Polymerase chain reaction (PCR) products of approximately 110 bp of 1RS rye chromosome fragment from 16 genotypes were obtained using specific primer pairs indicating the presence of translocation in these lines. The same genotypes were screened for the presence of dwarfing genes where 19 genotypes showed the presence of either the Rht-B1b or Rht-D1b allele having a semi-dwarf phenotype. Two of the genotypes (Kheri and Sufi) had wild type alleles in both the loci and two other genotypes showed the presence of double dwarf alleles (Rht-B1b + Rht-D1b). A 16.9 kDa HSP gene was characterized by validating a single-nucleotide polymorphism (SNP) linked with thermo tolerance in wheat. Thirteen of the 24 genotypes, which failed to amplify the specific PCR product due to the presence of an SNP, are expected to show tolerance for heat stress. Among the genotypes, Sonora-64, Balaka, Barkat, Aghrani and BARI Gom-24 (Prodip) tested positive for all the three markers evaluated (rye translocation, dwarfing genes and heat tolerance). These genotypes could be used to improve various stress tolerance attributes in wheat for future breeding programs.

Generation and analysis of expressed sequence tags (ESTs) of Camelina sativa to mine drought stress-responsive genes

Camelina sativa is an oil-producing crop belonging to the family of Brassicaceae. Due to exceptionally high content of omega fatty acid, it is commercially grown around the world as edible oil, biofuel, and animal feed. A commonly referred ‘false flax’ or gold-of-pleasure Camelina sativa has been interested as one of biofuel feedstocks. The species can grow on marginal land due to its superior drought tolerance with low requirement of agricultural inputs. This crop has been unexploited due to very limited transcriptomic and genomic data. Use of gene-specific molecular markers is an important strategy for new cultivar development in breeding program. In this study, Illumina paired-end sequencing technology and bioinformatics tools were used to obtain expression profiling of genes responding to drought stress in Camelina sativa BN14. A total of more than 60,000 loci were assembled, corresponding to approximately 275 K transcripts. When the species was exposed to 10 kPa drought stress, 100 kPa drought stress, and rehydrated conditions, a total of 107, 2,989, and 982 genes, respectively, were up-regulated, while 146, 3,659, and 1189 genes, respectively, were down-regulated compared to control condition. Some unknown genes were found to be highly expressed under drought conditions, together with some already reported gene families such as senescence-associated genes, CAP160, and LEA under 100 kPa soil water condition, cysteine protease, 2OG, Fe(II)-dependent oxygenase, and RAD-like 1 under rehydrated condition. These genes will be further validated and mapped to determine their function and loci. This EST library will be favorably applied to develop gene-specific molecular markers and discover genes responsible for drought tolerance in Camelina species.

Molecular identification and virulence analysis of AVR genes in rice blast pathogen, Magnaporthe oryzae from Eastern India

A set of 63 rice blast pathogen, Magnaporthe oryzae isolates from different cultivars in different rice growing regions of Eastern India was surveyed for the presence of nine known avirulence genes, Avr-Piz-t, Avr-Pita, ACE1, Avr-Pia, Avr-Pit, Avr1-CO39, Avr-Pi7, Avr-Pi15 and Avr-Pik with gene-specific molecular markers. These genes were detected with varying frequencies. Avr-Piz-t and Avr-Pik had the highest frequency (100 %) while Avr1-CO39 had the lowest (2 %). Frequencies of other Avr genes varied from 38 to 86 %. Spatial distribution analysis revealed almost uniform distribution of Avr genes in Eastern India. Out of 63 isolates, seven possessed the maximum of eight Avr genes, while a minimum of only two Avr genes were detected in another set of five isolates. Virulence analyses of 47 isolates revealed that many isolates possessing alleles of Avr genes were able to infect monogenic lines harbouring cognate R genes suggesting that such isolates might possess alternate mechanisms to escape host surveillance.

Analysis of effectiveness of R1-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of R1-nj expression.

R1-nj anthocyanin marker inhibition is highly frequent in tropical maize germplasm considerably affecting efficiency of haploid identification. Molecular markers reliably differentiating germplasm with anthocyanin color inhibitor have been identified in this study. The R1-Navajo (R1-nj) color marker facilitates easy and quick identification of haploid kernels at the seed stage during in vivo haploid induction process in maize. However, the Navajo phenotype can be completely suppressed or poorly expressed in some germplasm, making it impossible or inefficient to identify haploids at the seed stage. In this study, we characterized the expression of R1-nj marker in a large array of tropical/subtropical inbred lines, breeding populations and landraces by crossing with the R1-nj-based tropicalized haploid inducer. There was a high frequency of inhibition of the Navajo phenotype in the maize inbred lines, which are used in tropical breeding programs. Genome-wide association mapping showed that the C1 anthocyanin regulatory locus is the most significant genetic factor influencing inhibition of the Navajo phenotype. Molecular marker assays were designed based on polymorphism in the C1 vs C1-I alleles. Analysis of a set of 714 inbred lines demonstrated that a combination of two gene-specific markers--8 bp C1-I InDel and C1-I SNP--could predict with high accuracy the presence of anthocyanin color inhibition in the germplasm analyzed. Information generated in this study aids in making informed decisions on the constitution of source populations for doubled haploid (DH) line development in tropical germplasm, particularly those derived from elite maize lines from CIMMYT. The C1-I gene-specific molecular markers identified and validated will facilitate high-throughput and cost-effective evaluation of a large pool of germplasm for the presence of the dominant color inhibitor in maize germplasm.

Genomic variants of genes associated with three horticultural traits in apple revealed by genome re-sequencing.

The apple (Malus × domestica Borkh.) cultivar ‘Su Shuai’ exhibits greater disease resistance, shorter internodes and lighter fruit flavor compared with its parents ‘Golden Delicious’ and ‘Indo’. To obtain a comprehensive overview of the sequence variation in these three horticultural traits, the genomes of ‘Su Shuai’ and ‘Indo’ were resequenced using next-generation sequencing and compared to the genome of ‘Golden Delicious’. A wide range of genetic variations were detected, including 2 454 406 and 18 749 349 single nucleotide polymorphism (SNP) and 59 547 and 50 143 structural variants (SVs) in the ‘Indo’ and ‘Su Shuai’ genomes, respectively. Among the SVs in ‘Su Shuai’, 17 genes related to disease resistance, 10 genes related to Gibberellin (GA) and 19 genes associated with fruit flavor were identified. The expression patterns of eight of the SV genes were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of this study illustrate the genomic variation in these cultivars and provide evidence for a genetic basis for the horticultural traits of disease resistance, short internodes and lighter flavor exhibited in these cultivars. These results provide a genetic basis for the phenotypic characteristics of ‘Su Shuai’ and, as such, these SVs could serve as gene-specific molecular markers in maker-assisted breeding of apples.

Allele Characterization of Genes Required for rpg4-Mediated Wheat Stem Rust Resistance Identifies Rpg5 as the R Gene

A highly virulent form of the wheat stem rust pathogen Puccinia graminis f. sp. tritici race TTKSK is virulent on both wheat and barley, presenting a major threat to world food security. The recessive and temperature-sensitive rpg4 gene is the only effective source of resistance identified in barley (Hordeum vulgare) against P. graminis f. sp. tritici race TTKSK. Efforts to position clone rpg4 localized resistance to a small interval on barley chromosome 5HL, tightly linked to the rye stem rust (P. graminis f. sp. secalis) resistance (R) gene Rpg5. High-resolution genetic analysis and post-transcriptional gene silencing of the genes at the rpg4/Rpg5 locus determined that three tightly linked genes (Rpg5, HvRga1, and HvAdf3) are required together for rpg4-mediated wheat stem rust resistance. Alleles of the three genes were analyzed from a diverse set of 14 domesticated barley lines (H. vulgare) and 8 wild barley accessions (H. vulgare subsp. spontaneum) to characterize diversity that may determine incompatibility (resistance). The analysis determined that HvAdf3 and HvRga1 code for predicted functional proteins that do not appear to contain polymorphisms determining the compatible (susceptible) interactions with the wheat stem rust pathogen and were expressed at the transcriptional level from both resistant and susceptible barley lines. The HvAdf3 alleles shared 100% amino acid identity among all 22 genotypes examined. The P. graminis f. sp. tritici race QCCJ-susceptible barley lines with HvRga1 alleles containing the limited amino acid substitutions unique to the susceptible varieties also contained predicted nonfunctional rpg5 alleles. Thus, susceptibility in these lines is likely due to the nonfunctional RPG5 proteins. The Rpg5 allele analysis determined that 9 of the 13 P. graminis f. sp. tritici race QCCJ-susceptible barley lines contain alleles that either code for predicted truncated proteins as the result of a single nucleotide substitution, resulting in a stop codon at amino acid 161, a single cytosine indel causing a frame shift, and a stop codon at amino acid 217, or an indel that deleted the entire STPK domain. The three P. graminis f. sp. tritici race QCCJ-susceptible lines (Swiss landraces Hv489, Hv492, and Hv611) and the wild barley accession WBDC160 contain rpg5 alleles predicted to encode full-length proteins containing a nonsynonomous nucleotide substitution that results in the amino acid substitution E1287A. This amino acid substitution present in the uncharacterized C-terminal domain is not found in any resistant line and may be important to elicit the resistance reaction. These data suggest that rpg4-mediated resistance against many wheat stem rust pathogen races, including P. graminis f. sp. tritici race TTKSK, rely on the Rpg5 R gene; thus, rpg4- and Rpg5-mediated resistance rely on a common R gene and should not be considered completely distinct. The data also determined that Rpg5 gene-specific molecular markers could be used to detect rpg4-mediated wheat stem rust resistance for marker-assisted selection.

Molecular and phenotypic characterization of seedling and adult plant leaf rust resistance in a world wheat collection

Genetic resistance is the most effective approach to managing wheat leaf rust. The aim of this study was to characterize seedling and adult plant leaf rust resistance of a world wheat collection. Using controlled inoculation with ten races of Puccinia triticina, 14 seedling resistance genes were determined or postulated to be present in the collection. Lr1, Lr3, Lr10 and Lr20 were the most prevalent genes around the world while Lr9, Lr14b, Lr3ka and/or Lr30 and Lr26 were rare. To confirm some gene postulations, the collection was screened with gene-specific molecular markers for Lr1, Lr10, Lr21 and Lr34. Although possessing the Lr1 and/or Lr10 gene-specific marker, 51 accessions showed unexpected high infection types to P. triticina race BBBD. The collection was tested in the field, where rust resistance ranged from nearly immune or highly resistant with severity of 1 % and resistant host response to highly susceptible with severity of 84 % and susceptible host response. The majority of the accessions possessing the adult plant resistance (APR) gene Lr34 had a maximum rust severity of 0–35 %, similar to or better than accession RL6058, a Thatcher-Lr34 near-isogenic line. Many accessions displayed an immune response or a high level of resistance under field conditions, likely as a result of synergy between APR genes or between APR and seedling resistance genes. However, accessions with three or more seedling resistance genes had an overall lower field severity than those with two or fewer. Immune or highly resistant accessions are potential sources for improvement of leaf rust resistance. In addition, some lines were postulated to have known but unidentified genes/alleles or novel genes, also constituting potentially important sources of novel resistance.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9899-8) contains supplementary material, which is available to authorized users.

Characterisation of Latvia Fruit Crop Genetic Resources by Application of Molecular Genetics Methods

A large diversity of fruit crop accessions is maintained at the Latvia State Institute of Fruit- Growing, which consists of modern cultivars, landraces and selections from local breeding programmes, as well as germplasm that has resulted from scientific exchange and co-operation with other institutes. Presently, the germplasm collection comprises 2509 accessions of 17 fruit crops; 676 accessions are designated as national genetic resources. Conservation of germplasm itself has little value without characterisation and further utilisation of the stored plant material. To intensify these activities, DNA-based technologies have been implemented in the characterisation of germplasm. Two main groups of molecular markers have been utilised: non-specific markers and gene-specific (functional) markers, subsequently applicable for Marker Assisted Selection (MAS). Genotyping protocols based on SSR, RAPD and Methylation-sensitive amplification polymorphism (MSAP) markers have been developed for twelve fruit crops for use in plant material identification, True-to-Type verification and evaluation of genetic diversity and internal collection structure. In total, 790 accessions have been genotyped using any of the mentioned markers. These markers have been harmonised with the European cooperative programme for plant genetic resources working group (ECPGR WG) recommended sets to ensure international data exchange. Gene specific molecular markers have been applied to apple and pear (resistance to scab), strawberry (resistance to Gnomonia fragariae), sweet cherries and plums (self-incompatibility).

Development of gene-specific markers for acid soil/aluminium tolerance in barley (Hordeum vulgare L.)

Acid soil/aluminium toxicity is one of the major constraints on barley production around the world. Genetic improvement is the best solution and molecular-marker-assisted selection has proved to be an efficient tool for developing barley cultivars with acid soil/aluminium tolerance. In this study, barley variety Svanhals—introduced from CYMMIT (International Maize and Wheat Improvement Center)—was identified as acid soil/aluminium tolerant and the tolerance was mapped to chromosome 4H in 119 doubled haploid (DH) lines from a cross of Hamelin/Svanhals. The HvMATE gene, encoding an aluminium-activated citrate transporter, was selected as a candidate gene and gene-specific molecular markers were developed to detect acid soil/aluminium tolerance based on the polymerase chain reaction. Sequence analysis of the HvMATE gene identified a 21-bp indel (insertion–deletion) between the tolerant and sensitive cultivars. The new marker was further mapped to the QTL (quantitative trait loci) region on chromosome 4H for acid soil tolerance and accounted for 66.9 % of phenotypic variation in the DH population. Furthermore, the polymorphism was confirmed in other tolerant varieties which have been widely used as a source of acid soil tolerance in Australian barley breeding programs. The new gene-specific molecular marker provides an effective and simple molecular tool for selecting the acid soil tolerance gene from multiple tolerance sources.

Molecular analysis of native Manipur rice accessions for resistance against blast

The genetic diversity for resistance against blast, a major disease on rice, was assessed in the land race collections of Manipur, India, a region reported to be associated with origin of rice. The presence of eight well characterized genes, that is Pi-b, Pi-z, Piz-t, Pi-9, Pi-40, Pi-5, Pi-a and Pi-ta that were known to confer resistance to different isolates of blast were analyzed through polymerase chain reaction (PCR) assay using gene specific molecular markers. The characterization of one hundred and eight accessions revealed the presence of these genes in the range of two to seven confirming the rich diversity for this economically important trait in these native landraces. The genetic diversity for resistance appears to be associated with the high levels of incidence of blast in the hilly uplands of Manipur, a feature that characterizes the rice cultivation of this region. The natural gene pyramids having diverse combinations of resistant genes provide the breeders a great opportunity to transfer and deploy these genes to provide adequate security against this deadly disease of rice in India, a country characterized by highly varied agro-climates and diverse growing conditions. Key words: Manipur, genetic diversity, gene, blast disease.

Genetic analysis of starch paste viscosity parameters in glutinous rice (Oryza sativa L.)

Starch paste viscosity plays an important role in estimating the cooking, eating, and processing quality of rice. The inheritance of starch paste viscosity in glutinous rice remains undefined. In the present study, 118 glutinous rice accessions were collected, and the genotypes of 17 starch synthesis-related genes (SSRG) were analyzed by using 43 gene-specific molecular markers. Association analysis indicated that 10 of 17 SSRGs were involved in controlling the rapid visco analyzer (RVA) profile parameters. Among these, the PUL gene was identified to play an important role in control of peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), peak time (PeT), and paste temperature (PaT) in glutinous rice. Other SSRGs involved only a few RVA profile parameters. Furthermore, interactions between SSRGs were found being responsible for PeT, PaT, and BDV. Some of the RVA parameters, including PKV, HPV, CPV, CSV, and PaT, were mainly governed by single SSRG, whereas other parameters, such as BDV, SBV, and PeT, were controlled by a few SSRGs, functioning cooperatively. Further, three near-isogenic lines (NIL) of a japonica glutinous cv. Suyunuo as genetic background, with PUL, SSIII-1, and SSIII-2 alleles replaced with those of indica cv. Guichao 2, were employed to verify the genetic effects of the various genes, and the results were consistent with those obtained from the association analysis. These findings indicated that starch paste viscosity in glutinous rice had a complex genetic system, and the PUL gene played an important role in determining the RVA profile parameters in glutinous rice. These results provide important information for potentially improving the quality of glutinous rice.