S. Landjeva

Molecular markers: actual and potential contributions to wheat genome characterization and breeding

The demands for increasing global crop production have prompted the development of new approaches relying on molecular marker technologies to investigate and improve the plant genome. The merits of molecular markers make them valuable tools in a range of research areas. This review describes novel approaches based on modern molecular marker technologies for characterization and utilization of genetic variation for wheat improvement. Large-scale genome characterization by DNA-fingerprinting has revealed no declining trends in the molecular genetic diversity in wheat as a consequence of modern intensive breeding thus opposing the genetic ‘erosion’ hypothesis. A great number of important major genes and quantitative trait loci have been mapped with molecular markers. Marker-assisted selection based on a tight linkage between a marker allele and a gene(s) governing a qualitative or quantitative trait is gaining considerable importance as it facilitates and accelerates cultivar improvement through precise transfer of chromosome regions carrying the gene of interest. The implementations of molecular markers in wheat genotyping, mapping and breeding complemented by specific approaches associated with the complex polyploid nature of common wheat are analyzed and presented.

Evaluation of Genetic Diversity Among Bulgarian Winter Wheat (Triticum aestivum L.) Varieties During the Period 1925–2003 Using Microsatellites

Simple sequence repeats (SSRs) were used to study the genetic diversity within old and modern Bulgarian winter wheat (Triticum aestivum L.) varieties released in 20th century. A set of 91 varieties were screened by 19 wheat microsatellite markers (WMS), covering 17 wheat chromosomes, and one secalin-specific marker for rye chromosome arm 1RS. A total of 136 allelic variants were detected at 22 loci, ranging from 2 to 11, with an average of 6.8 alleles per marker. For 7 markers, null alleles were detected. The occurrence of rare alleles (frequency <2%) was observed for 13 markers. The polymorphism information content (PIC) values of the markers ranged from 0.10 (WMS0165 on 4AS) to 0.81 (WMS0437 on 7DL) with an average of 0.51. Approximately 74% of the varieties, mostly non-commercial, showed heterogeneity, with an average level of 10.1%. For the majority of markers, the relative frequencies of alleles varied considerably among different groups of varieties, revealing the effects of different selection between breeding centres. Some alleles, present in old genotypes, were lost, and new alleles have been introduced into modern varieties. Genetic diversity values over different periods of release were high, starting at 0.64 for varieties developed before 1960 to reach 0.71 in 1990s, revealing no declining trends in the diversity due to breeding activity. The cluster analysis discriminated all varieties (except for two) and revealed distinct groups of old and modern varieties, released from the main breeding centres in Northern, Southern and Western Bulgaria.

The contribution of the gibberellin-insensitive semi-dwarfing (Rht) genes to genetic variation in wheat seedling growth in response to osmotic stress

The effects of various gibberellin-insensitive semi-dwarfing (Rht) alleles and background genotypes on the growth response of wheat seedlings to simulated low water potential were investigated. Four sets of near-isogenic lines, each consisting of six members (Rht-Bla+Rht-D1a (rht), Rht-B1b, Rht-B1c, Rht-Dlb, Rht-B1b+Rht-D1b and Rht-B1c+ Rht-D1b), and one set of five members (rht, Rht-B1b, Rht-B1c, Rht-B1d and Rht-Dlb) were germinated in the presence of polyethylene glycol (PEG). The growth responses were assessed by measuring the lengths of the longest root, coleoptile and longest leaf (shoot) and calculating the root length : shoot length ratio and a tolerance index (TI). Seedling growth was significantly affected by the allelic status at the Rht loci, background genes and the water potential. The PEG treatment had major effects on root and shoot growth. Coleoptile growth was mainly affected by the Rht alleles. There were significant interactions of the Rht allele and variety on the growth response to low water potential. Genotypes with longer roots, coleoptiles and shoots when grown in water, as determined by the Rht allelic status (rht, Rht-B1b and Rht-Dlb) and varietal background, had the highest TI and maintained this advantage under stress, while genotypes with smaller seedlings (Rht-B1c and Rht-B1c+Rht-D1b) when grown in water were more strongly affected.

Physiological Response of Wheat Seedlings to Mild and Severe Osmotic Stress

In the present study the physiological status of two wheat (Triticum aestivum L.) cultivars subjected to polyethylene glycol-induced dehydration is evaluated. Wheat seedlings were exposed to either 8-d-long mild (15% PEG) or 24-h-long severe (30% PEG) osmotic stress by immersing their roots in PEG-supplemented Knop nutrient solution. Relative water content in the leaves and the levels of free proline, malondialdehyde, and hydrogen peroxide were chosen as indicative parameters corresponding to the degree of stress of the treated plants. Electrolyte leakage from leaf tissues of control and stressed plants was compared in terms of the common parameter Injury index used for characterizing cell membrane stability. In addition, a model test system was established for preliminary stress evaluation based on the kinetics of ion leakage. Short-term exposure to higher concentration of PEG was considered to be more harmful than prolonged mild stress as judged by RWC, proline and hydrogen peroxide accumulation, and injury index. The two cultivars demonstrated more obvious dissimilarities under conditions of prolonged mild stress than under severe stress.

Genetic diversity assessment of Bulgarian durum wheat (Triticum durum Desf.) landraces and modern cultivars using microsatellite markers

The genetic diversity in a Triticum durum Desf. collection, consisting of 102 Bulgarian landraces, nine Bulgarian and 25 introduced cultivars was studied using 14 highly polymorphic microsatellite markers. A total of 100 alleles were identified, with an average of 7.14 alleles per marker. The gene diversity values (He) of the markers for the total samples ranged from 0.23 (WMS357 and WMS631) to 0.77 (WMS46), with an average of 0.52. Within the landraces that were collected from 18 sites in Southern Bulgaria showed 2–11 alleles per locus with an average of 6.07. The microsatellite analysis suggests that the genetic diversity among landraces is lower compared to the diversity levels for durum wheat in countries close to the main centers of wheat domestication. Breeding activities have caused significant reduction of the allelic polymorphism, elimination of rare alleles, and increase in the number of common alleles and the frequency of dominating alleles.

Copper Toxicity Tolerance in Aegilops and Haynaldia Seedlings

The seedling response to high Cu concentrations (1 and 10 μM CuSO4 . 5 H2O) was studied in Aegilops triuncialis, Ae. geniculata, Ae. cylindrica and Haynaldia villosa. The negative effect of Cu on the root growth was recorded at both concentrations, while the shoot growth was inhibited at 10 μM. The most tolerant was Ae. triuncialis, followed by Ae. geniculata. Ae. cylindrica and H. villosa were more sensitive.

Seedling growth under osmotic stress and agronomic traits in Bulgarian semi-dwarf wheat: comparison of genotypes with Rht8 and/or Rht-B1 genes

Early-season drought is an adverse environmental factor affecting seedling emergence and crop establishment in winter wheat grown in continental climates. The different semi-dwarfing (Rht, reduced height) genes exert various effects on seedling growth, plant height and productivity. The occurrence of the major Rht genes in 69 Bulgarian bread wheat cultivars was postulated by molecular markers. Cultivars carrying Rht-D1b+Rht8 (1), Rht-B1b/d (6), Rht-B1b/d+Rht8 (19), or Rht8 (43) were identified. The three latter groups were compared in respect to germination and seedling growth in response to polyethylene glycol-induced osmotic stress, plant height and main yield components. Cultivars carrying only Rht8 produced longer roots, coleoptiles and shoots, and had smaller root-to-shoot length ratio in non-stress and in osmotic stress conditions compared with those carrying Rht-B1b/d or Rht-B1b/d+Rht8. The same cultivars produced shorter culms, shorter spikes with fewer spikelets, reduced number and mass of grain in the main spike, and reduced 50-grain mass. The Rht genic effects were confirmed on Rht near-isogenic lines. The preferential semi-dwarfing allele selection aiming to combine the advantages of Rht8 and Rht-B1b/d alleles in specific eco-climatic conditions and to improve breeding efficiency in wheat is discussed.

Effects of chromosome substitutions on copper toxicity tolerance in wheat seedlings

Substitution of wheat (Triticum aestivum L.) chromosomes 7A, 1D, 3A, 3B, 3D, 4A and 4D of cultivar Cappelle Desprez by their homologues of cultivar Bezostaya-1 increased the seedling tolerance to high concentrations of copper (1 μM CuSO4⋅ 5 H2O). Substitution of chromosome 1A had negative effects on seedling tolerance.

Frost tolerance in winter wheat cultivars: different effects of chromosome 5A and association with microsatellite alleles

Frost tolerance of ten Bulgarian winter wheat (Triticum aestivum L.) cultivars (Milena, Pobeda, Sadovo-1, Enola, Kristal, Laska, Svilena, Russalka, No301 and Lozen) and five foreign cultivars (Mironovskaya 808, Bezostaya-1, Rannaya-12, Skorospelka-35 and Chinese Spring) was studied in two experimental seasons following natural cold acclimation and in one experiment carried out in controlled acclimation conditions. Considerable intercultivar variability in plant survival was observed after freezing at −21 °C following sufficient cold acclimation, or at −18 °C following insufficient or controlled acclimation. In seven cultivars, the effects of chromosome 5A on frost tolerance were investigated in their F2 hybrids with chromosome 5A monosomic lines of cultivars with high, intermediate and low frost tolerance. The effects of chromosome 5A depended on the stress severity and the genetic background of the hybrids and varied even in cultivars of similar frost tolerance and vernalization requirements. Effects of other chromosomes besides 5A on frost tolerance were assumed. The analysis of six microsatellite loci located in the interval from centromere to Vrn-1 on of chromosomes 5AL, 5BL and 5DL showed that the major loci determining frost tolerance in Bulgarian winter wheats were Fr-A2 on chromosome 5AL, and, to a lesser extent, Fr-B1 on chromosome 5BL. A strong association of the 176 bp allele at locus wmc327 tightly linked to Fr-A2 with the elevated frost tolerance of cvs. Milena, Pobeda, Sadovo-1, Mironovskaya-808 and Bezostaya-1 was revealed. Relatively weaker association between frost tolerance and the presence of the 172 bp allele at locus Xgwm639 tightly linked to Fr-B1 was also observed.

Chromosome N-banding polymorphism in Aegilops geniculata Roth.

The chromosome morphology and the variation in the N-band distribution along the chromosomes were studied in 13 accessions of the wild wheat Aegilops geniculata Roth (2n=28, genome formula UUM°M°). The karyotype consisted of four metacentric, five submetacentric, three subtelocentric, and two satellited pairs of chromosomes. The N-banding technique stained differentially all 14 chromosome pairs. The most prominent bands were observed near the centromeres and in the intercalary regions of both arms. Telomeric bands were found in only seven chromosomes. All chromosomes produced a specific banding pattern, permitting their identification. Polymorphism for presence or absence of particular bands was observed among the different accessions. The variable bands were located predominantly at the terminal and subterminal chromosome regions, and were also unevenly distributed over chromosomes. This polymorphism resulted in up to eight distinct banding pattern variants for each of the Ae. geniculata chromosomes. Each accession in this study showed a unique overall karyotype, sharing 0 to 8 chromosomes of identical banding pattern with the other accessions. A generalized idiogram of Aegilops geniculata is presented.