Exploiting a wheat EST database to assess genetic diversity

Expressed sequence tag (EST) markers have been used to assess variety and genetic diversity in wheat (Triticum aestivum). In this study, 1549 ESTs from wheat infested with yellow rust were used to examine the genetic diversity of six susceptible and resistant wheat cultivars. The aim of using these cultivars was to improve the competitiveness of public wheat breeding programs through the intensive use of modern, particularly marker-assisted, selection technologies. The F2 individuals derived from cultivar crosses were screened for resistance to yellow rust at the seedling stage in greenhouses and adult stage in the field to identify DNA markers genetically linked to resistance. Five hundred and sixty ESTs were assembled into 136 contigs and 989 singletons. BlastX search results showed that 39 (29%) contigs and 96 (10%) singletons were homologous to wheat genes. The database-matched contigs and singletons were assigned to eight functional groups related to protein synthesis, photosynthesis, metabolism and energy, stress proteins, transporter proteins, protein breakdown and recycling, cell growth and division and reactive oxygen scavengers. PCR analyses with primers based on the contigs and singletons showed that the most polymorphic functional categories were photosynthesis (contigs) and metabolism and energy (singletons). EST analysis revealed considerable genetic variability among the Turkish wheat cultivars resistant and susceptible to yellow rust disease and allowed calculation of the mean genetic distance between cultivars, with the greatest similarity (0.725) being between Harmankaya99 and Sönmez2001, and the lowest (0.622) between Aytin98 and Izgi01

Contrasting patterns of the 5S and 45S rDNA evolutions in the Byblis liniflora complex (Byblidaceae)

To clarify the evolutionary dynamics of ribosomal RNA genes (rDNAs) in the Byblis liniflora complex (Byblidaceae), we investigated the 5S and 45S rDNA genes through (1) chromosomal physical mapping by fluorescence in situ hybridization (FISH) and (2) phylogenetic analyses using the nontranscribed spacer of 5S rDNA (5S-NTS) and the internal transcribed spacer of 45S rDNA (ITS). In addition, we performed phylogenetic analyses based on rbcL and trnK intron. The complex was divided into 2 clades: B. aquatica–B. filifolia and B. guehoi–B. liniflora–B. rorida. Although members of the complex had conservative symmetric karyotypes, they were clearly differentiated on chromosomal rDNA distribution patterns. The sequence data indicated that ITS was almost homogeneous in all taxa in which two or four 45S rDNA arrays were frequently found at distal regions of chromosomes in the somatic karyotype. ITS homogenization could have been prompted by relatively distal 45S rDNA positions. In contrast, 2–12 5S rDNA arrays were mapped onto proximal/interstitial regions of chromosomes, and some paralogous 5S-NTS were found in the genomes harboring 4 or more arrays. 5S-NTS sequence type-specific FISH analysis showed sequence heterogeneity within and between some 5S rDNA arrays. Interlocus homogenization may have been hampered by their proximal location on chromosomes. Chromosomal location may have affected the contrasting evolutionary dynamics of rDNAs in the B. liniflora complex

Search for microsatellite markers associated with water-stress tolerance in wheat through bulked segregant analysis

We used bulked segregant analysis (BSA) to identify microsatellite markers associated with water-stress tolerance in wheat. Two DNA pools (tolerant and sensitive) were, established from the selected F-2 individuals of crosses between water-stress-tolerant and -sensitive wheat parental genotypes on the basis of the paraquat (PQ) tolerance, leaf size, and relative water content. All three traits were previously shown to be associated with water-stress tolerance on segregating F2 progeny of the wheat crosses used in this study. Microsatellite analysis was then performed on the established DNA pools, using 35 primer pairs that included all of the chromosome group 5 (5A, 5B, 5D) markers, to detect microsatellite fragments that were present, absent, or both in the DNA pools and their parental lines. We identified one microsatellite fragment that was present in tolerant parent wheat and the tolerant bulk but absent in the sensitive parent wheat and sensitive bulk. We then followed the segregation of this marker in the tolerant F2 individuals. Use of this marker may significantly enhance the success of selection for PQ- and water-stress-tolerant genotypes in wheat breeding programs

Critical points of fish for the localization of single and low copy sequences in plant chromosomes

Fluorescence In Situ Hybridization (FISH) is a powerful means by which single and low copy DNA sequences can be localized on chromosomes. Although great progress has been made in the last few years and some reports already exist for the detection of single and low copy sequences of only a few kilobase pairs in length, the detection of these sequences is still difficult in plants. An overview is presented here on the important and unclear points in FISH for the localization of single and low copy sequences in plants

High percentage of regeneration and transformation in chickpea

Efficient tissue culture and gene transfer systems of Cicer arietinum L. cvs. Red chickpea, Canitez 87 and MB-10 varieties were established. The explants were taken from the shoot primordium part of the mature embryo. For callus induction MS+0.1 mg/l NAA+ 0.5 mg/l 2,4-D and for plant regeneration MS+0.2 mg/l IAA+ 0.5 mg/l Kn+ 0.2 mg/l 6-BAP containing media were used. Best regeneration response was for the MB-10 variety (85%). Young and wounded regenerated shoots were co-cultivated with A. tumefaciens 4404 and A. rhizogenes 9402 both containing pBI-121 plasmids. Explants cocultivated with A. tumefaciens produced crown gall and plantlets on selective media. However, applications with A. rhizogenes produced hairy roots. The existence of GUS and NPT II genes in the DNA isolated from plantlets and hairy roots was confirmed with Southern blot and digoxigenin labeling. The best transformation response was in Canitez 87 (12.7%) with A. tumefaciens and MB-10 variety (10.4%) with A. rhizogenes

Tolerance to paraquat is correlated with the traits associated with water stress tolerance in segregating F-2 populations of barley and wheat

To identify scorable marker traits that can be used in cereal breeding programs for selecting drought tolerant individuals, we investigated the correlation among the drought-associated traits in two F-2 populations derived from the crosses made between drought tolerant and sensitive barley and wheat parental genotypes. The parental genotypes of these crosses also differed by at least three other traits - paraquat tolerance, leaf size, and the relative water content. These three traits were scored in two F-2 populations of 80 individuals for each barley and wheat cross. Analysis of results indicated that the enhanced tolerance to paraquat was correlated with reduced leaf size and increased relative water content, two traits associated with water stress phenotypes of the drought tolerant barley and wheat parents. Our results suggested that the selection based on paraquat tolerance is technically less demanding and thus useful for rapid screening of individuals for enhanced drought tolerance in segregating populations

High frequency plant regeneration from nodal explants of Paulownia elongata

High frequency of plant regeneration from Paulownia elongata was obtained on Murashige and Skoog (MS) medium and Woody Plant Medium (WPM), with appropriate supplements of growth regulators. Leaves, leaves with petioles, internodes and nodes excised from 3-month-old non-aseptically grown P. elongata were used as explants. The highest shoot regeneration efficiency (93.7%) was obtained from the nodes of P. elongata on MS medium supplemented with 0.1 mg/ml naphthaleneacetic acid (NAA) and 1 mg/ml 6-benzylaminopurine (BAP). The highest root formation efficiency (100%) from the regenerated shoots was obtained on WPM supplemented with 1 mg/ml indolebutyric acid (IBA). Rooted plantlets were transplanted to soil with a survival efficiency of almost 100%. The regeneration system reported here could be useful for rapid multiplication of elite genotypes of P. elongata in a short period of time

Investigations on biotechnologically developed poplar clones as raw material for pulp and paper industry

In this review an overview of our research on biotechnologically developed poplar clones for application in paper and pulp industry was presented. Nine native clones of Populus tremula as well as five hybrid clones of P. deltoides x P. deltoides were used for establishment of plant tissue culture system. For this purpose 'Woody Plant Medium' (WPM), supplemented with 1 mg/l zeatine (WPMZ) and WPM supplemented with 0.5 mg/l indole-3-butyric acid (IBA) (WPMB), were used for shoot and root regeneration, respectively. Established tissue cultures of poplar clones offer the potential for manipulation with enzymes involved in the lignin biosynthetic pathway such as caffeic acid O-methyltransferase and lignin peroxidases. Poplar clones (89M011, 89M066, Dursunbey 2) with the highest shoot regeneration efficiencies (70-100 %) were used for Agrobacterium transformation with antisense caffeic acid O-methyltransferase (pOMT8) and cationic peroxidase (Shpx6a) genes. Tobacco (Nicotiana tabacum) was used as a model control system. Constructs used in the study were obtained from forage legume Stylosanthes humilis and showed a high homology to corresponding caffeic acid O-methyltransferase (COMT) and peroxidase genes of poplar. Our results showed that antisense pOMT8 and Shpx6a peroxidase genes down regulated the activity of related enzymes. In the pOMT8 transgenics composition of lignin was changed, without changes in amount of lignin. However, in Shpx6a transgenics decreases in amount of lignin (up to 20 %) was observed. In the tobacco experiments with two types of transgenes (pOMT8 and Shpx6a) reduction of related enzymes activities and reduction of lignin amount was occurred in several transgenic plants