Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 7 (2002) No. 2A

APPLICATION OF MOLECULAR MARKERS IN STUDIES ON PLANTS

SEPTEMBER 25-29, 2002, WARSAW, POLAND

PART I

Volume 7 (2002) pp 399
Title	PREFACE
Authors	The Organizing Committee
	Dear Colleagues! We are very pleased to inform you that the idea to organize an international meeting on molecular markers and their application in plant science was found highly interesting and important by many scientists from all over the world – the USA, Israel, India, Iran, Japan and most European countries. Thanks to the very fruitful co-operation that was established between the organizers, participants, referees and finally the publisher, we are pleased to present two successive issues of the Cellular & Molecular Biology Letters Journal, where the manuscripts announced for the conference “APPLICATION OF MOLECULAR MARKERS IN STUDIES ON PLANTS” and accepted for publishing by the international scientific board are enclosed. In total, more than fifty papers written by scientists from many European Countries, the USA and the Far East were reviewed and assembled according to the date of acceptance. Thus they were not divided into the sections of the meeting, where the presentations will appear. Although most of the papers are devoted to specific applications of various molecular systems, interested readers may also find more general papers on molecular mapping, molecular breeding, marker assisted selection, the evaluation of germplasm collections, molecular taxonomy and biotechnology. A broad range of techniques based both on DNA (RFLP, RAPD, AFLP, SSR, SNP etc.) and RNA (DDRT-PCR, microchips) technologies are discussed, giving insight into the state-of-the-art of plant science. Hopefully, this will make the material interesting both for specialists in a given field, and for those who wish to improve their knowledge or gain information on a specific point. Both issues of the CMBL journal are fully prepared, therefore you will be able to read the articles before the presentations begin. We believe that this will significantly stimulate discussion during the oral and poster sessions, and thus, make the conference more fruitful. Unfortunately, not all the participants presented their work in a form of paper. Hence, for your convenience, the appropriate abstracts are also available as a separate edition, in the conference materials available to registered participants only. We would like to express our thanks to the referees from all over the world who supported our efforts and without whom the materials would not have been ready on time. We would also like to thank Ms Małgorzata Nietubyć for her excellent editorial work. Finally, we are grateful to our sponsors both from Poland and abroad, whose financial support allowed us to decrease costs and partially sponsor some of the lecturers and students. Wishing you a fruitful and enjoyable conference.

Volume 7 (2002) pp 403 – 416
Title	NEW DNA MARKERS FOR DISCRIMINATION BETWEEN CLOSELYRELATED SPECIES AND FOR THE RECONSTRUCTION OF HISTORICAL EVENTS; AN EXAMPLE USING LIVERWORTS
Authors	Zofia Szweykowska-Kulińska*, Andrzej Pacak and Kamila Jankowiak
Abstract	A survey of fully-sequenced chloroplast genomes revealed that in land plants there are six tRNA genes that have introns. Moreover, the length of a particular tRNA gene intron remains relatively stable across species. However, in algae, the presence of chloroplast tRNA genes containing introns is exceptional. A survey of mitochondrial plant genomes revealed introncontaining tRNA genes are rather rare features, with the exception of tRNASer GCU genes in liverworts and peat-mosses. We isolated and sequenced one mitochondrial and three chloroplast intron-containing tRNA genes and a fragment of the mitochondrial coxIII gene containing the first intron from the following liverwort species: Pellia borealis, Pellia epiphylla-species N, Pellia epiphylla-species S and Porella baueri, Porella cordaeana, Porella platyphylla. We showed that, as in the case of higher plants, the rate of nucleotide substitution is lower in the mitochondrial genome than in the chloroplast genome. Moreover, the comparison of intron nucleotide sequences enabled us to show that in the case of one allopolyploid species, Pellia borealis, organelles were transmitted from one parent species, Pellia epiphylla-species N. In the case of another allopolyploid species, Porella baueri, organelles were also inherited from one parent species, Porella cordaeana. Therefore, organellar inheritance in liverworts seems to be uniparental. It remains clear that analysis of carefully chosen chloroplast and mitochondrial DNA sequences allowed us to reconstruct historical events.
Adress and Contact Informations	Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Międzychodzka 5, 60-371 Poznań, Poland * Corresponding author, E-mail: zofszwey@main.amu.edu.pl, Fax: (+4861)8292730

Volume 7 (2002) pp 417 – 422
Title	INTERVAL MAPPING OF QTLs CONTROLLING SOME MORPHOLOGICAL TRAITS IN PEA.
Authors	Lidia Irzykowska, Bogdan Wolko and Wojciech K. Święcicki
Abstract	A linkage map of pea was constructed based on a 104 RIL population derived from the cross combination Wt10245 x Wt11238. The map, which consisted of 204 morphological, isozyme, AFLP, ISSR, STS, CAPS and RAPD markers, was used for interval mapping of the QTLs controlling the stem length and internode number of pea. In the characterization of a given QTL, we included an identification of its position with reference to the flanking markers, an estimation of the part of variance explained by it, and a determination of gene action. Six QTLs per trait were identified as demonstrating linkage to ten intervals on five linkage groups. As many as seven QTLs influencing the analysed traits were mapped on linkage group II, indicating the important role of this region of the pea genome in plant height control.
Adress and Contact Informations	Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland

Volume 7 (2002) pp 423 – 429
Title	GENE AND GENOME MAPPING IN CEREALS
Authors	Andreas Börner
Abstract	The trait flowering time regulated by genes determining vernalisation and photoperiod sensitivity was used as an example for presenting data on comparative major gene and QTL mapping within the Triticeae. The major genes are shown to be members of homoeologous series. Furthermore it was demonstrated that in genome regions carrying major genes also QTLs for the same traits were detected.
Adress and Contact Informations	Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany

Volume 7 (2002) pp 431 – 436
Title	*THE GENETIC DIVERSITY OF APULIAN APRICOT GENOTYPES (Prunus armeniaca L.) ASSESSED USING AFLP MARKERS*
Authors	Luigi Ricciardi¹, Vito Giorgio², Claudio De Giovanni¹, Concetta Lotti³, Alessandra Gallotta² and Girolamo Fanizza¹
Abstract	Apricot is an important crop in Italy and, especially in Southern regions, in the last five years numerous plantings using new cultivars and appropriate cultural management have been established. The cultivars available were created in different environments (USA, France, New Zealand, etc), they then often show low adaptability to Italian conditions. However, in the South of Italy, it is still possible to safeguard and to exploit a considerable amount of the apricot genetic variation available in ecotypes often characterised both by useful bio-agronomic traits and by good environmental adaptation. These genetic materials could be used in breeding programs aimed at broadening the harvest period and obtaining high fruit quality and resistance to the main biotic and abiotic stresses.
Adress and Contact Informations	¹Department of Agroforestry, Environmental Biology and Chemistry, Section of Genetics and Plant Breeding, University of Bari, Amendola 165/A; 70126, Bari, Italy, ²Department of Vegetal Production Science, University of Bari, via Amendola 165/A; 70126, Bari, Italy, ³Agricultural Faculty, University of Foggia, via Napoli 26; 71100, Foggia, Italy.

Volume 7 (2002) pp 437 – 444
Title	GENETIC INTEGRITY OF EX SITU GENEBANK COLLECTIONS
Authors	Sabina Chebotar¹, Marion S. Röder¹, Viktor Korzun² and Andreas Börner¹
Abstract	The genetic integrity of four accessions of the cross-pollinating species rye (Secale cereale L.) was investigated. Seeds available from the first and most recent regeneration cycles, multiplied 8, 12 (twice) or 14 times were fingerprinted using microsatellite markers. In all four accessions the allele numbers and frequencies changed after regeneration. Alleles present in the original seed sample were not detectable in the regenerated populations, whereas on the other hand, alleles were found in the recent seed sample, which were not observed in the investigated plants of the original one.
Adress and Contact Informations	¹Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany, ²Lochow-Petkus GmbH, PF 1197, D-29296 Bergen, Germany

Volume 7 (2002) pp 445 – 448
Title	THE USE OF MOLECULAR MARKERS IN APPLE BREEDING FOR DISEASE RESISTANCE
Authors	Marta Stankiewicz, Emilian Pitera and Stanisław W. Gawroński
Abstract	Molecular markers have become a useful tool simplifying and speeding up breeding work. They are also helpful in the genetic analysis of complex agronomic traits. The investigations concern the use of already available SCAR markers for molecular analysis of breeding materials. The markers are the apple powdery mildew resistance gene Pl2, originating from Malus zumi, and the apple scab resistance gene Vf, from M. floribunda 821. The clone U 211 was found to be highly resistant to mildew under field conditions and transmitted a high level of resistance to the majority of its progeny. The presence of the Vf gene conferring resistance to scab was confirmed by molecular analysis. As the molecular markers for the Pl2 gene are not present in the DNA of U 211, it is probable that the clone U 211 is a new source of mildew resistance.
Adress and Contact Informations	Department of Pomology and Basic Natural Sciences in Horticulture, Warsaw Agricultural University – SGGW, ul. Nowoursynowska 166, 02-787 Warsaw, Poland

Volume 7 (2002) pp 449 – 455
Title	THE MAPPING OF QTLs FOR CHLOROPHYLL CONTENT AND RESPONSIVENESS TO GIBBERELLIC (GA3) AND ABSCISIC (ABA) ACIDS IN RYE
Authors	Paweł Milczarski and Piotr Masojć
Abstract	Genetic maps containing molecular markers are useful tools for the identification of genes underlying quantitative traits (QTLs). Three traits important for plant physiology, i.e. chlorophyll content, sensitivity to GA and sensitivity to ABA, were evaluated for 99 F2 families of the DS2 x RXL10 rye mapping population. The observed variation in the chlorophyll content in rye leaves was shown to be under the genetic control of four independent QTLs. They were located on the following chromosome arms: 1RL (QChc-1R.1), 3RS (QChc-3R.1), 4RL (QChc-4R.1) and 5RL (QChc-5R.1) The marker-assisted selection of recombinants comprising positively-acting alleles at these loci may significantly increase the chlorophyll content in rye leaves, which should result in a higher efficiency of photosynthesis. A decreasing of plant height in rye is one of the major aims of breeders selecting for lodging resistance. The polymorphism of genes controlling sensitivity to gibberellic acid may be the basis for a potential selection strategy. Three QTLs underlying the sensitivity of rye seedlings to GA were located on chromosomes 5RL (QGar-5R.1), 1RL (QGar-1R.1) and on 7RL (QGar-7R.1). The dwarfing allele of a Dw1 locus present in line RXL10 was mapped in the same position as the QGar-5R.1 gene. Sensitivity to ABA plays an important role in imposing dormancy in rye grain. Three QTLs affecting sensitivity to ABA were found. They were mapped on chromosomes 1RS (QAbr-1R.1), 2R in the centromeric region (QAbr-2R.1) and on 5RL (QAbr-5R.1). QAbr-5R.1, Dw1 and QGar-5R.1 are located in the same position on the distal part of the 5RL chromosome arm, which suggests that they belong to a single locus for a major regulatory gene controlling the development of rye plants.
Adress and Contact Informations	Department of Genetics and Plant Breeding, Agricultural University, Słowackiego 17, 71-434 Szczecin, Poland

Volume 7 (2002) pp 457 – 463
Title	AFLP-PROFILING OF LONG-TERM STORED AND REGENERATED RYE GENEBANK SAMPLES
Authors	Katarzyna Joanna Chwedorzewska¹*, Piotr Tomasz Bednarek¹, Jerzy Puchalski¹ and Paweł Krajewski²
Abstract	The aim of these studies was to analyse the genetic changes induced by natural aging during long-term seed storage of rye. For this purpose, the AFLP (Amplified Fragment Length Polymorphism) technique was applied. In the experiment, DNA variation was demonstrated in seven-day-old seedlings of four seed samples of cv. Dańkowskie Złote, showing different levels of viability following long-term storage. Among the 362 AFLP fragments analysed, 73 had significantly different frequencies in at least one of the series. Principle Coordinate Analysis (PCA) based on molecular data revealed differences between the progenies of naturally aged seed samples with variable initial viability. It was clearly shown that materials with low viability differed in structure from highly viable ones, and that the population changes exhibited in the first case are preserved through regenerations. Although changes that were observed for initially viable samples were not so significant, they still occurred – probably as a result of genetic shift.
Adress and Contact Informations	¹The Botanical Garden – the Centre for the Conservation of Biological Diversity of the Polish Academy of Sciences, Prawdziwka 2, 02-973 Warsaw, Poland ²Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, Poznań, Poland

Volume 7 (2002) pp 465 – 469
Title	THE GENETIC STRUCTURE OF TETRAPLOID AVENA: A COMPARISON OF ISOZYME AND RAPD MARKERS
Authors	Mohammed Benchacho^1,2, Rosana Guma², Marcelino Pérez De La Vega² and Pedro García²
Abstract	Isozymes were the first widely used molecular markers in plant population analysis. They yielded valuable information on the amount and the structure of genetic variability. DNA technology has provided new types of markers based on DNA sequence, which make it possible to study polymorphisms in a much greater proportion of the genome. This is the reason why the use of isozymes is less popular nowadays. This effect would be justified if all markers provided the same type of information on polymorphism and genetic relationships among populations; otherwise, it would be necessary to use different markers to obtain the complete picture of the genetic structure of populations and species. In this study, we compared data of isozyme and RAPD markers in the populations of two tetraploid species of wild oats: Avena barbata populations collected in Argentina, and Avena murphyi populations collected in Spain and Morocco. The samples were evaluated for 9 isozymatic systems and 10 primers. The structure of genetic variability was studied using Nei’s method, and the relationships between populations were estimated using Hedrick and Jaccard's similarities for isozymes and RAPDs, respectively. As expected, RAPDs were more polymorphic than isozymes, but the information obtained from both markers was weakly correlated. The various reasons for this observation are discussed, but our conclusion is that in order to study the structure of genetic variability, several types of markers should be used.
Adress and Contact Informations	¹Université Ibn Tofaïl, Faculté des Sciences, Département de Biologie; 14000, Kénitra, Morocco, ²Universidad de León, Facultad de Biología, Departamento de Genética; 24071 León, Spain * Corresponding author, E-mail: degpgg@unileon.es

Volume 7 (2002) pp 471 – 475
Title	THE GENETIC DIVERSITY OF COMPONENTS OF RYE HYBRIDS
Authors	Andrzej Rafalski¹, Lucjan Madej², Iwona Wiśniewska¹ and Małgorzata Gaweł¹
Abstract	The genetic variability between 5 open-pollinated varieties of rye (Secale cereale L) and between the components of rye hybrids was estimated using PCR-based marker analysis. The 22 maternal single crosses and 11 restorers were the components of hybrids investigated in the preofficial trials at the Plant Breeding and Acclimatization Institute in Radzików (PBAI), the Danko Breeding Co. Ltd (DBC) and in the Poznań Breeding Co. Ltd (PBC) during the 2001 growing season. The PCR system using semispecific primers targeting the intron-exon junction sequences of plant genes was applied for the evaluation of the genetic diversity of rye breeding materials. The genetic distances between varieties were relatively low; the coefficients of dissimilarity did not exceed 0.15. The highest average distance between maternal hybrids and restorers (0.28) was found in materials from PBAI, whereas in materials from PBC and DBC the average distances were lower and reached 0.21 and 0.20 respectively. The cluster analysis based on PCR data indicated that hybrid components that originated from different breeding centres exhibited different genetic characters.
Adress and Contact Informations	¹Dept. of Plant Biochemistry and Physiology, ²Dept. of Plant Breeding and Genetics, Plant Breeding and Acclimatization Institute, Radzików, 05-870-Błonie, Poland

Volume 7 (2002) pp 477
Title	THE GENETIC DIVERSITY OF BARLEY CULTIVARS FROM THE BALTIC STATES AND BELARUS, AS DETERMINED BY RAMPS
Authors	Donatas Žvingila and Violeta Popendikytė
Abstract
Adress and Contact Informations

Volume 7 (2002) pp 483 – 491
Title	THE PRODUCTIVITY CHARACTERISTICS OF SUBSTITUTED BARLEY LINES WITH MARKED CHLOROPLAST AND MITOCHONDRIAL GENOMES
Authors	Inna M. Goloenko, Natalia V. Lukhanina, Andrey M. Shimkevich, Elena A. Aksyonova, Nina G. Danilenko and Oleg G. Davydenko*
Abstract	We present the effects of cytoplasm substitution on five productivity traits in an alloplasmic barley collection. 60 lines combining 5 nuclear genomes of cultivated barley varieties and 12 plasmons of two barley species (H. vulgare, H. spontaneum) displayed various effects depending on definite nucleicytoplasm combinations. Only four cytoplasmic genomes (W1, W4, W5, W10) significantly modified the expression of the nuclear genes controlling productivity. RAPD-PCR analysis revealed that both the mitochondrial and chloroplast DNA of the W1, W5, and W10 lines have common molecular characters distinguishing them from the cytoplasmic genomes of the other lines. The cytoplasmic genetic factors influencing the expression of "productivity" genes remain elusive.
Adress and Contact Informations	Institute of Genetics and Cytology, Belarus Academy of Sciences, Staroborisovski tract, 34, 220141, Minsk, Belarus * Corresponding author, E-mail: soyanort@home.by

Volume 7 (2002) pp 493 – 498
Title	*APPLICATION OF GISH AND AFLP TECHNIQUES FOR IDENTIFICATION OF LOLIUM-FESTUCA* INTROGRESSIONS**
Authors	Maria Skibińska¹, Arkadiusz Kosmala¹, Michael W. Humphreys² and Zbigniew Zwierzykowski¹
Abstract	At present, breeding programmes aimed at combining advantageous traits within the Lolium-Festuca complex, are mainly focused on introgression procedures. One principal objective, is the transfer of genes conferring resistance to abiotic stresses from Festuca species (F. pratensis, F. arundinacea and F. glaucescens) into Lolium multiflorum and L. perenne germplasm. In our experiments, two different hybrids: triploid – L. multiflorum (4x) × F. pratensis (2x) and pentaploid – F. arundinacea (6x) × L. multiflorum (4x) were backcrossed twice onto L. multiflorum cultivars, and numerous BC2 progeny generated. BC2 plants from both combinations were tested in field and/or simulated conditions for winter hardiness and drought resistance. GISH (genomic in situ hybridisation) analyses were then performed on the most winter hardy and drought resistant plants to locate putative genes for stress resistance. Using resistant L. multiflorum genotypes with a single Festuca chromatin segment, it was possible to allocate AFLP (amplified fragment length polymorphism) markers specific to that segment. Markers associated with genes conferring stress resistance facilitate marker-assisted selection programmes to obtain new, more persistent grass cultivars. Preliminary results of GISH analysis, to identify Festuca chromosome segments in L. multiflorum introgression lines and to find segment-specific AFLP markers, are presented.
Adress and Contact Informations	¹Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland, ²Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, UK

Volume 7 (2002) pp 499 – 509
Title	THE APPLICATION OF MOLECULAR MARKERS IN THE PROCESS OF SELECTION
Authors	Piotr Masojć
Abstract	Molecular markers are modern diagnostic tools, which may help breeders to solve practical problems. They facilitate cultivar identification, the determination of genetic similarities among breeding stocks and enable the calculation of polymorphism level, heterozygosity or self-pollination rate. But the main expectation with respect to molecular markers is their potential use in marker-assisted selection (MAS). There are four major strategies of finding a molecular marker tightly linked to a target gene of agronomic importance. The first approach takes advantage of the nearly isogenic lines (NILs) which are differentiated only by the allelic sets in the gene of interest and in the adjacent chromosomal region. The second strategy involves bulked segregant analysis (BSA) for identification of markers linked to a single gene. The third involves the genetic dissection of more complex traits, which leads to the identification of quantitative trait loci (QTL) and their markers. The fourth strategy of marker identification includes computer databases (sequence and mapping data). The usefulness of these strategies is discussed in this paper. Some consequences of the application of BSA for gene tagging are described.
Adress and Contact Informations	Department of Genetics and Plant Breeding, Agricultural University, Słowackiego 17, 71-434 Szczecin, Poland

Volume 7 (2002) pp 511 – 522
Title	RNAi AND VIRAL VECTORS AS USEFUL TOOLS IN THE FUNCTIONAL GENOMICS OF PLANTS. CONSTRUCTION OF BMVBASED VECTORS FOR RNA DELIVERY INTO PLANT CELLS
Authors	Agnieszka Wojtkowiak^1,3, Anna Siek¹, Magdalena Alejska¹, Artur Jarmołowski², Zofia Szweykowska-Kulińska² and Marek Figlerowicz¹
Abstract	The sequencing of several complete genomes and the development of a DNA microarray technology are among the most important achievements of molecular biology. They gave the proper grounds for the development of modern functional genomics. However, there is one additional condition which needs to be satisfied to truely enable the study of how a genome works: a suitable method of selectively inducing and silencing the expression of each individual gene. The methods used so far have usually only permitted the influencing of gene expression through genetic manipulations at the DNA level (genetically modified plants). The discovery of RNA interference (RNAi) opens up completely new possibilities of research on the functioning of particular plant genes, without the necessity of altering the genome structure. In this case, interference takes place at the transcript level. Thus, at any given moment during plant development, the expression of a specific gene (or several genes) can be inhibited, even if it is important for the survival of the organism under study. To this end, a double-stranded RNA inducing the RNAi phenomenon has to be delivered into the plant cell. Here we describe the construction of four brome mosaic virus-based vectors, which, as our preliminary data indicate, can be used to transfer RNA into barley cells.
Adress and Contact Informations	¹Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland, ²Faculty of Biology, A. Mickiewicz University, Poznań, Poland, 3Faculty of Chemistry, A. Mickiewicz University, Poznań, Poland

Volume 7 (2002) pp 523 – 535
Title	*THE SEARCH FOR QTL IN BARLEY (HORDEUM VULGARE L.) USING A NEW MAPPING POPULATION*
Authors	Gerhard H. Buck-Sorlin
Abstract	Ninety-nine different lines of the Doubled-Haploid F2 winter barley population W766 ('Angora’ x 'W704/137') were genetically fingerprinted using AFLP, microsatellite, morphological and resistance markers. A preliminary map consisting of seven linkage groups is presented. The map contains a highly distorted region on the long arm of chromosome 3H reflecting preselection of the genotypes for resistance against barley mild mosaic virus. QTL analysis of morphological and phenological traits yielded 99 significant QTL, with most traits (66.3%) being represented by a single QTL. The distribution of significant QTL over the chromosomes was very uneven, the bulk being placed on the long arm of chromosome 3H and no QTL being found on chromosome 4H. This possibly points to the presence of a strong pleiotropic gene on 3H or of a group of related genes that mask weaker effects that were found on other linkage groups as subsignificant QTL. Using two examples of detected QTL (for tillering and grain number), it is shown how the findings of the QTL analysis could be incorporated into an existing morphological simulation model of barley using simple statistical methods.
Adress and Contact Informations	Institute of Plant Genetics and Crop Science, Department. of Taxonomy, Corrensstrasse 3, D-06466 Gatersleben, Germany

Volume 7 (2002) pp 537 – 546
Title	IN SILICO ANALYSIS ON FREQUENCY AND DISTRIBUTION OF MICROSATELLITES IN ESTs OF SOME CEREAL SPECIES
Authors	Rajeev K. Varshney¹, Thomas Thiel¹, Nils Stein¹, >Peter Langridge² and Andreas Graner¹
Abstract	During the last decade microsatellites or SSRs (simple sequence repeats) have been proven to be the markers of choice in plant genetics research and for breeding purposes because of their hypervariability and ease of detection. However, development of these markers is expensive, labour intensive and time consuming, in particular, if they are being developed from genomic libraries. In the context of large-scale sequencing and genomics programmes in various cereal species at different laboratories, a large set of expressed sequence tags (ESTs) is being generated, which can be used to search for microsatellites. Keeping in view the importance of such type of SSRs, available ESTs of some cereal species like barley, maize, oats, rice, rye and wheat were investigated for a study of abundance, frequency and distribution of various types of microsatellites. SSRs were present in about 7% to 10% of the total ESTs in the investigated cereal genomes. On the basis of surveying EST sequences amounting to 75.2 Mb in barley, 54.7 Mb in maize, 43.9 Mb in rice, 3.7 Mb in rye, 41.6 Mb in sorghum and 37.5 Mb in wheat, the frequency of SSRs was 1/7.5 kb in barley, 1/7.5 kb in maize, 1/6.2 kb in wheat, 1/5.5 kb in rye and sorghum and 1/3.9 kb in rice. The overall average SSR frequency for these species is 1/6.0 kb. Trimeric repeats are the most abundant (54% to 78%) class of microsatellites followed by dimeric repeats (17% to 40%). Among the trimeric repeats the motifs CCG are the most common in all the cases ranging from 32% in wheat to 49% in sorghum. When all these SSRs were analysed for assessing their potential to develop new markers, unique primer pairs could be designed for 30% to 70% of the total non-redundant microsatellites which are up to 3% of total ESTs in the studied species.
Adress and Contact Informations	¹Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D 06466 Gatersleben, Germany; ²Department of Plant Science, University of Adelaide, Waite Campus, PMB1 Glen Osmond, 5064 South Australia * Corresponding author, E-mail: graner@ipk-gatersleben.de

Volume 7 (2002) pp 547 – 558
Title	MORPHOLOGICAL AND AFLP VARIATION OF ELYMUS REPENS (L.) GOULD (POACEAE)
Authors	Magdalena Szczepaniak¹, Elżbieta Cieślak¹ and Piotr Tomasz Bednarek²
Abstract	Combined morphological and molecular techniques were used to characterize variation in Elymus repens. We studied the morphological variability of E. repens in relation to the degree of its genetic differentiation, in order to unravel the causes of conspicuous intraspecific morphological variation. Four populations of E. repens from different habitats were analyzed for 35 morphological characters, and their genetic differentiation was assessed by Amplified Fragment Length Polymorphism (AFLP). Four pairs of selective primers were used to detect a total of 279 AFLP bands, of which 104 (37.28%) were polymorphic between populations. Cluster analysis based on AFLP fingerprint data showed that individuals were arranged in population-specific groups. The analyses of variance (ANOVA and AMOVA) indicated significant morphological and genetic differentiation among populations (P<0.01). This study revealed low levels of AFLP variation, which suggests that conspicuous morphological variation of E. repens is caused by plasticity. E. repens is an evolutionarily young species, of hybrid origin, in which microevolutionary processes continue. This study showed that common analysis of genetic diversity and morphology is a powerful tool in low-level taxonomy.
Adress and Contact Informations	¹W. Szafer Institute of Botany, Polish Academy of Sciences, Department of Plant Systematics, ul. Lubicz 42, 31–512 Kraków, Poland, ²The Botanical Garden – the Centre for the Conservation of Biological Diversity of the Polish Academy of Sciences, ul. Prawdziwka 2, 02–973 Warszawa, Poland

Volume 7 (2002) pp 559 – 567
Title	*THE CYTOGENETICS AND MOLECULAR CHARACTERISTICS OF A TRANSLOCATED CHROMOSOME 1AS.1AL-1DL WITH A GLU-D1* LOCUS IN DURUM WHEAT**
Authors	Antonio Blanco¹, Alberto Cenci¹, Rosanna Simeone¹, Agata Gadaleta¹, Domenico Pignone² and Incoronata Galasso²
Abstract	Wheat quality depends directly on the grain protein content and protein composition. High and low molecular weight glutenin subunits play an important role in determining the visco-elastic properties of gluten. In an attempt to improve the breadmaking quality of hexaploid triticale, a fragment of wheat chromosome 1D, containing the Glu-D1 allele encoding the 5+10 subunits, was translocated to the long arm of chromosome 1A by Lukaszewski and Curtis [1]. The 1A.1D translocation chromosome was transferred to tetraploid wheat [2], making the Glu-D1 locus available for the improvement of durum wheat. The goal of this study was to evaluate using cytogenetics and molecular approaches the amount of chromatin introgressed in durum wheat. Fluorescence in situ hybridization with total genomic DNA (GISH) of Aegilops squarrosa L. indicated that the translocated chromosome 1A.1D had a terminal 1DL segment of about 35-40% of the recombinant arm length. Several pairs of microsatellite primers from chromosome 1A and 1D were used to genetically characterize the recombinant chromosome. The mapping data indicated that a 1AL segment, at least 150 cM long, was substituted by a 1DL segment with a minimal length of 72 cM, and that the translocation breakpoint was near the 1A centromeric region. The genetic and physical data highlight a substantial discrepancy between the recombinational and physical map distances. We are using a targeted strategy via the Ph pairing manipulation system to generate small intercalary 1D chromosome segments in a durum wheat background.
Adress and Contact Informations	¹Department of Agro-Forestry and Environmental Biology and Chemistry, University of Bari, Via Amendola 165/A, 70126 Bari, Italy; ²Germplasm Institute, C.N.R., Via Amendola 165/A, 70126 Bari, Italy * Corresponding author, E-mail: blanco@agr.uniba.it, Tel: 0039 080 5442992, Fax 0039 080 5442200

Volume 7 (2002) pp 569 – 576
Title	STUDIES ON CHANGES IN SPECIFIC RYE GENOME REGIONS DUE TO SEED AGING AND REGENERATION
Authors	Katarzyna Joanna Chwedorzewska, Piotr Tomasz Bednarek and Jerzy Puchalski
Abstract	The aim of this study was to identify the genetic changes in rye seeds induced by natural aging during long-term storage and successive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples (cv. Dańkowskie Złote), varying in their initial viability and having gone through one or three reproduction cycles, were analysed using specific PCR targeting of a secalin locus, and various repetitive fragments defined by the R173 sequence. A statistical analysis of the band frequencies for both secalin and R173.3 primer pairs revealed no changes in their frequencies. Similar data on R173.1 demonstrated significant changes between samples of different initial viability showing a lack of a band of the expected length (987 bp) in progeny originating from low viability seeds lots. These changes were inherited even after three regeneration cycles. Our results may indicate that long-term storage that leads to loss of viability also generates heritable changes in the preserved germplasm. However, it remains to be discovered where these changes occur and whether they are connected with coding or with non-coding DNA regions.
Adress and Contact Informations	The Botanical Garden – the Centre for the Conservation of Biological Diversity of the Polish Academy of Sciences, Prawdziwka 2, 02-973 Warsaw, Poland

Volume 7 (2002) pp 577 – 582
Title	SEMI-SPECIFIC PCR FOR THE EVALUATION OF DIVERSITY AMONG CULTIVARS OF WHEAT AND TRITICALE
Authors	Małgorzata Gaweł, Iwona Wiśniewska and Andrzej Rafalski
Abstract	The usefulness of a semispecific PCR system for the evaluation of genetic diversity among selected cultivars of wheat (^{Triticum aestivum L.}) and triticale (^{x Triticosecale Wittm.}) was investigated. A comparison was made of the diversity among cultivars of both species as revealed by primers targetting the semi-conservative sequences of the intron-exon junction. The main purpose of the study was the selection of the most informative primers from among the exon targetting (ET) and intron targetting (IT) primers of 12 to 18 bases in length. For triticale, the most satisfactory results were obtained using ET and IT primers of 18 bases in length. In wheat, complex and polymorphic banding patterns were generated by primers belonging to both groups, regardless of their length.
Adress and Contact Informations	Dept. of Plant Biochemistry and Physiology, Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland

Volume 7 (2002) pp 583 – 597
Title	GENERATION AND MAPPING OF AFLP, SSRs AND SNPs IN LYCOPERSICON esculentum
Authors	Saskia Suliman-Pollatschek¹, Khalil Kashkush¹, Hadas Shats¹*, Jossi Hillel² and Uri Lavi¹
Abstract	Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP), were applied to the tomato genome for assessment of polymorphism and for mapping. The polymorphism of AFLP was studied in twenty-one commercial tomato (L. esculentum) varieties. Four AFLP primer combinations produced 298 clear bands; an average of 75 bands per combination. SSR markers were generated from two sources: (1) size-selected genomic libraries screened with (AT)n, (CT)_n, (GT)_n, (ATT)_n and (CTT)_n probes. (2) GeneBank database. Primers were designed for 114 loci and used for genotyping 13 tomato varieties and three Lycopersicon species. Eighteen markers were used to evaluate the polymorphism among the commercial cultivars and were found to be a useful tool for cultivar identification. In-silico comparison of DNA sequences (ESTs and genes) of L. pennellii and L. esculentum, yielded 312 SNPs. Ten L. pennelli genomic fragments were sequenced and the comparison with L. esculentum yielded 22 SNPs. Another 19 SNPs were discovered by sequencing and comparing L. pennellii genomic DNA to L. esculentum DNA fragments containing SSRs. The average SNP frequency was found to be one in a few tens of base pairs. A total of 52 microsatellites, 159 polymorphic AFLP markers and six SNPs were mapped using the Introgression Lines generated by [1]. Map location and markers’ distribution are presented.
Adress and Contact Informations	¹ARO-Volcani Center, P. O. Box 6, Bet-Dagan 50250 Israel, ²Faculty of Agriculture, the Hebrew University, P. O. Box 12 Rehovot 76100, Israel * Present address: MBC, 5 Oppenheimer St. Science Park Rehovot P.O.B 4018 Nes- Ziona 70400, Israel; ** Present address: Department of Plant Sciences, The Weizmann Institute, Rehovot 76100, Israel. The first two authors are equal contributors to this paper

Volume 7 (2002) pp 599 – 606
Title	A MICROSATELLITE MARKER BASED STUDY OF CHROMOSOMAL REGIONS AND GENE EFFECTS ON YIELD AND YIELD COMPONENTS IN MAIZE
Authors	Sayyed Abolghasem Mohammadi¹*, Boddupalli Maruti Prasanna², Charu Sudan³ and Narsingh Narain Singh³
Abstract	The genetic basis of different traits and the effect of chromosomal regions on the expression of such traits is the primary interest of this study. In the present investigation, the effect of chromosomal regions on yield and its two primary components was studied, and two genetic parameters, additive effect and degree of dominance, were estimated based on a single-locus model using markers with significant effects on the traits (informative markers). Eight inbred lines from diverse geographical regions of India were crossed in all possible pairs, and F1s were evaluated for the yield and yield components in a replicated trial in two environments. Sequential path analysis was employed to find those yield components showing significant direct effects on yield with negligible multicollinearity. The parental lines were profiled using 56 polymorphic SSR (Simple Sequence Repeat) markers covering 10 chromosomes of maize. Stepwise multiple regression analysis was used to determine the informative markers on yield and its primary components detected through path analysis. Fourteen markers were found to have association with chromosomal regions showing significant effects on the total grain yield, 100-grain weight and total number of kernels per ear. Chromosome 1 with four informative markers revealed the highest genic effects on yield and its components. Markers bnlg594 and bnlg1360 on chromosome 10, and bnlg147 on chromosome 1 revealed the highest additive effects on the total grain yield, 100-grain weight and total number of kernels per ear, respectively. For the analyzed traits, overdominance occurred in all the loci and d/a values, varying from 8.60 for 100-grain weight to 1.40 for total grain yield.
Adress and Contact Informations	¹Dept. of Crop Production & Breeding, Faculty of Agriculture, University of Tabriz, Tabriz 51664, Iran, ²Division of Genetics, Indian Agricultural Research Institute, New Delhi 110012, India, ³Directorate of Maize Research, New Delhi, India * Corresponding author, E-mail: sa_mohammadi@yahoo.com

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