A. M. Figueiras

The use of ISSR and RAPD markers for detecting DNA polymorphism, genotype identification and genetic diversity among barley cultivars with known origin

Abstract.The potential of bulk analyses of RAPD and ISSR-PCR markers for fingerprinting purposes was evaluated using ten RAPD and ten ISSR primers. The phylogenetic relationships of 16 barley cultivars from different countries, and all having a known pedigree, were analysed using 353 PCR markers (125 RAPDs and 228 ISSRs). The band profiles generated were reproducible in spite of the different DNA extractions, PCR techniques, electrophoretic methods and gel scorings used. The RAPD primer S10 and four ISSR primers (811, 820, 835 and 881) were both able to distinguish all cultivars. A strong and quite linear relationship was observed between Resolving Power (Rp) of a primer and its ability to distinguish genotypes. The dendrograms obtained using these two molecular markers are in agreement with their known origin, showing clusters that separate very well the spring/winter and six-rows/two-rows cultivars. Thus, bulk analyses of RAPD and ISSR PCR markers provides a quick, reliable and highly informative system for DNA fingerprinting and also permit to establish genetic relationships which agree with, by other means, known origin of the cultivars.

Rapid identification of Triticeae genotypes from single seeds using the polymerase chain reaction

An easy and quick protocol has been developed for DNA analysis via PCR. Single cereal endosperm or small leaf pieces can be separately processed in several PCR reactions. The resultant PCR patterns are equivalents to those obtained with standard DNA extraction protocols using either specific or random primers. Intra-and inter-specific variability can be detected. This method allows the analysis of a large number of individuals in early stages prior to the plant sowing.

Chromosomal location of PCR fragments as a source of DNA markers linked to aluminium tolerance genes in rye.

To identify and locate rye DNA sequences homologous to three wheat c-DNAs (wali1, wali2 and wali5) whose expression is induced by aluminium (Al) stress, we designed three pairs of specific primers. They were used in the amplification of genomic DNA from wheat-rye disomic addition lines. The wali2 pair of primers amplified a 878-bp rye DNA fragment (rali2) located on chromosomes 4R and 7R that showed 79.37% homology with the corresponding wheat c-DNA. RAPD fragments were also used as genetic markers. We located 22 different RAPDs distributed on 11 different rye chromosome arms using wheat-rye disomic and ditelocentric addition lines. Thirteen of these markers were located on the chromosomes 3R, 4R and 6R, which also carry aluminium-tolerance genes. The OPA08415 and OPR01600 RAPD markers, located on the 6RL and 6RS chromosome arms, respectively, were converted to SCAR markers (SCA08415 and SCR01600) and linked to Alt1 gene (SCR01600-2.1 cM-Alt1-33.5 cM-SCA08415). We propose that the chromosomal location of RAPDs and SCARs using wheat-rye addition lines is a source of DNA markers linked to aluminium-tolerance loci and offers a valuable strategy in marker-assisted selection for the introgression of tolerance genes in wheat.

Maintenance of interchange heterozygosity in cultivated rye, Secale cereale L.

SummaryThe maintenance for several generations of interchange heterozygosity in both experimental and cultivated fields of rye is analysed. An experimental analysis of interchange heterozygotes has been carried out to ascertain whether only one or several translocations exist in the population. Interchange heterozygosity is discussed on the basis of the karyotypic orthoselection model. The maintenance of a constant frequency (20 per cent) of structural heterozygotes for several generations suggests the existence of an equilibrium. The possible mechanisms involved in such an equilibrium are discussed.

Phosphogluco mutase — a biochemical marker for group 4 chromosomes in the Triticinae

SummaryStructural genes for the isozymes of phosphogluco mutase (PGM) (EC 2.7.5.1) have been located on chromosome arms 4Aα, 4BL and 4DS of hexaploid wheat. These results support the homoeologies observed among these chromosome arms and also support the notion of conservation of gene synteny groups within the Triticinae.

Biochemical evidence of homoeology between Triticum aestivum and Agropyron intermedium chromosomes.

SummaryThe alcohol dehydrogenase (ADH), phosphoglucose mutase (PGM), glucosephosphate isomerase (GPI), glutamic oxaloacetic transaminase (GOT), malate dehydrogenase (MDH), leaf esterases (ESTL), leaf acid (ACPH) and endosperm alkaline (PHE) phosphatases, leaf peroxidases (PERL) zymogram phenotypes of Triticum aestivum, Agropyron intermedium, Triticum aestivum — Agropyron intermedium octoploids and six Agropyron intermedium chromosome additions to Triticum aestivum and two ditelocentric addition lines were determined. It was found that the six disomic chromosome addition lines and one ditelocentric chromosome addition line could be distinguished from one another and from the other possible lines on the basis of the zymogram phenotypes of these isozymes. The structural gene Acph-X1 was located on Agropyron chromosome L1, the genes Got-X3 and Mdh-X2 on chromosome L2, the gene Gpi-X1 on chromosome L3, the genes Adh-X1, Pgm-X1 and Phe-3 on chromosome L4, gene Perl-1 on chromosome L5 and the gene Estl-2 on chromosome L7 and chromosome arm L7d2. These gene locations provide evidence of homoeology between Agropyron chromosomes L1, L2, L3, L4, L5 and L7 and the Triticum aestivum chromosomes of homoeologous groups 7, 3, 1, 4, 2 and 6, respectively.

Chromosomal location of isozyme markers in wheat-barley addition lines

SummaryThe peroxidase (CPX, PER), α-amylase (α-AMY), acid and alkaline phosphatase (PHE, PHS) and esterase (EST) zymogram phenotypes of ‘Chinese Spring’ wheat, ‘Betzes’ barley and a number of presumptive ‘Betzes’ chromosome additions to ‘Chinese Spring’ were determined. It was found that five disomic chromosome addition lines could be distinguished from one another and from the other two possible lines on the basis of the zymogram phenotypes of these isozymes. The structural genes Cpxe-H1 and Cpxe-H2 were located in ‘Betzes’ chromosome 1, the Perl-H5 and Perl-H6 in chromosome 2, the α-Amy-H2 and α-Amy-H3 in chromosome 7, the Phs-H5 and Phs-H4 in chromosomes 1 and 3 respectively, the Phe-H2, Phe-H3 and Phe-H4 in chromosome 1, the Phe-H1 in chromosome 3, the Ests-H4, Este-H2 and Ests-H6, Este-H8 in chromosomes 1 and 3 respectively and the Estl-H10 and Estl-H2 structural genes were related to chromosomes 3 and 6 respectively. These gene locations provide evidence of homoeology between ‘Betzes’ chromosomes 1, 2, 3, 6 and 7 and the rye chromosomes 7, 2, 3, 6 and 5, respectively, and also between ‘Betzes’ chromosomes 1, 2, 3, 6 and 7 and the ‘Chinese Spring’ homoeologous groups 7, 2, 3, 6 and 5, respectively.

PCR derived molecular markers and phylogenetic relationships in theSecale genus

DNA from 22 different species, accessions, cultivars and lines included in theSecale genus were analyzed by the polymerase chain reaction (PCR), using as primers five pairs of oligonucleotides derived from specific sequences. A total of 42 amplified bands were considered, and some of them appeared to be potentially useful as molecular markers for some of the analyzed groups. These amplified bands were used to generate molecular phenograms inside theSecale genus.

Association of four isozyme loci with a reciprocal translocation between 1R/4R chromosomes in cultivated rye (Secale cereale L.)

SummaryThe progeny of four crosses between a structural heterozygote for a reciprocal translocation and a homozygote for the standard chromosome arrangement were analyzed in rye (Secale cereale L. cv “Ailés”) for the electrophoretic patterns of eight different leaf and endosperm isozymes and also for the meiotic configuration at metaphase I. The Pgi-1, 6-Pgd-2 and Mdh-1 loci are linked to each other and also to the reciprocal translocation. These loci have been located on chromosome 1R. The Mdh-1 locus is located in the interstitial segment of chromosome 1R, between the centromere and the breakpoint. The Pgm-1 locus has been located on chromosome arm 4RS and is linked to Pgi-1, 6-Pgd-2, Mdh-1 and the reciprocal translocation. The estimated distance between the Pgm-1 locus and the centromere is 14.98 ± 2.27 cM. Therefore, the reciprocal translocation involves the 1R and 4R chromosomes. Other linked loci detected have been Mdh-2b and Est-2 (7.40 ± 2.90 cM) and Got-3 and Est-2 (5.62 ± 3.07 cM). These three last loci are located on chromosome 3R and their order most probably is Mdh-2b — Est-2 — Got-3.

A map of rye chromosome 4R with cytological and isozyme markers.

The progeny of two crosses between a structural heterozygote for a reciprocal translocation (4RL/5RL) and a homozygote for the standard chromosome arrangement and of four crosses between standard chromosome homozygotes were analysed in rye (Secale cereale L. cv ‘Ailés’) for the electrophoretic patterns of five different leaf and endosperm isozymes (LAP, PGM, NDH, ADH and EPER). The presence or absence of the quadrivalents at metaphase I (MI) was also tested. Loci Adh-1, Pgm-1 and Ndh-1 were located on chromosome arm 4RS, and locus Eper-1 on chromosome arm 4RL. Locus Lap-2 was located on the 4RS chromosome arm. The estimated distances among the different linked loci support the following gene order: Eper1¨ (breakpoint-centromere)¨Lap-2¨ ¨Adh-1 ¨Pgm-1¨Ndh-1. These results provide evidence for the chromosomal location of Lap-2 locus on chromosome arm 4RS in cv ‘Ailés’. A high negative interference was detected between the zones delimited by centromere and Lap-2, and Lap-2 and Pgm-1 in plants with the 4RL/5RL translocation.