D. Kaemmer

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of streamlined’ laboratory techniques and data analysis to Developing Countries are discussed.

DNA fingerprinting of Ascochyta rabiei with synthetic oligodeoxynucleotides

SummaryThe ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea (Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA)4, (GTG)5, (CA)8, and (TCC)5, respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions.

Oligonucleotide Fingerprinting in Plants and Fungi

Synthetic oligonucleotides complementary to simple repetitive DNA sequence motifs are now routinely applied for multilocus DNA fingerprinting of humans and a large variety of animal species. Most recently, these probes have also been used successfully for the analysis of plant and fungal genomes. All simple motifs investigated to date (CA-, CT-, GATA-, GACA-, GAA-, GTG - GGAT- and TCC-multimers) are present and repeated to various extents throughout the plant and fungal kingdoms. Usually, these probes reveal intra- and interspecific genetic variability resulting in polymorphic or even hypervariable banding patterns. Depending on the combination of species and oligonucleotide probe, species- variety-, accession-, strain-or individual-specific “fingerprints” were obtained in plants and fungi. Somatic stability was observed. For their successful application to DNA fingerprinting, the optimal probe/species-combinations that give distinct banding patterns have to be developed empirically. Various applications of plant DNA fingerprinting using oligonucleotide probes are suggested: (1) characterization of the extent of genetic variability within races, (2) assessment of the “purity” of inbred lines, (3) selection of the recurrent parental genome in backcross breeding programs, (4) identification of crop cultivars and fungal strains, (5) characterization of fusion hybrids, (6) evaluation of the extent of somaclonal variation at the molecular level.

The potential of gene technology and genome analysis for cool season food legume crops: theory and practice

The potential of plant gene technology encompasses a multitude of different techniques ranging from the isolation of useful genes, their characterization and in vitro manipulation to the reintroduction of the modified constructs into target plants, where they are expressed at a rate that alters the phenotype of the plants. Genome analysis, on the other hand, aims at characterizing the genome architecture and function(s).Plant gene technology has catalyzed progress in plant breeding, as will be exemplified by a few examples, but has not yet been applied to food legume improvement on a large scale. Genome analysis, however, has a series of practical implications, as is illustrated by the successful introduction of DNA fingerprint and PCR fingerprint techniques to chickpea (Cicer arietinum L.) breeding and Ascochyta rabiei pathotyping. The present overview addresses both areas of plant molecular biology to illustrate their potential for food legume breeding.