Paolo Donini

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

Abstract. It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.

Assessment of EST- and genomic microsatellite markers for variety discrimination and genetic diversity studies in wheat

It is likely that in the near future sequence information from sequencing programmes and EST libraries will generate an abundance of genic microsatellite markers. This study is focused on the assessment of their likely impact and performance vis-à-vis their genomic counterparts. Microsatellites from two sources were used to assess the genetic diversity in 56 old and new varieties of bread wheat on the UK Recommended List. A set of 12 microsatellite markers generated from genomic libraries and 20 expressed sequence tag (EST)-derived microsatellites were used in the study, and the performance of both marker sets assessed. The EST-derived or genic microsatellites delivered fingerprints of superior quality, amplifying clear products with few stutter bands. Diversity levels as revealed bygenic microsatellites are similar to the few published results. The PIC values for the genic markers were generally lower than those calculated for the genomic microsatellites, though advantages of both marker classes for variety identification applications are discussed.

Development and analysis of multiplex microsatellite markers sets in common bean (Phaseolus vulgaris L.)

Multiplexing involves the simultaneous amplification of several loci in a single PCR reaction, and subsequent analysis of multiple molecular markers in a single gel lane. This study focuses on the usefulness of SSR (simple sequence repeat) multiplex-PCR, and how this method can be both highly informative and amenable to automation using a fluorescence-based, semi-automated DNA sequencing platform. We also discuss the relevance of this approach to the issue of the multiplex ratio of SSR markers and show that it is effective as an economic procedure for germplasm evaluation and evolutionary studies of plants. Various conditions of multiplex-PCR were examined and a method is proposed for developing multiplex sets of SSR markers in common bean (Phaseolus vulgaris L.). Seven multiplex sets, based on 30 SSR markers selected from GenBank sequences, were developed. The method was tested using a large number of samples from three common bean landraces. Technical aspects of the application and a description of the allelic constitution at various loci of all genotypes analysed are described. From a sample of 264 genotypes, selected from three landraces, we detected a total of 135 alleles, equivalent to 4.3 alleles per SSR. Null alleles were observed in each of the three landraces analysed. The procedures used in this study are applicable for the study of genetic diversity in common bean germplasm collections consisting of a significant number of accessions, and should be transferable to similar analyses of any species.

Use of AFLP in cereals research

Bands of interest can be excised from gels that have been dried onto 3MM filter paper (Whatman) or from dried silver stained gels17xCloning and mapping of variety-specific rice genomic DNA sequences: amplified fragment length polymorphisms (AFLP) from silver stained polyacrylamide gels. Cho, Y.G. et al. Genome. 1996; 39: 373–378Crossref | PubMedSee all References17, and re-hydrated overnight at 4°C. The DNA can then be re-amplified with the original primers and used as a probe, for direct sequencing or for cloning. Also, the AFLP technique can be modified so that one primer is obtained from a known sequence to detect sequence-specific amplification polymorphisms (S-SAP). This approach has been used successfully to map Bare-1 transposable elements throughout the barley genome18xGenetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Waugh, R. et al. Mol. Gen. Genet. 1997; 253: 687–694Crossref | PubMed | Scopus (330)See all References18.Increasingly, AFLP is being used for several specialist applications to assist the rapid isolation and characterization of interesting genes. AFLP-based mRNA fingerprinting has been used to isolate sequences mapping to deleted chromosome segments in cereals or other species19xAFLP-based mRNA fingerprinting. Money, T. et al. Nucleic Acids Res. 1996; 24: 2616–2617Crossref | PubMed | Scopus (68)See all References19 and cDNA-AFLP has been used to monitor the expression of genes20xVisualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber expression. Bachem, C.W.B. et al. Plant J. 1996; 9: 745–753Crossref | PubMedSee all References20. AFLP is also being used to screen superpools of plasmid DNA from 384 clones of bacterial artificial chomosomes (BACs; C. Goldstein and M. Dixon, unpublished), enabling the alignment of contigs and the rapid isolation of genes tightly linked to markers identified through high density mapping. The discriminating power of AFLP has also revealed the cryptic genome changes that could occur in transgenic rice plants generated by particle bombardment or intact cell electroporation21xMolecular analysis of the genome of transgenic rice (Oryza sativa L.). Plants produced via particle bombardment or intact cell electroporation. Arencibia, A. et al. Mol. Breed. 1998; 4: 99–109Crossref | Scopus (37)See all References21. Analysis of complex traits has so far been limited by mapping techniques with a low marker index (MI)22xThe comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Powell, W. et al. Mol. Breed. 1996; 2: 225–238CrossrefSee all References22. With AFLP the MI is more suitable, and AFLP has recently been applied to the analysis of quantitative traits in barley22xThe comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Powell, W. et al. Mol. Breed. 1996; 2: 225–238CrossrefSee all References22 and rice23xMapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping. Nandi, S. et al. Mol. Gen. Genet. 1997; 255: 1–8Crossref | PubMed | Scopus (110)See all References23.One of the major limitations of AFLP is the objective difficulty in identifying allelic variants at a specific locus, which has resulted in the technique being used almost exclusively for dominant markers. Recently, however, new software for image analysis of fluorescent PCR products has been developed by several companies including Keygene and Perkin Elmer. This will probably be developed for use with AFLP, which would enable AFLP to be used for scoring semi-dominant markers.As more maps of a given species are produced by various research groups, methods to integrate the different maps are clearly necessary to ensure that the same marker can be investigated. In a limited comparison within a single laboratory, accurately sized bands were found to be sufficient to identify homologous products between six different barley crosses5xHomology of AFLP products in three mapping populations of barley. Waugh, R. et al. Mol. Gen. Genet. 1997; 255: 311–321Crossref | PubMed | Scopus (140)See all References5. The reliability of this approach might be enhanced if more selective nucleotides were included to improve the discrimination of the polymorphic band. Further ambiguity could be eliminated by obtaining the sequence of the band.In conclusion, AFLP is a robust and reliable technique that has already proved its value in mapping and phylogenetic studies with a range of cereals. Increasingly, the technique is proving invaluable for the analysis of complex traits, for variety identification and for the rapid isolation of important genes. AFLP is rapidly becoming the preferred molecular technique for many types of investigation and will undoubtedly continue to find new applications in many areas of cereals research.Note: The AFLP technique is covered by patents and/or patent applications pending owned by Keygene n.v. Use of the technique for purposes other than research requires a license from Keygene n.v.

Molecular barley breeding

Breeding progress in barley yield in the UK is being sustained at a rate in the order of 1% per annum against a background of declining seed sales. Commercial barley breeders are largely concentrating upon the elite local gene pool but with genotypic evidence suggesting that there is still considerable variation between current recommended cultivars, even those produced as half-sibs by the same breeder. Marker Assisted Selection (MAS) protocols could be substituted for conventional selection for a number of major-gene targets but, in the majority of cases, conventional selection is more resource efficient. Results from current QTL mapping studies have not yet identified sufficiently robust and validated targets for UK barley breeders to adopt MAS to assist in the selection of complex traits such as yield and malting quality. Results from multiple population mapping amongst the elite gene pool being utilised by breeders and from association studies of elite germplasm tested as part of the UK recommended list trial process do, however, show some promise.

The impact of breeding on genetic diversity and erosion in bread wheat

This paper examines the fate of alleles and changes of genetic diversity in old ( ca 1930s) versus more modern ( ca 1990s) UK bread wheat varieties using 14 mapped DNA microsatellite (simple sequence repeat, SSR) loci and morphological markers. The allelic constitution of varieties belonging to three time periods (early, intermediate, late) was determined. While at certain loci one or more SSR alleles were gained between early and late periods, at others the allelic representation remained constant, although a shift in allelic frequencies could sometimes be detected. No locus showed a clear, net loss in the total number of alleles over the time period. In a further group of loci, there was neither clear gain nor loss, but rather a dynamic flux of alleles. A comparison of the allelic constitution of the UK variety set with a larger genetic pool (non-UK varieties) showed that some loci were rather similar in allelic constitution, while others possessed additional diversity. Certain SSR alleles appeared to be associated with old or modern varieties, possibly indicating associations with chromosome regions under selection pressure. The same exercise was conducted on the basis of 14 of the morphological characteristics recorded in the course of distinctness, uniformity and stability testing of varieties. Overall, this analysis generated a similar picture of changes in diversity to that obtained from the microsatellite data.