Knowledge of the genetic diversity within Hordeum is of utmost importance for practical as well as theoretic purposes. Little is known about which molecular methodology is most feasible for rapid molecular screening of this genetic diversity. However, four different approaches could be taken. These are:
1. Restriction Fragment Length Polymorphisms (RFLPs),
2. Randomly Amplified Polymorphic DNAs (RAPDs),
3. Variable Number Tandem Repeats (VNTRs), and
4. DNA Sequencing (PCR/Sequencing).
Each approach yields a different `type' of information and as yet, no direct comparisons have been made between techniques.
In the present project each molecular research group applies their preferred technique to a common set of material. The samples used are selected in close corporation with the Swedish research group that have access to the world's largest holdings of wild Hordeum germplasm and have close links with the European Hordeum genebank at Gatersleben, Germany. This strategy ensures close links with the Barley Core Collection (BCC), and hence also with classical descriptors.
A major objective of the project is to evaluate the four different molecular techniques on a common set of germplasm both with respect to speed and scale, and to make direct comparison between the quality of information obtained by each of the techniques, for practical, commercial, and financial reasons.
An estimate of the genetic diversity within the selected Hordeum germplasm will be obtained and linked with classical descriptors, such that guidelines and recommendations can be made when considering approaches to specific problems.
The final comparisons between all four molecular methodologies and the classical descriptors should take us a considerable step towards making the BCC the world's best characterized germplasm collection, and hence towards making the BCC of utmost importance for plant breeding.
Within cultivated barley data from RFLP markers give the clearest correlation to cultivar genealogy, separating spring from winter barley, and 2- from 6-rowed varieties. In general the genetic variation is greatest within the winter barleys. Whereas H. vulgare ssp. spontaneum as expected shows higher genetic diversity than cultivated barley, the modern cultivars do not exhibited a lower diversity than the older landraces. Hence, the overall loss of genetic diversity, which is often supposed to be the result of narrow breeding experiments, cannot be observed in barley. Data for numbers of microsatellite allelles also show a lowered genetic diversity of cultivated barley compared to H. vulgare ssp. spontaneum, but not compared to older landraces.
The related AFLP (Amplified Fragment Length Polymorphism) technique and multilocus SSR (Simple Sequence Repeat) fingerprinting give almost equally good resolution between winter and spring barleys, whereas neither microsatellite, RADP, nor DNA sequence data are able to produce quite the same clear results.
SSRs (or microsatellites, a technique not available at the start of the project) are the most polymorphic markers, but are less suited for assessing genetic relationships among cultivars. Rather they have their strength in population studies.
Multilocus SSR fingerprinting have revealed a high level of polymorphism in H. vulgare ssp. spontaneum, but no clear correlation to the geographic occurrence of accessions was found. Sequence data from the Adh1 locus showed only a limited amount of variation in H. vulgare ssp. spontaneum and again no correllation to geographic occurrence.
In spite of a high level of polymorphism revealed by RAPD markers, RAPDs suffer from problems of reproducibility, and we conclude that they in general constitute an inappropriate tool for screening genetic diversity. RFLP markers are particularly useful for assessing genetic relationships, but the technique suffer from being time-consuming. Multilocus SSR fingerprinting and AFLP are less time-consuming techniques, but their reproducibility has yet to be proberly tested.
To date DNA sequence data, even from introns of nuclear genes, reveals too little variation to be an appropriate tool at the lower taxonomic levels, i.e. below the species level. However, at higher taxonomic levels sequence data (eg. from Adh1, ITS, rbcL, rpoA) are most informative, and especially useful for phylogenetic studies, which are essential for assessing diversity in a proper perspective.
MAJOR SCIENTIFIC BREAKTHROUGHS:
We believe that we can provide the scientific community with valuable new knowledge in several areas:
1) We have designed a very rapid method for PCR amplification, bypassing the laborious DNA extraction procedures.
2) We have found several new microsatellite loci and designed sets of primers for amplification of each locus.
3) We have ascertained that RAPDs are of very dubious value for screening biodiversity. RFLPs, AFLPs, and multilocus SSR fingerprinting are much more useful for assessing genetic relationships of cultivars, but give indirect results.
4) We have developed nuclear and chloroplast DNA sequence data useful for phylogenetic studies of Hordeum and other grasses.
5) We have made important discoveries with respect to molecular evolution of the Adh1 gene.
Funding SchemeCSC - Cost-sharing contracts
750 07 Uppsala
28801 Alcala De Henares