Exploring and mapping genetic variation in wild barley, Hordeum spontaneum Tytti Kaarina Vanhala Promotor: Prof. Dr. Ir. P. Stam Hoogleraar in de plantenveredeling Samenstelling promotiecomissie: Prof. Dr. P.H. van Tienderen (Universiteit van Amsterdam) Prof. Dr. R.F Hoekstra (Wageningen Universiteit) Dr. H. Poorter (Universiteit Utrecht) Prof. Dr. Ir. P.C. Struik (Wageningen Universiteit) Dit onderzoek is uitgevoerd binnen de onderzoekschool Production Ecology and Resource Conservation Tytti Kaarina Vanhala Exploring and mapping genetic variation in wild barley, Hordeum spontaneum Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. Ir. L. Speelman, in het openbaar te verdedigen op vrijdag 10 september 2004 des namiddags te half twee in de Aula Tytti Kaarina Vanhala (2004) Exploring and mapping genetic variation in wild barley, Hordeum spontaneum Vanhala, T.K. –[S.1.:s.n.].Ill. PhD Thesis Wageningen University. –With ref.- With summaries in English, Dutch and Finnish ISBN : 90-8504-043-4 Contents Abstract 1 Chapter 1 General Introduction 3 Chapter 2 A genetic linkage map of wild barley (Hordeum spontaneum) 11 Chapter 3 Environmental, phenotypic and genetic variation of wild barley (Hordeum spontaneum) from Israel 25 Chapter 4 Association of AFLP markers with growth related traits in Hordeum spontaneum 43 Chapter 5 Quantitative trait loci for seed dormancy in wild barley (Hordeum spontaneum) 63 Chapter 6 Summarising discussion 75 Samenvatting 81 Tiivistelmä 87 References 95 Addresses of co-authors 107 Acknowledgements 108 Curriculum vitae 111 Abstract Wild barley represents an important genetic resource for cultivated barley, which has a narrowed gene pool due to intensive breeding. Therefore, it is imperative to study the genetics of different traits in wild barley, if it is to be used for cultivar improvement. This thesis describes studies of different aspects of diversity, as well as linkage, QTL and association mapping in wild barley. The natural populations that were studied were collected in environmentally diverse Israel. Using a cross between genotypes from contrasting habitats, a linkage map was produced. Due to unexpectedly high heterozygosity of one of the parents the map covered approximately 45% of the barley genome. This map was subsequently utilised in all other studies in this thesis. Genetic diversity was estimated within and between 21 populations. This was correlated with phenotypic and environmental diversities of the same populations. Genetic diversity was loosely connected with environmental diversity. In general, phenotypic diversity did not correlate with either one. Some markers were ecotype-specific; some of these were mapped and correlated significantly not only with environmental but also with phenotypic traits. Using the same populations, correlations between mapped markers and phenotypic traits were calculated. The significant associations were then compared with QTL found in an earlier study. Many of these associations mapped to the same regions as the QTL and therefore these two approaches of mapping traits complemented each other. Lastly, the map was used to locate QTL for dormancy in wild barley. Five QTL were found of which three map to regions where dormancy QTL has been found earlier in cultivated barley. For further research, another cross should be made to produce a map with larger coverage of the genome. The results of this thesis add to the characterisation of the diverse wild barley populations from Israel, which is important to the future improvement of cultivated barley. 1 2 Chapter 1 Many years have passed since those summer days Among the fields of barley See the children run as the sun goes down Among the fields of gold You'll remember me when the west wind moves Upon the fields of barley You can tell the sun in his jealous sky When we walked in the fields of gold Sting – Fields of gold 3 General Introduction Hordeum spontaneum, wild in Southwest Asia across a wide range of barley climates and soils. It is particularly Hordeum spontaneum (wild barley) is the common in the Near East Fertile Crescent ancestor of cultivated barley. It belongs (Zohary 1969). In general, wild barley is to the Poaceae- family of grasses and not tolerant to extreme low temperatures within it to the Triticeae-tribe. Triticeae is and is rarely found above 1500 m a temperate plant group mainly altitude. However, it is more drought concentrated around Central and resistant than the wild wheat and Southeastern Asia, although the species penetrates relatively deeply into the warm belonging to it are distributed around the steppes and deserts (Zohary & Hopf world. Triticeae includes many 1988). economically important cultivated cereals Wild barley and cultivated 2-rowed and forages but also about 350 wild barley have quite similar morphology. The species. The wild species are of great most notable differences are wild barley’s interest as potential gene donors for brittle rachis and its hulled grain. Six- commercial breeding. rowed barley has evolved during The genus Hordeum includes 30 domestication, the trait being controlled species (Von Bothmer 1992), both by a single gene on chromosome 2 annuals and perennials. Wild barley can (Komatsuda et al. 1999; Tanno et al. be winter or summer annual depending 2002). Wild barley is the only wild on the local climate. It is diploid (2n=14) Hordeum species that can produce fully and mainly inbreeding, although some fertile hybrids (with normal chromosome populations have a higher rate of pairing and segregation in meiosis) when outcrossing. On average this is 1.6%, crossed with cultivated barley. Hybrids varying from 0 to 9.6%, depending on can also be formed in nature when these the environment; it is significantly higher two occur at the same location (Asfaw & in the mesic than in the xeric regions Von Bothmer 1990). (Brown et al. 1978a). The collection of wild barley from The wild annual Hordeum species Israel was obtained between 1974 and grow in open habitats with comparatively 1976 (Nevo et al. 1979). During that low competition from other species (Von period over a thousand different Bothmer 1992). Wild barley is distributed accessions were collected from the whole over the eastern Mediterranean area and of Israel. Sampling was done from various habitats like road and mountainsides, 4 fields and deserts. Latitude, longitude, (PCR) to amplify DNA, many different altitude and different rainfall and types of molecular markers have been temperature variables were reported from developed. Wild barley from Israel has the nearby weather stations. This also been studied utilising several of these collection has been maintained in the PCR-based methods: RAPDs (Baum et al. Institute of Evolution, Haifa, Israel by 1997; Owuor et al. 1997, 1999), AFLPs prof. E. Nevo and his co-workers. During (Pakniyat et al. 1997, Turpeinen et al. successive years, more samples were 2003) and SSRs (Turpeinen et al. 2001). added to the collection; populations from RFLPs (Chalmers et al. 1992), Tabigha (Nevo et al. 1986a) and retrotransposon copy numbers (Kalendar Evolution Canyon (Nevo 1995). et al. 2000) and genome size (Turpeinen Accessions from this collection in Haifa et al. 1999) were also used in studying have been distributed around the world. wild barley diversity within Israel. However, because of the high number of Studies with wild and cultivated accessions available, there is hardly any barley have shown that there is more overlap on the material used in the variation within the wild than in the different studies. Because these samples cultivated barley (e.g. Saghai Maroof et have been collected from widely diverse al. 1995), although in some cases the habitats, the collection represents a rich opposite has been reported for some source of phenotypic and genetic isozymes and mitochondrial DNA (Nevo, variation, which can be used to study 1992). The larger genetic variation within ecophysiological issues both at the wild barley gives the opportunity to use phenotypic and genotypic level. In this variation for breeding purposes. addition, the wild barley accessions of The genetic diversity of wild this collection are a potential source of barley is correlated with its geographic variation to be tapped by plant breeders distribution on a large scale, between for the improvement of cultivated barley. countries. But it is not evident on the micro-geographic scale within Israel. For example, Song & Henry (1995) found that Variation in wild barley from some of the accessions that were identical Israel genetically were geographically widely The genetic variability of wild barley has scattered and of contrasting habitat. been described in many studies. So far, Resistance to biotic and abiotic the only research including all the stresses has been the main area of study collected accessions from Israel has been on the phenotypic variation in wild barley, conducted by using isozymes (Brown et as these are important targets for al. 1978b; Nevo et al. 1979). After the improvement in barley breeding (Ellis et invention of polymerase chain reaction al. 2000). Wild barley accessions from Israel have been screened for resistance 5 to several plant pathogens (e.g. marker alleles arises from linkage Moseman et al. 1983, 1990; Ordon et al. between marker loci and loci controlling 1997). Several lines were resistant to the trait. By identifying these Erysiphe graminis and Puccinia hordei as associations, the method allows the well as soil-borne mosaic-inducing viruses location of genomic regions on a marker (Moseman et al. 1983, 1990; Ordon et al. linkage map that most likely contain 1997). Other agronomically interesting genes involved in the trait (the QTL). The traits include a range of physiological and results of QTL mapping provide the most chemical traits (e.g. Ellis et al. 1993; likely position of the QTL, together with Forster et al. 1994; Nevo et al. 1993a, an estimate of the allele substitution 1992, 1985), abiotic stress tolerance effects (the additive effect) and so called (e.g.