Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense

Wild species constitute a potential source of genetic variation for cultivated species. Besides, they can be analyzed to answer the long-lasting questions concerning the origins, evolution and spread of major agricultural crops of the world. Recently, there has been considerable progress in plant genomics, leading to novel molecular breeding tools to reduce the costs and to simplify the assays. Plant genome research has been focused on the major crops and model species and a vast amount of genomic information has been accumulated. This information will provide an opportunity to use it as sources of information for thousands of minor grass species [1].

Hordeum chilense Roem et Schultes is a native South American diploid perennial wild barley (2n = 2x = 14), included in the section Anisolepsis [2]. It belongs to a heterogeneous group of South American Hordeum species and it is one of the species of the genus Hordeum with a high potential for cereal breeding purposes, given its high crossability with other members of the Triticeae tribe. H. chilense was used to obtain fertile amphiploids with wheat of different ploidy levels (diploid, tetraploid and hexaploid). These amphiploids were named Tritordeum and are the basic genetic material for using H. chilense genetic variability in wheat breeding [3]. H. chilense has agronomically interesting characteristics, like high carotenoid content, biotic and abiotic stress resistance, and variability for seed storage proteins [4]. A new cytoplasmic male sterility (CMS) source in bread wheat using the cytoplasm of H. chilense has also been reported [5].

The characterization of genetic variability in wild species like H. chilense and the development of tools to introduce it into cultivated crops are important plant breeding goals. The analysis of DNA sequence variation is of major importance in genetic studies. In this context, molecular markers are useful tools for assaying genetic variation, and have greatly enhanced the genetic analysis of crop plants. High development costs make it impractical to develop molecular markers directly from wild species like H. chilense. However, if markers developed in related crop species can be used, genetic analysis of H. chilense could be advanced rapidly [6].

Several types of molecular markers, including Random Amplified Polymorphic DNA (RAPD) and Sequence Characterized Amplified Regions (SCARs) [7, 8] have been developed de novo for H. chilense. Comparative genetic analysis has been established in the grass genomes [9] showing significant conservation of marker and gene order across the cereal crops. On this basis, Sequence Tagged Sites (STSs) and microsatellites or simple sequence repeats (SSRs) were transferred from wheat and barley to H. chilense [10, 11]. The SSR markers have been the molecular markers of choice for molecular breeders due to their codominant inheritance, multi-allelic nature and easy detection [12]. Recent studies have used the increase on the availability of large numbers of Expressed Sequence Tags (ESTs) in public databases, to the search for SSRs present in ESTs i.e. in barley [13], sugarcane [14], bread wheat [15], apricot and grape [16], rice [17] and Vaccinium [18].

Across-species transferability of SSRs derived from EST databases is greater than that of SSRs derived from enriched genomic DNA libraries, as they originate from expressed regions and therefore they are more conserved across a number of related species than non-coding regions [19]. They have shown to be useful for comparative mapping across species, comparative genomics, and evolutionary studies and they have been shown to posses a higher potential for inter-specific transferability than genomic SSRs [15, 16, 20–23]. On the other hand, they are expected to be less polymorphic within the species due to its conserved nature [19]. In summary, EST-SSR has provided a valuable source of new PCR-based molecular markers in cereal crops.

We have tested the transferability of 82 EST-SSR markers developed in barley [24, 25], and their potential use as new molecular tools for introgression, variability and phylogenetic analysis of the H. chilense genome. The chromosomal locations of the transferred and polymorphic EST-SSRs were assigned using wheat chromosome addition lines [26].

The grass family (Poaceae) is formed by 600 genera and between 9,000 to 10,000 species of grasses. This family comprises the most important cultivated crops like wheat, barley, rye, and rice [27]. H. chilense belongs to the Poaceae family, Triticeae tribe, genus Hordeum. Previous cytogenetic work suggested that H. chilense chromosomes are more similar to the D- than to the A- or B-genomes of wheat [28]. The phylogenetic relationships of H. chilense with respect to Triticum and Hordeum have not been studied in detail so far. For this reason, the set of EST-SSR primers that successfully amplified H. chilense and showed polymorphism was further tested for transferability to other species of the Poaceae family with the aim to investigate phylogenetic relationships.

Genomic SSRs have been extensively used for mapping, genetic diversity analysis, and plant breeding, but have a lower rate of transferability across species when compared to the EST-SSR markers. Thus, the latter are a better choice for application in cross-species phylogenetic studies [35], and are also valuable tools for plant breeding, germplasm collection conservation [36], and to measure genetic diversity [37]. More than 500.000 ESTs are presently available for barley and represent an invaluable resource for the development of SSR markers [24, 25]. To systematically exploit potentially useful, albeit less studied wild species, like H. chilense it would be desirable to use these EST-SSR markers as anchors to the cultivated species genome.

A set of barley genomic SSR markers [38] has been previously tested in H. chilense [11]. As expected, the level of transferability (66%) of barley EST-SSR markers found in the present work is higher than for the neutral SSRs (54%). Additionally, the level of polymorphism detected within H. chilense with the EST-SSRs (26%) is also higher than with the genomic SSRs (6%).

Our results confirm the high cross-species transferability of the set of 165 barley EST-SSRs tested in wheat, rye and rice [13]. Varshney et al. [13] observed 78.2% amplicons in wheat, 75.2% in rye and 42.4% in rice, demonstrating the high potential of EST-SSR markers for comparative mapping among these species. In the present study, we tested the subset of 21 EST-SSR markers showing higher level of transferability [13] and polymorphism in H. chilense lines across genera for phylogenetic inference. We observed 100% amplification of the selected barley EST-SSR markers in the genome of H. murinum, 90% amplification in the genome of T. durum, 81% amplification in the genomes of T. aestivum, T. urartu and T. tauschii and 76% amplification in the genome of B. distachyon. Our results confirm the general observation that the rate of EST-SSRs transferred across species or genera decays as the species or genera are more phylogenetically distant [19].

On the other hand, the sensibly higher transfer rate to B. distachyon than to rice confirms the usefulness of Brachypodium as a model species for barley and wheat and gave us the species of choice for rooting the phylogenetic tree in the present study. Our results showed a higher transferability of barley EST-SSRs to H. chilense than to other wild barley species reported previously [24], where 80% of examined barley markers were successfully amplified in wild barley accessions and about 60% of the primers yielded amplification products in wheat and rye. The high transferability among Triticeae species and genera has also been reported for wheat EST-SSR markers. For instance, Tang et al. [39] tested 243 wheat EST-SSR markers, from which 216 (88.9%) produced amplicons in wheat, 211 (86.8%) in barley, 187 (77.0%) in rice and 166 (68.3%) in maize. Zhang et al. [40] reported the transferability of bread wheat EST-SSRs to closely related Triticeae species, ranging from 76.7% for A. tauschii to 90.4% for T. durum. The rates were lower for more distant relatives such as barley (50.4%) or rice (28.3%). Similar results were obtained by Yu et al. [22], who found that a total of 53% of the wheat EST-SSR markers produced amplicons in barley.

The location of barley EST-SSR on the H. chilense chromosomes was determined by the amplification of the available wheat addition lines. They were all found in the same linkage group as barley, thus corroborating the conserved nature of these markers and their potential use in comparative genomics among species. Conserved chromosome locations, together with strong selection criteria (including similar molecular weight range to the donor species and conserved locus stuttering patterns) make these markers' transferability robust enough for their practical application. Fifteen barley EST-SSR produced amplicons of different size in wheat and H. chilense, and four did not amplify in wheat. Therefore, 19 barley EST-SSR markers are useful for wheat-H. chilense introgression analysis.

Wheat EST-SSRs have been recently demonstrated useful for phylogenetic analysis among the Triticeae species [23]. One of the aims of this study was to infer phylogenetic relationships among the H. chilense genome, the cultivated barley genome and the wheat genomes using barley EST-SSRs (Figure 4). In the obtained dendrogram, the nodes were significantly supported by bootstrap analysis, indicating that there were subgroups that could be clearly separated.

As expected, the H. chilense genome was situated in the clade of the Hordeum species, despite the reported cytogenetic similarity with the D genome of wheat [1]. The species H. murinum (Xu-genome) was closer to H. vulgare (I-genome) than to H. chilense (H-genome). Several other studies have also grouped species of the I- and Xu-genomes [41], using sequences from three nuclear regions DMC1, EF-G and ITS; and the vrs1 locus [42].

In the clade of Triticum species, the hexaploid and tetraploid wheats allelic data were separated into their genomes. A closer association was observed between the B- and D-genome and tetraploid species were closely related to hexaploid species, which is in agreement with a previous analysis based on wheat EST-SSR data [23].

Our study shows the utility of barley EST-SSR for the genetic analysis of H. chilense, with a remarkably high level of polymorphism within this species. It highlights a reliable and efficient way of obtaining microsatellite markers for wild relatives of a major crop. The transferred markers have shown to be useful for phylogenetic studies among the Triticeae species, and to anchor the H. chilense genome within the wheat-barley framework using Brachypodium as a root genome. The availability of additional sets of mapped EST-derived SSR markers for barley and other Triticeae genomes will assist the development of molecular maps for H. chilense and its integration into the genomic network of grass species.