Characterization of novel genic SSR markers in Linum usitatissimum (L.) and their transferability across eleven Linum species

Braulio J. Soto-Cerda*^#1 · Hector Urbina Saavedra¹ · Cristell Navarro Navarro¹ · Paula Mora Ortega¹

¹Centro de Genómica Nutricional Agro-Acuícola, Unidad de Genómica y Bioinformática, INIA-Carillanca, Temuco, Chile

*Corresponding author: braulio.soto@cgna.cl

^#Present address: Cereal Research Center, Agriculture and Agri-Food Canada, Winnipeg, Canada.

Financial support: This work was supported by CGNA, Centro de Genómica Nutricional Agro-Acuícola, Araucanía-Chile.

Keywords: EST-SSRs, expressed sequence tag, flaxseed, polymorphic loci, wild relatives.

Little is known about the evolutionary relationships among Linum species, basically because of the lack of transferable molecular markers. Currently, expressed sequence tags available in public databases provide an opportunity for the rapid and inexpensive development of simple sequence repeat (SSR) markers in wild flax species. In this regard, fifty expressed sequence tag-derived microsatellite markers (EST-SSRs) were evaluated for polymorphism and transferability in 50 Linum usitatissimum cultivars/accessions and 11 Linum species. Among them 23 EST-SSRs were polymorphic in L. usitatissimum, while 2-4 alleles were detected (average 2.26 per locus). The polymorphism information content value ranged from 0.08 to 0.55 (average 0.38). Forty one genic markers (95.3%) produced strong amplicons in at least two of the 11 Linum species. The percentage of cross amplification ranged from 34.1% to 92.7% in L. tauricum and L. bienne, respectively. Moreover, the rate of transferability was associated positively with the botanical section. Our results suggest that the high degree of EST-SSRs transferability to Linum species can be a useful enhancement of the current database of SSR markers for future genetic and evolutionary studies.

Linum is the largest genus of the Linaceae family comprising nearly 200 species (Heywood, 1993) divided into six botanical sections: Linum, Linastrum, Cathartolinum, Dasylinum, Syllinum and Cliococca (Winkler, 1931). This genus has more than 100 species that exhibit a wide range of diversity in morphological characters and life forms (Gill, 1987). Their uses can vary from industrial to ethnobotanic applications. For instance, Linum usitatissimum is the most important species of the genus due to its industrial applications in fiber, oil and functional foods production. Furthermore, L. bienne the wild progenitor of cultivated flax is a potential donor of new alleles for L. usitatissimum genetic improvement. With regard to the evolutionary relationships among species of the genus Linum efforts have been carried out based on morphological characters, chromosome numbers and karyological studies (Gill, 1987; Muravenko et al. 2010).

Nowadays, molecular characterization of cultivated flax as well as studies of potential evolutionary pathways of wild flax species have been assessed using Amplified Fragments Length Polymorphism (AFLP) (Everaert et al. 2001), Random Amplified Polymorphic DNA (RAPD) (Fu, 2006) and Inter Simple Sequence Repeat (ISSR) markers (Wiesnerova and Wiesner, 2004; Uysal et al. 2010). However, this goal has been partially achieved using the current molecular tools. Comparison across taxa is still lacking since many marker systems are more or less species specific. When they are amplified across different species not necessarily are compared the same genomic regions which can conduct to erroneous conclusions (Pashley et al. 2006). Although, Genomic Simple Sequence Repeats (G-SSRs) have been developed in cultivated flax (Roose-Amsaleg et al. 2006; Deng et al. 2010; Soto-Cerda et al. 2010) their transferability to related species is generally low because they commonly occur in noncoding regions of the genome which are not highly conserved (Kim et al. 2008). Moreover, homoplasy is another limitation of G-SSRs in evolutionary studies where identical band sizes may not be identical by descent (Thiel et al. 2003). Thus, one of the major challenges in the molecular analyses across the Linum species is the generation of informative and transferable molecular markers. On the contrary, Expressed Sequence Tag-derived microsatellite (EST-SSRs) or genic SSRs are popular because they are derived from transcription products and conserved coding sequences; as a consequence they exhibit a high rate of transferability to other species (Eujayl et al. 2004; Varshney et al. 2005; Ellis and Burke, 2007; Hisano et al. 2007; Iñiguez-Luy et al. 2008). In addition, they are cost-effective, co-dominant and useful for studying functional diversity, comparative mapping and interspecific linkage map construction (Varshney et al. 2005). Recently, Cloutier et al. (2009) reported the only EST-SSRs collection developed in cultivated flax which were included in the first linkage map in L. usitatissimum (Cloutier et al. 2010). Subsequently, Fu and Peterson (2010) utilized part of these genic SSRs to characterize their transferability to Linum species. It is noteworthy that this EST-SSRs collection provides new molecular tools for Linum studies. Nonetheless, they are still limited for performing a wide genome comparison among the species of the genus Linum. Therefore, in order to achieve a comprehensive understanding of the genetic diversity and genome evolution of the genus Linum as well as to use wild relatives in linseed breeding, more informative genic SSRs should be developed.

The objective of this study was to evaluate the informative value and transferability of 50 new genic SSR primers in 50 L. usitatissium cultivars/accessions and 11 Linum species, so that they can be use for future molecular characterizations in Linum species.

Plant material and DNA extraction

A total of 50 L. usitatissimum cultivars/accessions and 11 species of genus Linum, divided in 4 botanical sections were selected for screening polymorphism and transferability of the EST-SSR primers, respectively. Thirty four cultivars/accessions of cultivated flax and the eleven Linum species were provided by Dr. Axel Diederichsen, Plant Gene Resources of Canada. Sixteen Chilean linseed cultivars/accessions were provided by the Germplasm Bank of the Agricultural Research Institute of Chile INIA, Carillanca (Table 1). DNA was extracted from fresh, young leaf tissue using the CTAB method (Doyle and Doyle, 1987). In cultivated flax, DNA was isolated from a single plant per cultivar/accession. On the other hand, in Linum species 5 plants were bulked. The quality and final concentration was estimated by spectrometry using a MBA 2000 Perkin Elmer spectrometer.

Sequence database and EST-SSRs detection

A total of 7960 Expressed Sequence Tag (EST) sequences of L. usitatissium available by May 1, 2009 were retrieved from NCBI database. The identification of sequences containing repeats, redundancy and primers design was performed in accordance with Soto-Cerda et al. (2010). The parameters for designing the primers were: length 18-27 bp with 20 being optimum, Polymerase Chain Reaction (PCR) product size of 100-300 bp, optimum annealing temperature of 60ºC and GC content of 20-80%. A subset of 50 primer pairs was custom synthesized by Sigma-Aldrich (St. Louis, MO), giving priority to trinucleotide motifs.

PCR amplification and electrophoresis

All PCR amplifications were carried out following the PCR conditions developed by Soto-Cerda et al. (2010). PCR products were separated by 6% 8M urea polyacrylamide gel electrophoresis and visualized by the silver staining method (Bassam et al. 1991). Fragment sizes for each locus were determined using 50 bp DNA ladder (Promega, USA). The quality of the SSR loci was expressed as the Polymorphism Information Content (PIC) as described by Botstein et al. (1980). In addition, transferability was considered successful when the amplicons were in the size range predicted for L. usitatissimum.

Distribution of EST-SSRs and polymorphism

Of the 7960 EST sequences examined, 494 genic SSRs were found in 436 non-redundant sequences (5.4%) which account for an average density of 1 SSR every 9.51 Kb. In general, it is estimated that 2-5% of all plant-derived ESTs harbor SSRs (Varshney et al. 2005; Pashley et al. 2006), matching our results in the expected range. Among them the most abundant motifs were trinucleotides (57.1%) followed by tetra- (17.2%), penta- (11.7%), hexa- (7.1%), mono- (5.9%) and di- (1.0%). This result is in accordance with other studies in Arabidopsis (Morgante et al. 2002), Festuca arundinacea (Saha et al. 2004), durum wheat (Wang et al. 2007) and cultivated flax (Cloutier et al. 2009) where trimeric repeats were more abundant. Unlike other plant species such as Lactuca sativa (Simko, 2009) and L. usitatissimum (Cloutier et al. 2009) dinucleotide motifs were underrepresented. One possible explanation for such difference is that the majority of the ESTs analyzed in our study derived from a stem phloem fiber BAC library which did not cover the whole genome of L. usitatissimum. For instance, Cloutier et al. (2009) used 10 cDNA libraries to generate cultivated flax ESTs representing tissues from bolls, stem, globular embryos, heart stage embryos, seed coat at torpedo stage; thereby it is guaranteed homogenous genome coverage. Furthermore, tri- and hexanucleotide SSRs seem to be under positive selection since they do not cause frameshift mutation in ESTs coding region owing to they are multiples of three, the number of nucleotides in codons. This is not the case of dinucleotide motifs whose number could be under negative selection (Varshney et al. 2005). 

Forty three primer pairs amplified expected size alleles (86%) while 23 (53.5%) were polymorphic in L. usitatissimum. This is higher than EST-SSRs reported in wheat (25%), sorghum (45%) and cultivated flax (40.7%) (Eujayl et al. 2002; Cloutier et al. 2009, Yonemaru et al. 2009), but lower than another study in L. usitatissimum (62.2%) (Roose-Amsaleg et al. 2006). Differences in polymorphism might be attributable to the genomic origin of the SSRs developed by Roose-Amsaleg et al. (2006) considered more polymorphic than EST-SSRs. Nonetheless, they are not directly comparable since their evolution is explained by different factors in transcribed and non-transcribed sequences. These new genic SSR primers were identified with the prefix LM “Linum Microsatellite” (Table 2). No multi loci EST-SSR primers were detected, in total 23 reproducible loci (Figure 1a). The total number of alleles was 52, ranging from 2 to 4, with an average of 2.26 alleles per locus. Commonly, EST-SSRs are reported to show lower allele variation than G-SSRs (Simko, 2009). Indeed, Roose-Amsaleg et al. (2006) and Deng et al. (2010) reported in G-SSRs an average of 3.32 and 3.45 alleles per locus, respectively. On the other hand, Cloutier et al. (2009) reported similar results between the two studies based on EST-SSRs (2.3 alleles per locus) which would confirm the assumption exposed above. In this study we prioritized trinucleotide EST-primers since in G-SSRs have been demonstrated that they are more polymorphic (Song et al. 2002). However, our results showed low polymorphism in EST-SSRs carrying trimeric repeats. Similarly, the low variability of trimeric EST-SSR loci was reported in Oryza sativa (Cho et al. 2000) and Pinus taeda (Liewlaksaneeyanawin et al. 2004). On the contrary, dimeric EST-SSRs with high numbers of repeats seem to have high polymorphism, as do genomic SSRs. The relationship between polymorphism and the number of repeats has been reported for EST-SSRs in barley (Thiel et al. 2003) and G-SSRs in cultivated flax (Soto-Cerda et al. 2010). PIC values ranged from 0.08 to 0.55 with an average of 0.38. These results are quite similar to those reported by Cloutier et al. (2009) (average 0.35). Nevertheless, when trimeric PIC values were compared between the two studies our results were higher (0.38 versus 0.33). On the contrary, when the average PIC value of the EST-SSRs was compared with G-SSRs, this tend to be lower, i.e., L. usitatissimum 0.56 (Deng et al. 2010) Musa acuminata 0.48 (Wang et al. 2010), common bean 0.45 (Hanai et al. 2007) and Brassica oleracea 0.48 (Iñiguez-Luy et al. 2008). Nonetheless, 69.56% of genic SSRs showed PIC values up the average, suggesting that informative SSRs can be obtained from L. usitatissimum ESTs.

EST-SSRs transferability across the Linum species

Forty one EST-primer pairs (95.3%) produced strong amplicons in at least two of the eleven species of the genus Linum. The percentage of transferability ranged from 34.1% to 92.7% in L. tauricum and L. bienne and L. pallescens, respectively (Table 2). Among botanical sections the average transferability was 72.4% Linum, 58.5% Dasylinum, 58.5% Linastrum and 52.4% Syllinum. Interestingly, in the section Linum the lowest rate of cross-amplification was observed in L. narbonense (41.5%). This species markedly differs from the other species of the section by the number and size of chromosomes, their C-banding pattern and location of ribosomal genes (Muravenko et al. 2010). In addition, by morphology and size, L. narbonense chromosomes are more similar to chromosomes of L. hirsutum which agree with botanists that L. narbonense systematic position needs to be refined (Muravenko et al. 2010) as suggested by the low level of EST-SSRs transferred to this species. Our results indicate a positive correlation between the rate of transferability and the botanical section. For instance, Roose-Amsaleg et al. (2006) and Fu and Peterson (2010) reported 85.7% and 97% of transferable G-SSR and EST-SSR loci in L. bienne (n = 15, Linum), respectively. This indicates that this species shares a high sequence similarity and genome background with cultivated flax. Indeed L. bienne is known as the wild progenitor of cultivated flax from previous morphological, cytological and molecular characterizations (Samadi et al. 2007; Diederichsen and Fu, 2008; Uysal et al. 2010), and it is considered the primary gene pool for crossing with cultivated flax (Diederichsen and Fu, 2008; Cloutier et al. 2009). Both species produce interfertile F₁ progenies (Gill and Yermanos, 1967), share the same chromosomes number, and have similar chromosomes morphology and C-banding pattern (Muravenko et al. 2003). With regard to L. pallescens (n = 9, 15, Linum) there also exist reports of hybridization with L. usitatisiimum with normal meiosis and fertile F₁ progenies (Gill and Yermanos, 1967). When we observed L. grandiflorum (n = 9, Linum) this disclosed 78% of cross-amplification. Interestingly, this species has been used for the induction of dihaploid embryos in L. usitatissimum improvement (Diederichsen and Richards, 2003). Hybridization between inter botanical sections have also been reported; i.e. L.usitatissimum (n = 15, Linum) x L. hirsutum (n = 9, Dasyllinum) and L.usitatissimum (n = 15, Linum) x L. strictum (n = 9, Linastrum) (Jhala et al. 2008). These species disclosed 58.5% and 78% of transferability, respectively. Although plant morphology and karyotype are important factors in taxonomy classification, the primary criterion is the ability to cross hybridize and produce fertile offsprings (Diederichsen and Richards, 2003). It stated that the level of transferability success reduces as the evolutionary distance and sexual compatibility between the source and the target species increases (Liewlaksaneeyanawin et al. 2004; Simko, 2009). This statement supports the number of transferable EST-SSRs to these five species of the genus Linum which might indicate that similar functional sequences are shared with L. usitatissimum to induce chromosome pairing in artificial hybridizations. Examples from tomato (Tanksley and McCouch, 1997), lettuce (Jeuken and Lindhout, 2004) and rice (Nguyen et al. 2003) represent successful gene introgressions from wild relatives which is a real option for linseed breeding. The remarkable level of transferability supports previous findings in other plant species (Saha et al. 2004; Varshney et al. 2005; Pashley et al. 2006; Fu and Peterson, 2010). These results confirm the usefulness of EST-SSRs since they are derived from transcribed and conserved regions of the DNA, allowing cross-species transferability (Eujayl et al. 2004; Sim et al. 2009). In addition, the four botanical sections belong to the genus Linum; for which higher rates of transferability of EST-SSR loci is expected within genus (Eujayl et al. 2004; Pashley et al. 2006) as confirmed by ≈ 65% of average cross amplification in our study. Twenty six primers (63.41%) showed polymorphism inter-species, disclosing 85 total putative bands ranging from 2-5 with an average of 2.07 per locus (Figure 1b). On the other hand, 34.1% of them disclosed multi allele/loci compared to L. usitatisimum amplicons which might entail intra-species variation as a consequence of bulked samples. By the contrary, other possible reason could be the high rate of conservation of EST sequences, suggesting amplifications from either orthologous or paralogous copies (Saha et al. 2004; Sim et al. 2009), particularly in polyploidy species such as L. narbonense (Muravenko et al. 2010). Similarly, Yu et al. (2004) reported that 39% of EST-SSRs detected multiple loci in wheat (hexaploide). As a result, to elucidate this question sequence analysis should be carried out in the future. In fact, sequencing PCR amplicons of EST-SSRs have shown high similarities, especially within genus (Zhang et al. 2005; Pashley et al. 2006). Our results suggest that EST-SSRs might help to elucidate the evolutionary relationships of Linum species, providing genetic information from cultivated flax to its wild relatives and vise versa (Sim et al. 2009). Among polymorphic loci in L. usitatissimum, 69.6% showed polymorphism across Linum species. Furthermore, 55.5% of the monomorphic loci in L. usitatissimum were informative in the other Linum species, indicating that monomorphic EST-SSRs developed for one species might display allele variation in other related taxa (Cloutier et al. 2009).

This study confirms that by data mining was possible to develop inexpensive, informative and transferable EST-SSRs in L. usitatissimum and related species. The high rate of transferability validates their potential application for fingerprinting, functional diversity, comparative mapping and Marker Assisted Selection (MAS) with emphasis on those interfertile species such as L. bienne and L. pallescens. From our point of view is an urgent need that more EST-SSRs being developed since the current number is still lacking for whole genome analysis in the genus Linum.

The authors are grateful to Dr. Axel Diederichsen and Dr. Gastón Muñoz for supplying the germplasm utilized in this study.

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