Evaluation of cross-genus transferability of SSR markers from other legumes to two closely related Onobrychis (Fabaceae) taxa

Microsatellite markers previously developed for other leguminous species were tested for cross-genus transferability and evaluated for their potential usefulness in providing an improved assessment of the genetic relationships between two closely related taxa belonging to Onobrychis genus (Fabaceae). Candidate microsatellite markers were tested for polymorphism and replicability in sixteen populations of O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and O. montana. Out of the 23 SSRs, there were identified seven polymorphic loci. In total 32 alleles were detected and the number of alleles per locus varied from two to six. PIC values ranged from 0.375 to 0.6454, and four SSRs displayed a PIC > 0.5. Relative uniform rates of genetic diversity were obtained. In case of O. montana DC. subsp. transsilvanica (Simonk.) Jáv. the observed and expected heterozygosity ranged from 0.100 to 0.952 and from 0.219 to 0.525, respectively, while for O. montana ranged from 0.166 to 0.750 and from 0.083 to 0.375, respectively. Seven polymorphic SSRs with clear and reproducible amplification were identified. These markers proved to be very efficient for unambiguous population discrimination based on both geographic and taxonomic criteria. Hereafter, these SSR markers can be used as tools for evolutionary studies in Onobrychis genus, as well in providing knowledge on patterns of the species phylogeography.

Microsatellites (or Single Sequence Repeats -SSRs) are codominant markers characterized by high levels of polymorphism, thus being widely recognized as very powerful and informative in both animal and plant species (Ellegren, 2004). The hypervariable nature of SSRs produces allelic variations even among very closely related varieties. Therefore, they are considered the markers of choice for the characterization of core collections and for the management of germplasm collections (Kumar et al., 2023). One of the characteristics that make these markers particularly interesting in genetic diversity studies is their high rate of transferability to closely related species (Gupta et al., 2003;Simko, 2009). Nevertheless, significantly low values of crosstransferability have been observed for genomic SSRs, which are known to be more polymorphic and located in less conserved regions of the genome (Peakall et al., 1998;Sourdille et al., 2001).
The main purpose of this study was to test the cross-genus transferability of several SSR markers into O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and O. montana and provide a preliminary evaluation of their usefulness for assessing the genetic relationships between the two taxa.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Sampling and DNA extraction Ten populations belonging to O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and six populations of O. montana were sampled from the Alps and the Carpathians Mountains (Table 1). More details on the sampling strategy, on the populations and on the DNA extraction can be found in Băcilă et al. (2015). Table  Table Table  Table 1 1 1 1.   Each primer pair had to be optimized, as poor amplification or unspecific bands were otherwise present. Following amplification and analysis of gel patterns, only seven SSR primer pairs were selected, fluorescently dyed (6-FAM) and used in subsequent reactions. For the amplification of these seven microsatellites, four different PCR programs were used in order to obtain a clear and reproductible amplification (Table 2). The PCR products were purified with Sephadex -Sephacryl (1:1) (GE Healthcare Bio-Sciences AB, USA) and then diluted 50 times. 1.5 μL of dilution were added to 10 μL mix of HiDi formamide and GeneScan 500 ROX Size Standard (Applied Biosystems, Thermo Fisher Scientific, USA) and subjected to capillary electrophoresis on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, USA). The characteristics of the seven primer pairs are presented in Table 3. Table 3  Table 3  Table 3  Table 3. Characteristics of seven microsatellite loci used for cross-transferability in Onobrychis sp.

Data analysis
Alleles scoring was performed with GeneMapper v.4.0 software (Applied Biosystems, Thermo Fisher Scientific, USA).
PowerMarker v.3.25 (Liu and Muse, 2005) was used to calculate the total number of alleles, gene diversity and polymorphism information content (PIC). Descriptive statistics as: number of alleles and observed [Ho] and expected heterozygosities [He], were estimated per population using GenAlEx 6.5 (Peakall and Smouse, 2006). A frequency matrix was generated and subsequently used within SplitsTree v.4.10 (Huson and Bryant, 2006) to compute Unweighted Pair Group Method with Arithmetic Mean (UPGMA) phylogenetic tree based on the Shared Allele distance and the Neighbor-Net method. Bootstrap values were calculated from 1000 replicates.

Results
Results Results 23 SSRs were tested in O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and O. montana and consistent amplification was obtained for 18 of them (71.26%), while the rest provided multiple nonspecific bands. However, due to lack of polymorphism and low reproductibility, only seven SSR (Table 3) were selected for the subsequent characterization of the Onobrychis sp. populations. A total number of 32 alleles were detected, each SSR amplified 2-6 alleles, and the average number of alleles per SSR was 4.571. PIC values ranged from 0.375 to 0.6454, with an average of 0.5089 (Table 4). Only four SSRs (MtBA27D09, MtBB22G10, PV-at001, and MTIC272) displayed a PIC > 0.5, and therefore were considered informative. Relative uniform rates of genetic diversity were obtained, ranging from the lowest value of 0.5 (AG81, BG178, and BM141) to the highest value of 0.7 (MTIC272). The gene diversity and PIC values pointed out that MTIC272 represented the most informative locus in the two Onobrychis species analysed (Table 4). Table  Table Table  Table 4  Ho and He ranged in case of O. montana DC. subsp. transsilvanica (Simonk.) Jáv. from 0.100 to 0.952, and from 0.219 to 0.525, respectively, while for O. montana, they ranged from 0.166 to 0.750 and from 0.083 to 0.375 (Table 5). Table 5  Table 5  Table 5  Table 5. Genetic characterization of seven polymorphic microsatellite loci tested across sixteen populations of Onobrychis sp. Ho = observed heterozygosity; He = expected heterozygosity. The UPGMA analysis (data not shown, manuscript in preparation) managed to clearly differentiate all the 16 populations of Onobrychis, exhibiting taxonomic and geographic delineation.

Discussion Discussion Discussion
The rate of SSR cross-genera transferability was 18 out of 23 tested markers (71.26%). This value was lower than 81%, as previously reported by Demdoum et al. (2012), but higher than other related data (Eujayl et al., 2004). The intra-genus amplification rate was considered to be around 50% (Peakall et al., 1998), but this value quickly declined inter-genera. Zhang et al. (2007) found 18-22% transferability from Medicago to Trifolium, while Peakall et al. (1998) reported only 1-3% transferability of Glycine's SSR to other leguminous genera.
However, a narrow proportion of microsatellites was found to be polymorphic in Onobrychis (38.8% out of the 18 transferable SSRs). Several markers showed multiple bands that could not be eliminated by calibrating the PCR conditions. The generation of multiple products during cross-species amplification may occur by mutation, rearrangements and duplications in the flanking region and/or changes in the number of repeats (Peakall et al., 1998), similar results being reported by Gutierrez et al. (2005) in their study of EST-SSR in leguminous. Eventually, only seven SSR loci were selected on the base of polymorphism and reproducibility and they were subsequently used for characterization and genetic diversity evaluation of 16 populations of O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and O. montana. These seven markers showed medium PIC values (average 0.5089) ( Table 3). The number of alleles per locus ranged from 2 to 6 (Table 3), lower than previously reported by other studies (4-14) (Falahati-Anbaran et al., 2007). Since the studied Onobrychis species are diploid or tetraploid species (O. montana DC. subsp. transsilvanica (Simonk.) Jáv. 2n=14, L ve, 1975;and respectively O. montana 2n=28;L ve, 1984), the number of detected alleles seemed to be low. A possible explanation is the PCR amplification bias, which could cause the loss of the less frequent alleles and predominant detection of the most common alleles, therefore leading to an under estimation of the number of alleles per loci in each population (Peakall et al., 1998). Although the level of polymorphism exhibited by the seven employed microsatellites was relatively low and only four of them (MtBA27D09, MtBB22G10, PV-at001, and MTIC272) were informative (PIC > 0.5), it was possible to differentiate all the analysed populations by taxonomic and even geographic criteria.

Conclusions Conclusions Conclusions
Within the studied group represented by 16 populations of O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and O. montana the rate of SSR cross-genera transferability was 18 out of 23 tested markers (71.26%). Subsequently, only seven SSR loci were selected on the base of polymorphism and reproducibility. A total number of 32 alleles were detected, the average number of alleles per SSR being 4.571. Relative uniform rates for PIC and genetic diversity were obtained, pointing out that MTIC272 represented the most informative microsatellite. Although the level of polymorphism of the seven analysed microsatellites was relatively low, they managed to clearly differentiate all the analysed populations based on taxonomic and geographic criteria.

Authors' Contributions Authors' Contributions Authors' Contributions Authors' Contributions
The contributions of authors to the manuscript are as follows: conceptualization: IB, GC; field work: GC; data curation: IB, AC, ZRB, DȘ; formal analysis: IB and DȘ; funding acquisition: IB; investigation: IB; methodology: IB; project administration: IB; writing -original draft: IB; writing -review and editing: IB, AC, ZRB, GC and DȘ. All authors read and approved the final manuscript. 7 Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable. 8