Microsoft Word - 11549 NSB Bacila 2023.06.13.docx Received: 13 Apr 2023. Received in revised form: 08 Jun 2023. Accepted: 13 Jun 2023. Published online: 19 Jun 2023. From Volume 13, Issue 1, 2021, Notulae Scientia Biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. The journal will continue to appear quarterly, as before, with four annual numbers. SHSTSHSTSHSTSHST Horticulture and ForestryHorticulture and ForestryHorticulture and ForestryHorticulture and Forestry Society of TransylvaniaSociety of TransylvaniaSociety of TransylvaniaSociety of Transylvania Băcilă I et al. (2023) Notulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia Biologicae Volume 15, Issue 2, Article number 11549 DOI:10.15835/nsb15211549 Research ArticleResearch ArticleResearch ArticleResearch Article.... NSBNSBNSBNSB Notulae Scientia Notulae Scientia Notulae Scientia Notulae Scientia BiologicaeBiologicaeBiologicaeBiologicae Evaluation of crossEvaluation of crossEvaluation of crossEvaluation of cross----genus transferability of SSR markers from other genus transferability of SSR markers from other genus transferability of SSR markers from other genus transferability of SSR markers from other legumes to two closely related legumes to two closely related legumes to two closely related legumes to two closely related OnobrychisOnobrychisOnobrychisOnobrychis (Fabaceae) taxa(Fabaceae) taxa(Fabaceae) taxa(Fabaceae) taxa Ioan BĂCILĂ1, Dana ŞUTEU1,4*, Ana COSTE1, Zoltán R. BALÁZS2,3,4, Gheorghe COLDEA1 1National Institute of Research and Development for Biological Sciences, Institute of Biological Research, Department of Experimental Biology, 48 Republicii St., 400015 Cluj-Napoca, Romania; ioan.bacila@icbcluj.ro; dana.suteu@icbcluj.ro; ana.coste@icbcluj.ro; gheorghe.coldea@icbcluj.ro 2Babeş-Bolyai University, Faculty of Biology and Geology, Department of Molecular Biology and Biotechnology, 1 Kogălniceanu St., 400084 Cluj-Napoca, Romania; zoltan.balazs@ubbcluj.ro 3Babeș-Bolyai University, Faculty of Biology and Geology, Center for Systematic Biology, Biodiversity and Bioresources - 3B, 1 Kogălniceanu St., 400084, Cluj-Napoca, Romania 4Babeș-Bolyai University, Doctoral School of Integrative Biology, 1 Kogălniceanu St., 400084 Cluj-Napoca, Romania; dana.suteu@icbcluj.ro (*corresponding author) AbstractAbstractAbstractAbstract 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. Keywords:Keywords:Keywords:Keywords: cross-genus transferability; leguminous; microsatellite; Onobrychis; polymorphism https://www.notulaebiologicae.ro/index.php/nsb/index Băcilă I et al. (2023). Not Sci Biol 15(2):11549 2 IntroductionIntroductionIntroductionIntroduction The Onobrychis genus includes about 206 species (POWO, 2023), cross-pollinated, diploid (2n = 14, 16) or tetraploid (2n = 28) (Mohsen and Nasab, 2010), perennial or annual herbs or shrubs. The genus extends throughout the Europe (excepting Scandinavia and the British Isles), Central and Eastern Asia, and North Africa (POWO, 2023). Within the genus, Onobrychis montana DC. subsp. transsilvanica (Simk.) Jáv. (Ciocârlan, 2009; Sârbu et al., 2013) (≡ Onobrychis transsilvanica (Nyárády and Nyárády, 1957); ≡ Onobrychis montana DC. var. transsilvanica (Simk.) Beck (Borza, 1949) is an endemic taxon in the Romanian Carpathian chain. It shares close, yet controversial, taxonomic relationships and a strong morphological resemblance with the allopatric species Onobrychis montana DC. Our previous study (Băcilă et al., 2015) represented the first attempt to provide some molecular insights for this Carpathian endemic taxon with the use of AFLP and cpDNA markers. Because these markers failed to clearly resolve the distinction between O. montana and O. montana DC. subsp. transsilvanica (Simonk.) Jáv., other molecular markers, more informative ought to be identified. 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 cross- transferability 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). We selected and tested for transferability and polymorphism 23 expressed sequence tag -EST-SSRs originated from other leguminous species: Glycine max, Medicago sativa, Medicago trunculata, and Phaseolus vulgaris (Peakall et al., 1998; Yu et al., 2000; Gaitán-Solís et al., 2002; Julier et al., 2003; Gutierrez et al., 2005; Zhang et al., 2007). Previously, Demdoum et al. (2012) successfully cross-amplified 14 of these markers in O. pyrenaica Sennen, O. argentea Boiss. and O. viciifolia Scop., while the remaining nine markers were noted by Avcı et al. (2014) as polymorphic in 58 Onobrychis species from Turkish flora. 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 MethodsMaterials and MethodsMaterials and MethodsMaterials 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). Băcilă I et al. (2023). Not Sci Biol 15(2):11549 3 TableTableTableTable 1111. Sampled populations of O. montana and O. montana DC. subsp. transsilvanica (Simonk.) Jáv.: taxon, numbering, population code, country of origin (Ro – Romania; Fr – France; Po – Poland; Sk – Slovakia; Mne – Montenegro), mountain range, sampling locality, geographic coordinates (partially reproduced from Băcilă et al., 2015) TaxonTaxonTaxonTaxon NoNoNoNo Population Population Population Population codecodecodecode CountryCountryCountryCountry RangeRangeRangeRange Locality/MassifLocality/MassifLocality/MassifLocality/Massif Coordinates Coordinates Coordinates Coordinates (Longitude °E/ (Longitude °E/ (Longitude °E/ (Longitude °E/ Latitude °N)Latitude °N)Latitude °N)Latitude °N) O. montana DC. subsp. transsilvanica (Simonk.) Jáv. 1 OTRM Ro SW Carpathians Piatra Iorgovanului Peak, Retezat Mts. 45°16’55.96” 22°50’45.09” 2 OTR Ro SW Carpathians Piule Peak, Retezat Mts. 45°18’25.7” 22°54’31.4” 3 OTM Ro SE Carpathians Cearcănu Peak, Maramureşului Mts. 47°38’57.96” 24°49’54” 4 OTCh Ro SE Carpathians Toaca Peak, Ceahlău Mts. 46°59’35.3” 25°57’57.3” 5 OTGH Ro SE Carpathians Ocsem Peak, Giurgeu- Hăşmaş Mts. 46°40’41” 25°50’11” 6 OTC Ro SE Carpathians Zăganu Peak, Ciucaş Mts. 45°29’22” 25°58’39.1” 7 OTPC Ro SE Carpathians Piatra Craiului Mică Peak, Piatra Craiului Mts. 45°33’10.3” 25°15’47.6” 8 OTB Ro SE Carpathians Caraiman Peak, Bucegi Mts. 45°24’56.7” 25°29’51.71” 9 OTBV Ro SE Carpathians Postăvaru Peak, Bârsei Mts. 45°33’58.88” 25°33’02.22” 10 OTF Ro SE Carpathians Jgeabul Văros Peak, Făgăraş Mts. 45°36’20.82” 24°35’37.68” O. montana 11 OMAC Fr Alps Col d’Izoard, Cottian Alps 44°49’36” 6°43’48” 12 OMA Fr Alps Col du Lautaret, Dauphiné Alps 45°04’09.13” 6°24’05.23” 13 OMJ Fr Alps Colomby de Gex, Jura Mts. 46°19’38.75” 6°0’4.35” 14 OMAD Mne Dinaric Alps Durmitor, Dinaric Alps 43°06’26.75” 19°0.1’10.38” 15 OMTW Po W Carpathians Wawoz Krakow, High Tatras 49°10’27.24” 20°08’11.97” 16 OMBT Sk W Carpathians Saddle between Mt. Muran and Mt. Novy, Belianske Tatras 49°14’55” 20°11’00” SSR fingerprinting 23 microsatellites (original code names: MtBA01B04R2, MtBA27D09F1, MtBB36F05F1, MtBA04C08R1, MtBB22G10F1, MtBC47B06F1, MtBB44F02R1, AG81, BI74, AL79, BG178, AL46, AW265, AW567861, PV-at001, BM141, MTIC326, MTIC272, MTIC230, MTIC21, BM175, BM152, BM137) developed by Peakall et al. (1998), Yu et al. (2000), Gaitán-Solís et al. (2002), Julier et al. (2003), Gutierrez et al. (2005), and Zhang et al. (2007) for other leguminous species were tested for transferability in O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and O. montana. Băcilă I et al. (2023). Not Sci Biol 15(2):11549 4 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). Table 2Table 2Table 2Table 2. PCR programs used for SSR amplifications. PVat001, MtBB22G10, MtBA27D09, AG81, PVat001, MtBB22G10, MtBA27D09, AG81, PVat001, MtBB22G10, MtBA27D09, AG81, PVat001, MtBB22G10, MtBA27D09, AG81, BG178, BM141, and MTIC272BG178, BM141, and MTIC272BG178, BM141, and MTIC272BG178, BM141, and MTIC272 represent the original names of the markers (see also Table 3 for references) PCR PCR PCR PCR stepsstepsstepssteps PVat001PVat001PVat001PVat001 MtBB22G10MtBB22G10MtBB22G10MtBB22G10 MtBA27D09, MtBA27D09, MtBA27D09, MtBA27D09, AG81AG81AG81AG81 BG178, BM141, BG178, BM141, BG178, BM141, BG178, BM141, MTIC272MTIC272MTIC272MTIC272 Initial denaturation 94 °C, 2 min 94 °C, 3 min 94 °C, 3 min 95 °C, 5 min Denaturation 94 °C, 45 sec 94 °C, 45 sec 94 °C, 45 sec 94 °C, 30 sec Annealing temperature 50 °C, 45 sec 50 °C, 1 min 51 °C, 1 min 50 °C, 30 sec Elongation 72 °C, 1 min 72 °C, 1.5 min 72 °C, 1.5 min 72 °C, 1 min Repet steps 2-4 35x 35x 40x 35x Final elongation 72 °C, 5 min 72 °C, 10 min 72 °C, 10 min 72 °C, 10 min 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 3Table 3Table 3Table 3. Characteristics of seven microsatellite loci used for cross-transferability in Onobrychis sp. LocusLocusLocusLocus Primer sequence (5’Primer sequence (5’Primer sequence (5’Primer sequence (5’----3’)3’)3’)3’) Allele size Allele size Allele size Allele size range (bp)range (bp)range (bp)range (bp) ReferenceReferenceReferenceReference MtBA27D09 F:GAAGAAGAAAAAGAGATAGATCTGTGG R: GGCAGGAACAGATCCTTGAA 100-326 Gutierrez et al., 2005 MtBB22G10 F: CCAGTGGCAGCTACGGTACTA R: GAGACGGAGGAGAAGTTGCTT 149-161 Gutierrez et al., 2005 AG81 F: ATTTTCCAACTCGAATTGACC R: TCATCAATCTCGACAAAGAATG 134-184 Peakall et al., 1998 BG178 F: ACCCACTCAACTCAACACACAC R: TTCTCCTTGACCAACCTTGATT 184-187 Zhang et al., 2007 PV-at001 F: GGGAGGGTAGGGAAGCAGTG R: GCGAACCACGTTCATGAATGA 157-266 Yu et al., 2000 BM141 F: TGAGGAGGAACAATGGTGGC R: CTCACAAACCACAACGCACC 103-487 Gaitán-Solís et al., 2000 MTIC272 F: AGGTGGATGGAGAGAGTCA R: TCATGAATAGTGGCACTCAA 132-210 Julier et al., 2003 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) Băcilă I et al. (2023). Not Sci Biol 15(2):11549 5 phylogenetic tree based on the Shared Allele distance and the Neighbor-Net method. Bootstrap values were calculated from 1000 replicates. Results 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). TableTableTableTable 4444. Number of alleles, PIC, and gene diversity values for seven SSR loci analysed in Onobrychis sp. SSR locusSSR locusSSR locusSSR locus No. of allelesNo. of allelesNo. of allelesNo. of alleles Gene diversityGene diversityGene diversityGene diversity PICPICPICPIC MtBA27D09 6 0.6500 0.5957 MtBB22G10 4 0.6618 0.6033 AG81 4 0.5000 0.3750 BG178 2 0.5000 0.3750 PV-at001 6 0.6486 0.5931 BM141 6 0.5000 0.3750 MTIC272 4 0.7000 0.6454 MeanMeanMeanMean 4.5714.5714.5714.571 0.59430.59430.59430.5943 0.50890.50890.50890.5089 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 5Table 5Table 5Table 5. Genetic characterization of seven polymorphic microsatellite loci tested across sixteen populations of Onobrychis sp. Ho = observed heterozygosity; He = expected heterozygosity. LocusLocusLocusLocus O. montana O. montana O. montana O. montana DC. subspDC. subspDC. subspDC. subsp. transsilvanica . transsilvanica . transsilvanica . transsilvanica (Simonk.) (Simonk.) (Simonk.) (Simonk.) Jáv.Jáv.Jáv.Jáv. Onobrychis montanaOnobrychis montanaOnobrychis montanaOnobrychis montana HHHHoooo HHHHeeee HHHHoooo HHHHeeee MtBA27D09 0.366 0.183 0.611 0.305 MtBB22G10 0.550 0.275 0.166 0.083 AG81 0.100 0.05 0.333 0.167 BG178 0.952 0.525 0.667 0.333 PV-at001 0.066 0.033 0.222 0.111 BM141 0.433 0.216 0.277 0.139 MTIC272 0.450 0.225 0.750 0.375 The UPGMA analysis (data not shown, manuscript in preparation) managed to clearly differentiate all the 16 populations of Onobrychis, exhibiting taxonomic and geographic delineation. Băcilă I et al. (2023). Not Sci Biol 15(2):11549 6 DiscussionDiscussionDiscussionDiscussion 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, LÖve, 1975; and respectively O. montana 2n=28; LÖve, 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. ConclusionsConclusionsConclusionsConclusions 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’ ContributionsAuthors’ ContributionsAuthors’ ContributionsAuthors’ 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. Băcilă I et al. (2023). Not Sci Biol 15(2):11549 7 Ethical approvalEthical approvalEthical approvalEthical approval (for researches involving animals or humans) Not applicable. AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements We are grateful to Andreas Tribsch, Nadir Alvarez, Rolland Douzet, Zbigniew Mirek, Liviu Filipaș, Mihai Puşcaş, Adrian Ilie Stoica, and Tudor Ursu for their valuable help in collecting the plant material. This work was financially supported by a grant from the Romanian National Authority for Scientific Research, CNDI–UEFISCDI, project number PN–II–RU–PD–2012–3–0005; 15/26.04.2013, as well as by the Core Project BIORESGREEN, subproject BioClimpact no. 7N/03.01.2023, code 23020401. Conflict of InterestsConflict of InterestsConflict of InterestsConflict of Interests The authors declare that there are no conflicts of interest related to this article. ReferencesReferencesReferencesReferences Avcı S, Ilhan E, Erayman M, Sancak C (2014). 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