Giorgia Carletti · Luigi Cattivelli · Lorenzo Vietto · Giuseppe Nervo

Abstract Salix L. (willow) is the largest genus of the family Salicaceae and plays an important role in riparian habitats, wetlands and in shrub tundra. Due to the different implications for the species belonging to this family, it is fundamental to identify molecular tools characterizing relevant clones. A set of six multilocus and multiallelic simple sequence repeat (SSRs) markers are presented, leading to 390 polymorphic fragments considered as single dominant markers and able to discriminate successfully 92 S. alba L.from 24 Salix spp. The polymorphic fragments have been used to perform genetic diversity studies, and to investigate population structures and cluster analysis in a germplasm collection. The results highlight the capability of the six SSRs to be powerful genetic resources in applied forestry research, both to distinguish S. alba clones from Salix spp.and to perform genetic population studies for breeding programs.

Keywords Microsatellites · Salix · Genetic diversity

Introduction

Salix (willow) is the largest genus of the family Salicaceae which includes approximately 450 species (Argus et al.2010) distributed across the temperate to arctic regions of the Northern Hemisphere, only entering tropical regions along mountain ranges. Willows have been introduced worldwide such as in Europe (Argus 1997), in North America in northern Mexico (Argus et al. 2010), and in China (Fang et al.1999). Salix spp. are mostly shrubs, playing strategic roles in riparian habitats, wetlands and in the tundra biome. During the last century, interest on the environmental applications of the genus, such as for biomass production and bioremediation (Karp et al. 2011) led to the increase in resources for conserving Salix genetic diversity. Various molecular markers, such as RAPDs, SSRs, AFLPs, have been used to assess genetic diversity of this genus in germplasm collections (Trybush et al. 2008; Gupta et al. 2009; Singh et al.2013), and for germplasm characterization. However, insuff icient information is available. In this study, 116 Salix spp.(92 S. alba and 24 interspecific hybrids) (composed by individual collected in several European countries) were genotyped with six multilocus and multiallelic simple sequence repeats (SSRs) for genetic diversity studies. The data were used to investigate population structure and to generate informative parameters such as the diversity genetic index,polymorphism information, and the degree of genetic variation. The results will permit the discrimination of Salix alba genotypes in a Salix spp. natural collection, and to perform genetic diversity studies within all the 116 Salix accessions.

Materials and methods

Plant material and DNA extraction

The total genomic DNA of the 116 Salix plants (92 Salix alba and 24 Salix spp., Table 1), grown and maintained in an open field at the CREA-Research Centre for Forestry and Wood, Casale Monferrato (AL), Italy, was isolated from fresh tissue following the modified CTAB method of Lauron-Moreau et al. (2013). DNA concentrations were determined using Nanodrop (Invitrogen, city, country)and quality checked on 1% agarose gel. All samples were genotyped with six variable SSR loci previously published(Smulders et al. 2001; Barker et al. 2003; Singh et al. 2013).PCR amplifications were carried out in 10 μL volumes containing 10 ng genomic DNA, 1 × PCR buffer, 1.5 mM MgCl 2 , 0.4 mM dNTP mix, 0.25 μM forward primer, 2.5 μM reverse primer, 1U Dream Taq DNA polymerase (ThermoFisher Scientific, Waltham, Massachusetts, US). The forward primer was end-labelled with 6-carboxyfluorescein(6-FAM) or hexachloro-carboxyfluorescein (HEX) for the visualization of PCR products on capillary DNA analyzer.To identify the major number of alleles, amplification was obtained performing a touchdown PCR program consisting in an initial denaturation at 94 °C, 2 min; 10 cycles of touchdown PCR including: 94 °C for 40 s; first cycle annealing temperature of 65 °C followed by 10 cycles, reduced by 1.0 °C/cycle, then 72 °C for 45 s, followed by 25 cycles at 94 °C for 40 s; 55 °C for 1 min; 72 °C for 2 min, with a final extension at 72 °C for 20 min. The microsatellites were analyzed on GA3500 (Applied Biosystem, Foster City,California, US) automated capillary electrophoresis using GeneScan ROX 500 as standard size. Visualizations and sizing of the PCR fragments were performed using the GeneMapper software version 4.1 (Applied Biosystem, Foster City, California, US). The polymorphic SSRs gave 38 to 131 alleles each. The four Salix SSRs reported allele sizes from 76 to 185 bp (SB38), 75 to 592 bp (SB80), 75 to 345 bp(SB100), and 68 to 395 bp (SB 85), while two Populus SSRs showed allele size from 80 to 223 bp (PMGC1) and 75 to 237 bp (WPMS20) (Table 2). Each allele was scored for its presence/absence (1/0) in each individual. Polymorphic information content (PIC) values were calculated for each SSR primers according to Eq. 1:

Table 1 Salix germplasm collection identified by name, clone number and country of origin

where P ij is the frequency of theith pattern revealed by the j th primer summed across all patterns revealed by the primers (Botstein et al. 1980).

Structure population, genetic diversity and cluster analysis

The genetic structure of Salix spp. was examined using the STRU CTU RE 2.3 software package (Pritchard et al. 2000)to determine the most likely number of independent genetic clusters (K). Setting an admixture model and independent allele frequencies (Falush et al. 2003), the best number of groups (K) was evaluated from 1 to 15, with 10 independent runs per K value. Each run was executed using 5 × 104burn-in periods and 10 5 MCMC repetitions after burn-in for population clustering were used. The optimal K value was identified from the maximum value of DK (Evanno et al.2005) as implemented in STRU CTU RE HARVESTER 0.6.93 (Earl and von Holdt 2012). CLUMPP 1.1.2 (Jakobsson and Rosenberg 2007) was used to summarize the results of the optimal K value based on the pairwise similarity average of individual assignments across runs through Greedy’s method and G’ statistics. ARLEQUIN 3.5.1.2 (Excoffier and Lischer 2010) was used to estimate the basic descriptive molecular diversity statistics and the Analysis of Molecular Variance (ANOVA) to calculate the partitioning of genetic variation between the two populations and within the total germplasm collection. Tajima’s D neutrality tests were performed using the ARLEQUIN software to detect evidence for deviation from a neutral equilibrium model of evolution in the population.

Genetic similarity analysis among all individuals, through the analysis of principal coordinates (PCoA) and the construction of a dendrogram (UPMGA), was carried out using PAST software. The two principal components were plotted to evaluate the dispersion of the data and the p-distance was estimated to compare the mean intra- and interspecific genetic distances among the three subpopulations.

Results

Evaluating the SSRs informativeness

Genotyping 116 clones with 6 SSRs produced 390 polymorphic fragments. Allele size range, number of amplicons, PIC values, nucleotide sequence accession number of markers are reported in Table 2.

SB80 gave the highest number of alleles (131), whereas PtPMGC2163 showed the lowest number of amplicons (38).The highest PIC value was associated with the SB38 marker located on chromosome 9 of the Populus genome with a PIC value of 0.727.

The six microsatellites carried out 84 haplotypes within the 92 S. alba individuals, showing a considerable genetic diversity among haplotypes (0.9974 ± 0.0023). The S.alba var. Belders and S. alba clone SE86-004 belonged to the same haplotype, such as S. alba_S-59-004 and S.alba_SI63-010, S. alba_SI64-010 and S. alba_SI62-096,S. alba_SI63-007, S. alba_SI68-015, S. alba_SE62-007, S.alba_SE67-007.

Population genetic structure, PCoA analysis and dendrogram cluster analysis

The Bayesian approach in the software program STRU CTU RE, through the application of Evanno’s method, was employed to evaluate genetic structure of the collection. The results suggest the optimal number of K = 2, implying the existence of two genetically homogeneous groups (Fig. 1 a,b). Subsequently, the intragroup analysis led to K = 7 as reported in the seven colored subpopulations of alleles(Fig. 1 c). All S. alba were identified from 1 to 92. Individuals from 1 to 29 shared a higher prevalence of alleles represented by the color rose; individuals 53-63, 68-77 and 80-86 shared alleles identified with the color yellow, and the alleles of genotypes 64-67, 78-79, 87-88, 91 belonged for > 90% to green. All the genotypes from 93 to 116, representing Salix spp. individuals, were structured as a distinct and homogeneous group and all were mainly characterized by alleles reported with red color, except for individual 95.

ashs g e n o m e R ef er en ce s p e c i e s Salix burjatica Salix burjatica l us t r i c h o c a rpa rpa Popu Salix burjatica l us ni g r a l us t r i c h o c a in Salix and Popu Popu in l in/F ts Z f a m i ly p r o t e i n-li ke a u x i n-r e s p o n s i v e f a m i ly p r o t e i n otif, the nucleotide sequence accession x in re si st-a n t 1 f a m i l y p r o t e i n e ti ca l p r o t e i n ashington, W Annotation f W coding Tubu Populu 7)R ec ep to r k i n a s e UR No SA Au region Hypoth niversity o 0009s16570 e qu en ce A c c e s s i o n N o. i n P o p u l u s g e n o m e 0017s02110 0019s11160 0006s09940 r 13)hr 10)(C tide S R_PThr 9)R_PThr 1 R_R_PThr 19)PThr 6)35 cleo 93 (Ch PO o. in S a l i x g e n o m e N u(C(C PO PO 4020(C AJ2972 ooperative at the U(C PO GQ c e N uc le ot id e S e q u e n c e A c c e s s i o n N.1 ism information content (PIC), the repeat m.1.1.1 enetics C 92 4426 97 4426 96 4426 00 4427 AF AF AF AF--olecular G oplar M C: P Repetition u ni t s e q u e n(tg)n(ccg)n(tc)n(ccg)n(ttctgg)n(ag)n MG 0.21 0.73 0.3−0.64 0.69 0.52 orph f a l l e l e s (b p)P IC S iz e r a n g e s o8 5 95 92 olym 45 37 23 e are reported. P 76-1 68-3-5 75 75-3 75-2 80-2 n size, the p t al n u m b e r o f alleles s genom 62 61 56 43 38 To 130 Range of a l l e l e s f o r c l o n e 2-20 2-13 3-29 2-15 ge of amplico 1-8 1-8 arker, the ran n and A CTT T C A the annotation on Populu G GA A A A TC CG TC G A GA C C T AA C CA C TC T GA T CA G TA T TT G TC T GT T CA A GT T AC T AT G CA G AC T A CG G TG C GG G TT C AC lifi ed for each m s A TT A AC G TT T GTT GT T AT C TT C TC A AG G GG T TC G AT e localizatio A CA ber of alleles ampG AG er sequences SR Details of the six S G GA T GA T AA A AT C CC C AG A CT A TC A TT G GT A TA osom A CT A TG C AA T AA T TA C AG T TG G AG A TG G AG A CA A TT G TC T TA G GT C AC G TA G CG C TT G AA G AC A TC T TG F-CC es, the chrom Prim R-CT R-GT F-CT F-TA R-AT F-AT R-AT F-GT R-AT F-CA R-CG The total num pulu Table 2 20 2163 tate, U s genom SA e r n a m e Prim 38 5 0 0 0 SB-8 SB-8 SB-1 SB MS WP GC PM in Po ington S

Fig. 1 a Results from Structure Harvester analysis for K = 2 and K = 7. b and c Structure of population of the willow gene pool based on Bayesian inference among 116 genotypes analyzed with 6 microsatellite markers. Each individual is shown as a vertical line divided into segments representing the estimated membership proportion in the two (b) and seven (c) ancestral genetic clusters inferred with STRU CTU RE. Individuals from 1 to 92 belong to Salix alba, individuals from 93 to 116 represent Salix spp

Fig. 2 PCoA graph of Salix alba and Salix spp. collection showing an evident clusterization in two groups

As an alternative to the Bayesian method, a Principal Coordinates Analysis was carried out (PCoA, Fig. 2). The first two principal components, able to discriminate all Salix alba from Salix spp., collectively explained 29.8%of the total genetic variance in the Salix gene pool. Using the genetic distance matrix of values between −1 and +1,where 1 represented the perfect genetic identity and −1 the major distance, all the individuals were compared. Within S.alba, the genotype pairs closest/similar were S. alba_SI82-001- S. alba_SI63-007, S. alba_SI82-001- S. alba _SI68-015, S. alba_SI82-001- S. alba_SE62-007, S. alba_SI82-001- S. alba_SE67-007, showing a genetic similarity index of 0.934, In contrast, the two most distant genotypes were S. alba _SE62-008 and S. alba_SE66-038 wit6 h a genetic similarity index of −0.084. Among the Salix spp. subgroup,the closest genotypes were S. nigricans and S. koreensis with a genetic distance index of 0.628, while the most distant were, curiously, the two S. babylonica individuals (one cultivated in China and the other in Italy), with an index value of −0.072. Extending the analysis to all 116 individuals, S.mongolica and S. **alba _SI64-003 showed the major distance (genetic similarity index of −0.116).

In addition to the structure population analysis and the PCoA approach, to evaluate the genetic diversity a dendrogram was created, based on the correlation matrix among the alleles scores and the Neighbor Joining (NJ) algorithm of similarity. Figure 3 conf irmed the seven different clouds identified by the analysis of structure population in which the Salix germplasm collection was divided.

Genetic diversity analysis

The six SSRs markers yielded a total of 390 alleles, with an average of 65 alleles per locus ranging from 38 (PMGC2163)to 131 (SB80) (Table 2), leading to 108 haplotypes (84 S.alba and 24 Salix spp.). The allele diversity investigation based on Nei’s index was performed within the S. alba collection, within Salix spp. individuals and between S. alba and Salix spp.

There were 256 loci (65.6%) and 249 (63.8%) in S. alba and Salix spp., respectively, resulted in polymorphic and used for genetic diversity analysis. The gene diversity over loci, (calculated with pairwise differences method), was estimated at 0.9974 ± 0.0023. A major degree of heterozygosity occurred in Salix spp. (observed heterozygosity(Ho) = 0.11101, expected heterozygosity (He) = 0.11864),compared to S. alba (observed heterozygosity = 0.09332,expected heterozygosity = 0.07898), but we did not assist to high value of He and Ho (> 0.3) as expected and reported in other allogamous plants (Perdereau et al. 2014; Wang et al. 2014).

ANOVA among populations revealed the percentage of variation of 21.3%, while within the populations, the percentage of molecular variation was 78.7%, considering a Fixation Index (FST, Rousset 1997) of 0.21316. The neutrality test, developed with Tajima’s test, reported a Tajima’s D value of −1.47675 and −1.75968 in Salix spp. and S. alba,respectively.

Discussion

Population structure, PCoA

The Salix germplasm collection in this study is a mix of close and of distantly related willows, polyploid and diploid dioecious trees which considered each allele (390 alleles obtained by genotyping the individuals with 6 SSRs), as a single dominant marker. This set of markers provided highly informative data used to discriminate S. alba genotypes from Salix spp. and allowed an investigation of gene diversity and genetic structure of this Salix germplasm.

Several studies have analyzed the genetic information of consecutively selected populations of Salix spp. using molecular markers aimed to improve breeding strategies(Singh et al. 2013; Lauron-Moreau et al. 2013). However,such a study to identify a minimum set of microsatellites able to capture S. alba genotypes within a Salix spp. germplasm collection has not been carried out.

The most frequent S. alba SSR is SB-85 containing an allele (amplicon of 81 bp) present in 65 genotypes (70.6%)and another allele (amplicon of 88 bp) present in 69 genotypes (75%).

Fig. 3 Dendrogram graph using a correlation matrix and NJ algorithm of similarity. Two major groups (Salix spp and Salix alba) and seven subpopulations are shown, as identified after structure population and PCoA analysis

In general, a high level of diversity was observed among alleles. In other studies using a similar genotype panel,the mean number of polymorphic alleles per locus ranged between 9 and 1 (Singh et al. 2013) and between 8 and 2(Bozzi et al. 2015). In this work, the number of alleles was more conspicuous. These results addressed the high polymorphic regions in S. alba due to the multiallelic loci prof ile from 38 (PMGC2163) to 131 (SB80), with an average of 65 alleles per locus.

The evidence of structured populations among several Salix genotypes has not been previously reported. Two subpopulations, (using Principal Component Analysis, PCoA),representing S. alba and Salix spp. are shown in Fig. 2. This was also conf irmed by performing the structure of population analysis which identified two major subpopulations(K = 2), and seven minor subpopulations (K = 7), conf irmed by a dendrogram generated by Neighbor-Joining algorithm.Each genotype represented in Fig. 1 c is the result of several subpopulations (different colors), showing the high number of natural crosses and hybridizations. Only a few number of genotypes, such as numbers 4, 10, 38, belong to only one subpopulation (one color), showing the absence of rearrangements in the regions amplified by the six SSRs.

Genetic diversity analysis

Given the breeding system of willow species, (obligated outcrosser), genetic variation within populations is usually as high as the genetic differentiation between populations, whereas gene flow between species is fairly moderate (Barcaccia et al. 2003). The genetic parameters of the Salix collection in this study are summarized in Table 2.A major degree of heterozygosity occurred (Loci = 249,Ho = 0.11101, He = 0.11864), compared to S. alba(Loci = 256, Ho = 0.09332, and He = 0.07898), although there were not high values of He and Ho as expected. The neutrality test reported negative Tajima’s D values both for Salix spp. and for S. alba (−1.47675 and −1.75968, respectively), explaining low average heterozygosity. We address the low heterozygosity identified within the germplasm collection to the methodological approach used in this work.Considering each allele as a single marker, the codominant aspect of SSRs was bypassed, losing information about the degree of heterozygosity in the natural population. The ANOVA revealed a lower percentage (～ 21%) of variation divided among populations, about 79% of the variation was attributed to differences within populations. This result is in agreement with Hamrick and Godt (1996) who explained that outcrossing woody plants tends to be more genetically diverse with less genetic differentiation among populations.Actually, there are no elements in this study to identify the biological aspects causing variation in the population, but it is evident that the different origins and the geographical distribution do not influence the genetic variability in this germplasm collection.

Conclusion

This study identifies genetic resources useful in Salix applied research for both clone identification and genetic population studies. A minimum set of multilocus and multiallelic SSRs has been described for their ability to discriminate S. alba germplasm from Salix spp. germplasm. Genotyping a natural population of 116 Salix clones with the 6 SSRs, the population structure, the genetic diversity and the cluster analysis showed the different clusterization of S. alba individuals compared to Salix spp, conf irming the importance of these markers in Salix breeding programs.