Abstract
There is a need for classifying and conserving local apple cultivars from two main regions in Bosnia and Herzegovina (B&H). Consequently, 71 local apple accessions (31 from Sarajevo and 40 from eastern Bosnia) were evaluated with a set of 10 simple sequence repeats (SSRs). These accessions were compared with 37 reference cultivars (24 traditional B&H and 13 international cultivars maintained at the ex situ collection Srebrenik) to determine synonyms, homonyms, and possible introgression of foreign genotypes into the local apple germplasm. Using 10 primer pairs of microsatellites, we were able to amplify 135 alleles for the 71 local apple accessions. Detection of more than two different alleles per locus was observed for 34 accessions. Fourteen different homonyms and 12 synonyms were identified among all the apple cultivars analyzed. Analysis of molecular variance (AMOVA) revealed a significant genetic differentiation between most of the groups analyzed but not between accessions from Sarajevo and eastern Bosnia. Bayesian method and admixture analysis of the allele frequency allowed classification of all accessions analyzed and found that they fell into two main groups [reconstructed panmictic populations (RPPs)]. Strong genetic differentiation between these two groups was detected using AMOVA (fCT = 0.130; P < 0.001). Analysis of the genetic structure indicates that overall, approximately half of the local apple cultivars from Sarajevo and eastern Bosnia (52% and 45%, respectively) grouped in the RPP1 consisting mainly out of international reference cultivars, whereas the other half grouped in the RPP2 with traditional B&H reference cultivars. Both neighbor joining (NJ) cluster analysis based on Bruvo genetic distance and factorial correspondence analysis (FCA) confirmed the results of the genetic structure analysis. The molecular data show that both apple accessions from Sarajevo and from eastern Bosnia represent an interesting source of diversity, which needs to be conserved.
Traditional apple cultivars in B&H have significantly higher genetic and morphological diversity than modern international cultivars grown in this country (Gasi et al., 2010, 2011). Because genetically heterogeneous material represents a potential source of positive pomologic traits and resistance to biotic and abiotic stress (Kellerhals et al., 2004), local traditional B&H apple cultivars may be useful for inclusion in international apple breeding programs and therefore must be conserved. To conserve the aforementioned genetic resource, the main ex situ collection in the country was established in the fruit tree nursery Srebrenik (Spionica, B&H, lat. 44°45′45″N, long. 18°29′49″E) in 2000. Aside from local traditional B&H cultivars, the established collection maintains many international cultivars widely grown in the region, which often serve as reference genotypes in apple germplasm studies. Genetic assessment of 39 accessions, 24 well-known traditional B&H cultivars and 15 modern international cultivars, maintained at the ex situ collection Srebrenik has recently been performed using molecular markers (Gasi et al., 2010). The molecular analyses revealed a clear differentiation between traditional B&H cultivars and some of the most widely cultivated modern cultivars in the region, confirming the importance of conserving B&H apple germplasm. Unfortunately, there are high costs associated with maintaining a large ex situ collection and in a post-conflict country with limited resources, there is an even greater need for increased efficiency and elimination of redundancies by avoiding duplication and mislabeling.
In 2010 and 2011, an effort was made to fill the gaps in the apple collection Srebrenik. During the original collecting missions, completed in 2000, which resulted in establishment of the collection, two geographical areas of Bosnia and Herzegovina were omitted: the capitol Sarajevo with the surrounding area and large parts of eastern Bosnia. The mentioned areas had at that time been significantly depopulated as a result of the war and only in recent years have most of the original inhabitants returned to their homesteads. To ensure that the existing ex situ apple collection contains as much as possible of the diverse B&H apple germplasm, we surveyed the Sarajevo region and parts of eastern Bosnia for local apple cultivars. This was done with the help of local agricultural experts and the traditional knowledge gathered from the farmers living in surveyed parts of the country. The results of the survey indicate a presence of a large number of local cultivars that are grown in the aforementioned regions. However, without knowing the genetic identity of these genotypes, it is impossible to estimate if local cultivars from the surveyed areas can be considered part of traditional B&H apple germplasm or if they are mostly mislabeled international cultivars. Because DNA markers are much more efficient in revealing mislabeled plant accession compared with traditional pomological characterizations (Nybom and Weising, 2010), we chose this approach in our study. SSR molecular markers have shown great promise as tools for managing Malus ex situ germplasm collections as well as for collection and preservation strategies of these genetic resources (Hokansson et al., 1998). Examining ex situ collections with microsatellite markers has so far been performed in a number of studies (Garkava-Gustavsson et al., 2008; Gharghani et al., 2009; Guarino et al., 2006; Guilford et al., 1997; Hokanson et al., 2001; Pereira-Lorenzo et al., 2006; Ramos-Cabrer et al., 2007; Urrestarazu et al., 2012; Van Treuren et al., 2010), which has greatly contributed to more efficient management and use of the analyzed collections. Aside from managing established collection, SSR markers have also proved very useful in determining synonyms and genetic relationships of local apple cultivars found on farms (Bassil et al., 2009; Pereira-Lorenzo et al., 2008; Routson et al., 2009).
The objectives of this study therefore were: 1) to compare genetic profiles of local apple cultivars from Sarajevo and eastern Bosnia with a database constructed for previously characterized traditional and international apple cultivars in B&H using a set of 10 SSR markers; and 2) to evaluate the need for conservation of local apple cultivars from Sarajevo and eastern Bosnia.
Materials and Methods
A total of 71 accessions were sampled from a number of backyards and small farms in Sarajevo and eastern Bosnia (31 from Sarajevo and 40 from eastern Bosnia) (Table 1). The apple accessions, all grafted on generative rootstocks, are maintained in good condition by farmers and their fruits are used for a wide range of purposes (fresh consumption, manufacturing of traditional desserts, and production of apple brandy). Farmers identified each accession as a local, traditional B&H cultivar. In addition, 37 apple accessions (24 well-known traditional B&H cultivars and 13 international cultivars widely grown in the region) maintained at the ex situ collection Srebrenik and genotyped in a previous study by Gasi et al. (2010) using the same set of SSR markers were included as reference cultivars (Table 2). Traditional B&H genotypes used as reference cultivars in this study are part of the apple germplasm introduced in Bosnia and Herzegovina during the reign of the Ottoman and later Austrian–Hungary Empire (Bubić, 1952). The mentioned germplasm has extensively been selected by farmers but has yet to be included in plant breeding programs established in the region.
Seventy one apple accessions (31 sampled from farms in Sarajevo and 40 from farms in eastern Bosnia) analyzed in this study using 10 simple sequence repeat markers.z
Twenty-four previously genotyped traditional Bosnia and Herzegovina (B&H) and 13 international apple accessions maintained in the ex situ collection Srebrenik used as reference cultivars in this study.
Simple sequence repeat analyses.
Tissue samples (leaves) for DNA analyses were collected in the spring of 2011 immediately after bloom from a single tree for each accession located in Sarajevo and eastern Bosnia. DNA extraction was performed with a Qiagen DNeasy® Plant Mini Kit (Qiagen, Valencia, CA) according to the protocol included in the kit. Ten primer pairs used for SSR amplifications, previously published by Gianfranceschi et al. (1998) and Liebhard et al. (2002), were chosen in cooperation with a research team from Wageningen University and Research Center and Central gene bank of Netherlands. Polymerase chain reaction (PCR) amplification of SSR sequences was performed in a Veriti TM Thermal Cycler (Applied Biosystems, Foster City, CA) using fluorescent-labeled primers, which enabled the detection of PCR products using an ABI 3130 Genetic Analyzer (Applied Biosystems). All PCR amplifications were performed as described in Gianfranceschi et al. (1998). The PCR product was diluted with ddH20 (1:50), then added to 8.75 μl HiDi and 0.25 μL Genescan 500 LIZ size standard. Data were analyzed using the software package GeneMapper 4.0 (Applied Biosystems).
Biostatistical analyses.
SSR profiles obtained by genotyping 71 accessions from Sarajevo and eastern Bosnia were added to the existing microsatellite database constructed in a previous study on apple accessions maintained at the main ex situ apple collection in B&H. All accessions analyzed were divided into four groups for biostatistical purposes: Sarajevo accessions, eastern Bosnia accessions, international reference cultivars, and traditional B&H reference cultivars.
Population genetics software SPAGeDI 1.2 (Hardy and Vekemans, 2002) was used for calculating allele frequencies, gene diversity (Nei, 1978), and F statistics (Weir and Cockerham, 1984). Analyses of molecular variance (Exoffier et al., 1992), based on the stepwise mutation model (Ohta and Kimura, 1973), was performed using GenoType software with 1000 permutations. Genetic distance between accessions (Bruvo et al., 2004) based on a two-phased mutation model (Di Rienzo et al., 1994) and Shannon-Weaver’s information index was calculated using GenoDive software. Both programs are part of the GenoType/GenoDive package (Meirmans and Van Tienderen, 2004).
Neighbor-joining cluster analysis, based on the mentioned genetic distance, was performed in MEGA 5 software (Molecular Evolutionary Genetics Analysis) (Tamura et al., 2011). A multivariate analyses, FCA, based on allele frequencies was performed using Genetix 4.02 (Belkhir et al., 2001), which meant excluding the triploid genotypes, as proposed by Pereira-Lorenzo et al. (2008).
To examine population structure and assign individual genotypes to either traditional B&H or international reference cultivars apple germplasm, we used the Bayesian model-based cluster procedure within Structure Version 2.2.3 (Pritchard et al., 2000), which is suitable for analysis of diploids and polyploids. We computed K (unknown) RPPs of individuals testing K (log-likelihood) = 1 to 5 for 108 accessions assuming that sampled cultivars were from unknown origin anonymous trees. Assignment of one cultivar in a RPP was provided by a probability of membership qI chosen at 80% according to similar studies (Breton et al., 2008; Pereira-Lorenzo et al., 2008; Urrestarazu et al., 2012). Tests were done based on an admixture model in which the allelic frequencies were correlated and a burn-in period of 200,000 and 500,000 iterations for data collection was applied (Urrestarazu et al., 2012).
Results and Discussion
Simple sequence repeat polymorphism.
Of 71 accessions from Sarajevo and eastern Bosnia, 64 showed a unique SSR profile (Table 3). When the reference cultivars, genotyped in a previous study (24 well-known traditional B&H cultivars and 13 international cultivars widely grown in the region), were included, 14 different homonyms and 12 synonyms were detected among the 108 accessions. Five accessions from eastern Bosnia and three from Sarajevo were already present in the Srebrenik collection under different names. Two accessions registered as local cultivars in the Sarajevo region had in fact the same SSR profile as the international reference cultivar Jonagold, indicating the presence of international genotypes in Sarajevo local apple germplasm. After excluding synonyms and duplicates, 96 different genotypes remained in the new database.
Simple sequence repeat (SSR) profiles (allele sizes expressed in base pairs) of 71 apple accessions from Bosnia and Herzegovina (31 sampled from farms in Sarajevo and 40 from farms in eastern Bosnia) analyzed using 10 SSR markers.
Detection of more than two different alleles per locus was observed for 34 accessions (18 accessions from Sarajevo and 16 from Eastern Bosnia), or 48% of 71 analyzed local cultivars. Seven of the mentioned accessions had only one locus with third allele, whereas most others had several SSR loci with a third allele. Although flow cytometry is most commonly applied for identifying triploids, because none of the used primer pairs displayed the ability to amplify more than one locus in our study, a presence of a third allele indicates a triploid state. A detected percentage of triploids is considerably higher than that reported for the local Spanish apple cultivars (29%) (Ramos-Cabrer et al., 2007), as well as compared with the data from the previous molecular study on B&H apple germplasm (21%) (Gasi et al., 2010).
Ten primer pairs of microsatellites managed to amplify 135 alleles or an average of 13.5 alleles per locus for the 71 apple accessions sampled from Sarajevo and eastern Bosnia, which represents a 30% increase compared with the results of the previous study on apple germplasm in Bosnia and Herzegovina (10.4 alleles per loci) (Gasi et al., 2010). Number of detected alleles per locus ranged from seven alleles for CH01H02 to 22 alleles for CH02C02a (Table 4). Average number of alleles per locus obtained in this study was also higher than that reported for the Italian, Danish, and Swedish material as well as foreign and local cultivars from northwestern Spain (Garkava-Gustavsson et al., 2008; Guarino et al., 2006; Larsen et al., 2006; Pereira-Lorenzo et al., 2006). Of the two investigated B&H regions, a higher number of alleles was amplified among apple accessions from eastern Bosnia (12.4) compared with Sarajevo accessions (10.5). Lowest numbers were detected for international reference cultivars (6.3).
Allele size range (bp) for all the analyzed apple accessions, number of alleles per locus, and gene diversity (Nei, 1978) based on 10 simple sequence repeat loci for 31 apple accessions from Sarajevo, 40 apple accessions from eastern Bosnia, 71 accession genotyped in this study, 24 traditional Bosnia and Herzegovina (B&H), and 13 international reference apple cultivars from ex situ collection Srebrenik.
Analyses of the allele frequency for all the loci revealed SSR alleles specific for each of the four groups of accessions examined. A total of 23 alleles was exclusively present among the accessions from eastern Bosnia, whereas nine alleles were only detected among accessions registered in Sarajevo. Nine different alleles were specific for traditional B&H reference cultivars, whereas only three alleles were specific for the international reference cultivars.
Gene diversity for individual markers ranged from 0.62 for CH02C02b to 0.93 for CH02C02a. Average gene diversity estimated in this study was 0.80 (0.79 for Sarajevo and 0.80 for eastern Bosnia accessions), higher than the values published by Hokanson et al. (2001) (0.62), Liebhard et al. (2002) (0.74), and Guarino et al. (2006) (0.73). Gene diversity for 71 accessions from Sarajevo and eastern Bosnia was identical to the one calculated for 109 traditional Spanish cultivars (0.80) (Pereira-Lorenzo et al., 2006) and slightly lower than the values reported by Urrestarazu et al. (2012) (0.82) who analyzed 538 apple cultivars in northeastern Spain. Shannon-Weaver’s information index (I) showed diversity to be highest in eastern Bosnia accessions (I = 1.59) and the lowest in international reference cultivars (I = 1.11). Traditional B&H reference cultivars and accessions from Sarajevo had similar values for this index (I = 1.38 and I = 1.39, respectively). It is important to note that this measure is suitable only when comparing groups of similar size (similar number of analyzed genotypes). Based on the number of amplified alleles, specific alleles, and gene diversity, accessions from eastern Bosnia seem to represent the most genetically diverse group of all four analyzed.
Values for the inbreeding coefficient Fit, calculated for the 71 apple accessions sampled from Sarajevo and eastern Bosnia, ranged from –0.159 for CH01H10 to 0.182 for CH05E03 with an average value of –0.047 for all loci (Table 5). The average Fit value calculated for all 108 analyzed accessions (international and traditional B&H reference cultivars included) was also very low (–0.009). Both values were much lower than those previous published for Bosnian, Spanish, and Iranian apple accessions (Gasi et al., 2010; Gharghani et al., 2009; Pereira-Lorenzo et al., 2008). Because high Fit values indicate heterozygosity deficiency, a lower Fit value obtained in this study points to no loss of heterozygosity among the analyzed accessions. Very low values obtained for Fis coefficient (–0.050 for Sarajevo and eastern Bosnia accessions and –0.028 for all four groups of accessions) is in concordance with the calculated Fit inbreeding coefficient.
F-statistics (Weir and Cockerham, 1984; estimated with SPAGeDI 1.2) for 71 apple accessions from Sarajevo and eastern Bosnia and for all 108 analyzed accessions (reference cultivars included) based on 10 simple sequence repeat loci.
The differentiation between apple accessions from Sarajevo and eastern Bosnia (Fst value) (Weir and Cockerham, 1984) for all 10 loci was very low (0.002; P value nonsignificant), possibly indicating a common genetic structure of the genotypes sampled from these two regions. Fst value calculated for all 108 analyzed accessions (international and traditional B&H reference cultivars included) was significant (0.019; P < 0.0001) but much lower than that reported by the previous studies, which examined differentiation between traditional and international apple cultivars in Bosnia and in Spain (Gasi et al., 2010; Pereira-Lorenzo et al., 2006, 2008). Further examination of genetic differences among the four groups of apple accessions was performed using AMOVA. AMOVA calculated for all 108 analyzed accessions indicated that most of the variance was retained within the groups of accession (99%), whereas 1% of the total diversity was attributed to the differences among the analyzed groups of accessions (Table 6). The largest percentage of variance between the groups was detected among apple accessions from Sarajevo and international reference cultivars (fCT = 0.051; P < 0.01) as well as among apple accessions from eastern Bosnia and international reference cultivars (fCT = 0.058; P < 0.01).
Analysis of molecular variance (AMOVA) based on the 10 simple sequence repeat loci of 71 apple accessions corresponding to two areas in Bosnia and Herzegovina (B&H) (Sarajevo and eastern Bosnia) as well as 24 traditional B&H and 13 international reference apple cultivars from the ex situ collection Srebrenik and to the two reconstructed populations (RPPs) defined by Structure (Pritchard et al., 2000).
The smallest percentage of variance between the groups was detected among apple accessions from Sarajevo and eastern Bosnia (fCT = 0.001; P value nonsignificant), which is in concordance with the low Fst values calculated for these groups. Nonsignificant fCT was also calculated among apple accessions from eastern Bosnia and traditional B&H reference cultivars, indicating that both of the groups, at least in part, belong to the same germplasm. Although the percentage of variance among apple accessions from Sarajevo and traditional B&H reference cultivars was low (2.9%), it was significant (P < 0.05), indicating that a significant part of Sarajevo accessions belongs to the international apple germplasm.
Genetic relationships.
Bayesian analyses, which were done within Structure, revealed that almost all of the international reference cultivars (92%) grouped in RPP1, whereas most traditional B&H reference cultivars (71%) were in RPP2. Local apple cultivars from Sarajevo and eastern Bosnia grouped in both RPPs. The first RPP included 53 genotypes, all of them with qI greater than 80%. Of those, 12 were international reference cultivars, seven traditional B&H reference cultivars, 16 apple accessions from Sarajevo, and 18 accessions from eastern Bosnia. The second RPP included 55 genotypes, 53 of them with qI greater than 80%. Of those, 16 were traditional B&H reference cultivars, 15 apple accessions from Sarajevo, and 22 accessions from eastern Bosnia. Overall, approximately half of the local apple cultivars from Sarajevo and eastern Bosnia (52% and 45%, respectively) grouped in the RPP consisting mainly out of international cultivars, whereas the other half grouped in the RPP with traditional B&H reference cultivars. Structure analysis therefore confirmed the conclusion based on the AMOVA results, that the local accessions analyzed in this study represent a mixture of traditional B&H and international germplasm. The variance among RPPs (13%; P < 0.001) (Table 6) was significant, although slightly lower than reported by Pereira-Lorenzo et al. (2008) (14.41%) for RPPs constructed with Spanish and international cultivars but higher than the values obtained by Gasi et al. (2010) (9.2%) in a previous study on apple germplasm in B&H.
NJ cluster analysis was used to group all 108 examined accessions (Fig. 1). The tree was constructed based on a genetic distance model, which takes into consideration mutation characteristics of microsatellites and allows genotypes with different ploidy level to be included in analyses (Bruvo et al., 2004). Although the separation of most of the traditional B&H and international reference cultivars was evident, accessions from Sarajevo and eastern Bosnia were dispersed throughout the entire dendrogram. A large number of the mentioned accessions formed clusters exclusively with traditional B&H reference cultivars, whereas a slightly smaller number grouped very tightly with international reference cultivars indicating introgression of foreign genotypes into the local germplasm and/or presence of mislabeled international cultivars. In Figure 1, all cultivars with qI over the minimum threshold of 80% were assigned to one of the two RPPs. It is interesting to note that although NJ cluster analysis based on Bruvo genetic distance represents a different biostatistical approach than the Bayesian model-based cluster procedure in Structure, the results of both analyses largely concurred. Namely, only three accessions from the RPP1 clustered tightly with genotypes from RPP2, whereas only one accession from RRP2 grouped in a cluster containing RPP1 accessions (Fig. 1).
Neighbor joining cluster analysis based on polymorphisms of 10 simple sequence repeat loci in 71 local apple cultivars and 37 reference apple cultivars (written in capital letters) using Bruvo genetic distance (Bruvo et al., 2004). In brackets are the reconstructed populations (RPP1 or RPP2) to which each cultivar is assigned with probability of membership qI greater than 80%.
Citation: HortScience horts 48, 1; 10.21273/HORTSCI.48.1.13
To get a clearer picture of the relationships between the analyzed groups of accessions, a multivariate approach based on FCA analysis was used. The three-dimensional plot (Fig. 2) revealed a clear differentiation between the traditional B&H and international reference cultivars, whereas the groups of accessions from Sarajevo and eastern Bosnia overlapped. Because the genetic differentiation between these groups is negligible (nonsignificant values for Fst and P in AMOVA), this was completely expected. Apple accessions from both Sarajevo and eastern Bosnia generally clustered closer to traditional than to the international reference cultivars, whereas some overlapping between eastern Bosnia and traditional B&H reference group was noted. Both of these observations are in concordance with the results of ANOVA (Table 6). Factorial correspondence analysis was also done for the two RPPs and it revealed a strong genetic differentiation between RPP1 and RPP2 (Fig. 3), which is in concordance with the results of AMOVA.
Multivariate analysis (factorial correspondence analysis) of simple sequence repeat data for 37 apple accessions from two areas in Bosnia and Herzegovina (B&H) (13 from Sarajevo and 24 from eastern Bosnia) as well as 19 traditional B&H and 12 international reference apple cultivars from ex situ collection Srebrenik (diploids analyzed only).
Citation: HortScience horts 48, 1; 10.21273/HORTSCI.48.1.13
Multivariate analysis (factorial correspondence analysis) of simple sequence repeat data for reconstructed populations (RPPs) calculated using Structure (Pritchard et al., 2000) (only diploid genotypes with qI greater than 80%). Ninety-two percent of the international reference cultivars grouped in RPP1, whereas 71% of traditional Bosnia and Herzegovina (B&H) reference cultivars were in RPP2. Local apple cultivars from Sarajevo and eastern Bosnia grouped in both RPPs.
Citation: HortScience horts 48, 1; 10.21273/HORTSCI.48.1.13
Conclusions
Molecular data indicated that both groups of apple accessions analyzed (from Sarajevo and from eastern Bosnia) represent an interesting source of diversity, which needs to be conserved. Although introgression of international reference cultivars in local apple germplasm was detected, both groups contain a large number of diverse traditional B&H apple cultivars, which have so far not been preserved, and inclusion of all this material in the ex situ collection is imperative. In this process, priority should be given to all the local apple accessions from Sarajevo and eastern Bosnia, which grouped in the RPP2. Accessions maintained in the ex situ collection can then be investigated for advantageous agronomic traits.
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