Morphological and Molecular Characterization and Relationships of Turkish Local Eggplant Heirlooms

A total of 38 eggplant genotypes, of which 32 were heirloom accessions collected from different regions of Burdur province five were different local genotypes from other provinces, and one was a cultivar, were used as reference in this study. The phylogenetic relationships among these heirlooms were evaluated using 40 morphologic descriptors and five randomly amplified polymorphic RAPD markers. The horizontal dendrograms were created by using UPGMA with both morphologic and molecular data. Burdur heirloom accessions showed high genetic diversity based on morphological and molecular data. The genetic similarity rates ranged from 0.29 to 0.91 according to the morphological data, and ranged from 0.84 to 0.98 according to the molecular data. Molecular data generated by RAPD method, compared to morphological data, were insufficient to reveal genetic diversity. Therefore, in order to confirm genetic variations, studies based on other molecular methods are necessary. The regional genetic populations include a wide eggplant genetic diversity which can be good source for the breeding studies performed in the future.


Introduction
Eggplant, which was called as the king of vegetables (Daunay and Janick, 2007), is an indigenous plant of India (Weese and Bohs, 2010).It has been well known since BC III and cultivated for 1,500 years in Asia (Kashyap et al., 2003).It is cultivated as a perennial in tropical areas, while it is cultivated as annual in subtropical areas (Kowalska, 2008).Eggplant is placed in Solanum genus and includes wide genotypic and phenotypic variation (Fukuoka et al., 2010).Eggplant is thought to have been developed from the wild ancestor, Solanum insanum has small, round, green, thick-skinned and bitter taste fruits (Barchi et al., 2010).Cultivation S. insanum of had been performed in China, India and Thailand (Daunay et al., 2001).Large fruiting eggplants were cultured in India in early time, and small fruiting was cultured at IV century in China and at IX century in Africa (Sekera et al., 2007).First cultivated eggplants were described as high tall plants, with big spines on the calyx, small, bitter fruiting and with high seed content in fruits (Swarup, 1995).Mutation, natural pollination and hybridization, together with selection gave rise to genetic diversity, as well as in decreasing of prickles and bitterness at fruit, changing of fruit shape, size and color (Frary et al., 2007).Genetic diversity accumulated and many different heirlooms emerged in countries where it was cultivated (Prohens et al., 2003).Entrance of the eggplant to Turkey was carried out by the silk-road.The genetic diversity accumulated in producing areas and by trade of eggplant throughout centuries in Anatolia (Janick, 2001).Eggplant cultivation was done in open field until the second half of the 1970s in Turkey and then cultivation under protected cultivation started.The eggplant cultivation in greenhouse was begun with local varieties.However, the growers preferred hybrid F1 varieties which have the cylindrical and dark purple or black colored fruiting and using of them has become commonly in a short period (Ekiz and Boyaci, 2001).Also, the increase of use F1 hybrids in the open field cultivation was observed in recent years.Steadily decrease was seen in the cultivation of local varieties compared to the hybrids (Cericola et al., 2013).The genetic diversity is low among the genotypes which have dark purple-black fruits (Muñoz-Falcón et al., 2009).In recent years, one of the important problems faced by eggplant breeding programs, as well as in other species, is a narrowing of the genetic base.To create variations, time-consuming and expensive methods are needed, including mutation breeding, interspecific hybridization and biotechnological approaches.The genetic variation contained by heirlooms among is seen in the previous studies (Demir et al., 2010;Muñoz-Falcón et al., 2008, 2009;Prohens et al., 2003Prohens et al., , 2008Prohens et al., , 2011)).Therefore, collection and characterization of genetic resources is required for the improvement of new varieties.In this respect, locations of collected eggplants in Burdur province were represented on the map (Fig. 1) and their geographic coordinates were defined (Table 2).

Morphological observations
For each accession, the seeds were sown in seedling treys containing peat moss.Seedlings in 4-5 leaves stages were planted in glasshouse.Twenty plants were planted for each genotype.Morphological observations were performed according to 40 descriptors chosen among the International Board for Plant Genetic Resources Institute (IBPGRI), International Union for the Protection of New Varieties of molecular characterization is more reliable then morphological characterization (Li et al., 2010).
Here we investigate the genetic relationships between Turkish heirlooms using morphological and molecular data is presented here.

Materials and methods
Morphological and molecular studies were conducted at Bati Akdeniz Agricultural Research Institute, Antalya, Turkey in 2010-2013.
In total 38 materials were used in the study of these.32 were local heirlooms collected from Burdur province during survey studies.Materials origin, providing method and place/company of origin are given in Table 1.Long purple commercial variety (YRL 68) and heirlooms originating in other province (YRL 1,YRL 3,YRL 6,YRL 57,and YRL 79) were used as reference cultivars for classification of Burdur province heirlooms.

Survey
The survey studies were performed at nine different locations in Burdur province in September both in 2010 and 2011.In total 32 materials were collected from different farms.Every record represents the average of three readings which were taken from the equatorial region, spaced equidistantly.
DNA extractions and PCR analysis DNA extractions from young leaves were performed according to a modified Doyle and Doyle (1990) method by using CTAB protocol (Mutlu et al., 2008).RAPD analysis was performed according to Demir et al. (2010).The amplification reactions were released containing 20 ng DNA, 0.5 unite Taq polymerase, 2.5 µL 10X buffer, 3.5 µL 25 mM MgCl2, 2 µL 2.5 mM dNTPs, 2 µL RAPD primer for RAPD-PCR.DNA was amplified in a thermal cycler.It was programmed for an initial 5 min denaturation step at 94 °C, followed by 35 cycles of a 30 s denaturation step at 94 °C, 1 min annealing at 35 °C, 45 s extension step at 72 °C, followed by a final 8 min extension step at 72 °C.
For molecular analysis data, each genotype was identified for each primer based on the presence (1) and absence (0) of bands.In order to show the variations STANDARDIZATION module was used.Correlation matrix adapted to SIMINT module was used to determine of correlation coefficient.The dendrograms were drawn using the clustering method UPGMA via the SHAN module.The cophenetic correlation coefficient was calculated with Mantel method to evaluate the efficiency of clustering.

Results
A high morphological diversity was observed among eggplant genotypes characterized by quantitative and qualitative descriptors.Some of the phenotypic observation data related with important variable traits (calyx prickles, fruit size, weight, shape and curvature, skin color, groove etc.) are reported in Table 4.The fruits calyx of all genotypes had prickles.However, nearly 50% of them had lower numbers of prickles.Most of the genotypes had between 20-70% of their fruit length covered by the calyx.The degree of fruit curvature in 50% of the genotypes was slight.Nearly 30% percent of the genotypes had fruits with grooved appearance.Dominant fruit color was mostly purple and distribution of this color was 50% regular, 22% mealy, 18% mottled, and 2% striped according to the genotypes.Fruit flesh color was generally white and hole in the fruit was mostly absent.The lowest total soluble solid content (brix value) was 2.8.The highest brix value (6.1) was determined in YRL 15 which was collected from Cine village The average diameter of the fruits ranged from 32.67 to 73.22 mm.Fruit length and fruit weight average ranged between 10.9 and 23.3 cm, 0.100 and 0.235 kg, respectively.
Skin color characteristics of the eggplant heirlooms are shown in Table 5. Fruit color varied greatly among different genotypes.Regarding the skin Lightness (L*), higher values were obtained from genotypes which were mostly, cream, yellowish, orange-yellowish or goldenred in color (L* > 70).When L* values were ranged from 48 to 58 together with h° values ranged from 80 to 112, it is observed that these genotypes' skin color were generally greenish-yellow, chartreuse or green in color.Regarding the b* values, very low values near or below zero mostly indicated that the skin color was cyan (if h ° > 285); blue (if h ° > 240); purple (if h ° > 285); magenta (if h ° > 350).It is observed from the research, that fruits which had lower h ° values (1.05 < h ° < 12.60) were found to be red in color.Chroma values were varied from 3.82 to 24.26.
Totally 40 basic morphological descriptors were used for to determine the phylogenetic relationships among the Burdur local heirlooms.The Eigen value was 84.A 2-way Mantel test (Mantel, 1967) method was performed.Approximate Mantel t-statistic test were t = 10.0925,p = 1.0000.The matrix correlation (r) was 0.72.The similarity rates according to the coefficient similarity of genotypes ranged between 0.29 and 0.91.
Two major groups were revealed using the dendrogram generated by the UPGMA method using morphological data (Fig. 2).First group (Group A) was consisted of YRL 75, YRL 65, YRL 61 and YRL 59.These genotypes showed low genetic similarity with reference genotypes and cultivar (Long purple).Second group was divided into three subgroups.Group B consist of YRL 19 which showed high genetic similarity with reference cultivar YRL 68 (Long purple).The highest genetic similarity was observed in Group D between genotypes YRL 47 and YRL 51.The fruits of eggplant Burdur heirlooms belonging to the Group C are shown in Fig. 3.A total of 65 amplified RAPD bands were generated.Twenty nine bands were polymorphic and the mean percentage of polymorphism was 44.61%.OPO-10 primer produced the maximum numbers of bands (18).Although the OPB-07 primer produced the minimal number of bands (8), it revealed a 100% polymorphism (Table 6).The OPH-2, OPB-07, OPO-10 and OPL-16 primers' PCR products and their band patterns are shown in Fig. 4a-d.The phylogenetic relationships among 38 genotypes were evaluated using 29 polymorphic loci of the genomic DNA generated using randomly amplified polymorphic DNA (RAPD) technique.The approximate Mantel t-test statistic were t = 8.9263, p = 1.0000.The matrix correlation (r) was found to be 0.64.The similarity rates according to the coefficient similarity of genotypes ranged between 0.84 and 0.98.
Using molecular data two major groups emerged in the dendrogram generated with UPGMA method (Fig. 5).First group branched into two subgroups and were showed in brackets as Group A and Group B. Group A was consisted of YRL 15 and YRL 27.Group E included most of the genotypes had high similarities with reference cultivar.The highest genetic similarity was observed in Group D with 98% percentage similarity index between genotypes YRL 6 and YRL 44.YRL 1 and YRL 27 were detected as the most distantly genotypes from each other in the cluster.Fruits of Burdur eggplant heirlooms YRL 6 and YRL 44, which showed highest genetic similarity with 98% percentage are shown in Fig. 6.Also, the fruits of Burdur heirlooms which grouped together in cluster E in the UPGMA dendrogram using molecular data and reference culture are shown in Fig. 7.

Discussions
Eggplant has a wide genetic diversity in the regions where it is cultivated, although they are not native to the region (Muñoz-Falcón et al., 2008).In spite of the fact that Turkey is not the place origin of eggplant, wide genetic diversity has been reported in Turkey (Demir et al., 2010;Tümbilen et al., 2011aTümbilen et al., , 2011b)).It is clearly evident in this study data that Burdur, which is a small geographical region, had a rich genetic diversity.Also, a wide genetic variability was determined in both Spain and Jordan local genotypes (Prohens et al., 2003).Local genotypes can contribute to enhancing the gene pool used in breeding studies and to help increase heterosis (Muñoz-Falcón et al., 2009).In recent years, some factors like cultivation of commercial varieties instead of heirlooms, construction of buildings on agricultural land, and innovation in cultivation methods have led to erosion of plant genetic resources (Cericola et al., 2013).Therefore, there is a need collecting and identification of local heirlooms before they disappear (Muñoz-Falcón et al., 2008).Some characters that contributed to genetic diversity were as flowering dates, the number of seeds per fruits, fruit features, and the growth pattern of plants.These features are controlled by several genes in eggplant (Frary and Doğanlar, 2003).Solanum melongena accessions could characterize these descriptors like bigger and flabby fruits, less flowers/inflorescence, few fruits/plant and higher acidity etc. compared to the wild relatives (Polignano et al., 2010).Consistent with previously works, a higher diversity for most morphological descriptors was recorded in the collection of Burdur local heirlooms identified in this study.Fruit color can be cream, green, red, reddish-purple, dark purple or black, and some varieties produce fruit which is where the genetic variation necessary for future varietal improvement and for addressing future breeding challenges will be found.
Molecular markers linked with agronomic traits are useful tools for marker assisted selection and mapping candidate genes studies in breeding programs (Nunome et al., 2009; Wang et al., 2010).Some of the RAPD markers used were determined to have relationship with coloring of stem, calyx and fruiting in eggplant (Frary et al., 2003).This provides a great advantage for identifying features affecting by ecological conditions (Biswas et al., 2009;Nunome et al., 2001).Relationships among eggplant materials have been studied by molecular studies for use by eggplant breeders (Furini and Wunder, 2004).As mentioned above the RAPD markers revealed as a potential useful tool for determination of genetic diversity.It was found that RAPD analysis in eggplant with four primers had been adequate to identify of genetic diversity (Biswas et al., 2009).Also, RAPD markers were more effective than ISSR for revealing genetic diversification as reported by Ali et al. (2011).Tiwari et al. (2009) previously reported that even two of the 29 RAPD primers were found to be sufficient for identification of local cultivars.All RAPD markers used in the study have produced polymorphic bands as expected.However, if fruit criteria are taken into consideration for distinguishing in the dendrogram and grouping, it is not enough for the breeders.More informative DNA markers can be used to provide better progress in eggplant breeding studies (Stàgel et al., 2008).Simple Sequence Repeats (SSRs) methods were found more successful for distinguishing closely related eggplant cultivars (Hurtado et al., 2012;Muñoz-Falcón et al., 2009;Prohens et al., 2008).The approach of using SSR markers instead of RAPD markers can provide better results in the phylogenetic relationships studies.Muñoz-Falcón et al. (2009) reported that if the morphological and molecular data are considered together, they can be provide sufficient and useful information for the breeders.Similarly to the findings of other studies, we suggest that the molecular evidences need to be supplemented by morphological data to validate the phylogenetic relationships among the genotypes.It is very important to note that the genetic variations can't be detected by only showing a dendrograms generated by molecular data.

Conclusions
The local populations are of great importance for the breeders so that they adapted well to their cultivated areas.There is a need collecting and identification of these heirlooms before integrated to the breeding programs.The aim of the study was to investigate the diversity among heirlooms cultivated in Burdur province.A high genetic diversity was determined widely among them.These materials can be of a potential value for the breeders.

Fig. 1 .
Fig. 1.Geographic map of district and village in Burdur province of Turkey including collection places

Fig. 2 .
Fig. 2. UPGMA dendrogram showing phylogenetic relationships of local Burdur eggplant heirlooms together with reference cultivars using morphological data

Fig. 3 .
Fig. 3.The fruits of Burdur eggplant heirlooms belonging to the Group C

The 101 Table 1 .
Origin, providing method and place/company of origin of the materials used in the study

Table 2 .
Geographical coordinates of local heirloom eggplant collected in Burdur province of Turkey , blue, purple, or intermediate colors between adjacent pairs of these basic colors.The L*, a*, and b*, C and h ° values obtained from six samples of each eggplant accession.

Table 3 .
Descriptors used for characterization and evaluation of eggplant accessions used in the study

Table 4 .
Some of the phenotypic descriptors related important variable traits of eggplant

Table 5 .
Skin color characteristics of eggplant X: Means represent three 10-fruit samples ± SD.