Biochemical Systematics and Ecology 59 (2015) 45e53
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Transferability of Cucurbita SSR markers for genetic diversity assessment of Turkish bottle gourd (Lagenaria siceraria) genetic resources Mehtap Yildiz a, *, Hugo E. Cuevas b, Suat Sensoy c, Ceknas Erdinc a, Faheem S. Baloch d a
Department of Agricultural Biotechnology, Faculty of Agriculture, Yuzuncu Yil University, 65080, Van, Turkey USDA-ARS, Tropical Agriculture Research Station, 2200 Pedro Albizu Campos Avenue, Mayaguez, PR 00680, USA Department of Horticulture, Faculty of Agriculture, Yuzuncu Yil University, 65080, Van, Turkey d Department of Field Crops, Faculty of Agricultural and Natural Sciences, Abant Izzet Baysal University, Bolu, Turkey b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2014 Accepted 2 January 2015 Available online
The genetic diversity present in crop landraces represents a valuable genetic resource for breeding and genetic studies. Bottle gourd (Lagenaria siceraria) landraces in Turkey are highly genetically diverse. However, the limited genomic resources available for this crop hinder the molecular characterization of Turkish bottle gourd germplasm for its adequate conservation and management. Therefore, we evaluated the efficacy of 40 SSR markers from major cucurbit crops (Cucurbita pepo L. and Cucurbita moschata L.) in 30 bottle gourd landraces, together with 16 SRAP primer combinations. In addition, we compared the genetic relationship between bottle gourd and 31 other cucurbit accessions (11 Cucurbita maxima, 3 C. moschata, 5 C. pepo subsp. ovifera, 10 C. pepo and 2 Luffa cylindrica). Twentyseven Cucurbita SSR markers showed transferability to bottle gourd. SSR markers amplified 59 alleles, in bottle gourd genome with an average of 1.64 alleles per locus. Together, SSR and SRAP markers amplified 453 fragments across the 61 accessions, and clearly discriminated L. siceraria and L. cylindrica from the other cucurbit species. Genetic diversity analysis separated edible cucurbit from ornamentals, while population structure analysis classified L. siceraria in two subpopulations defined by fruit shape, rather than geographical origin. The results indicated that the genomic resources available for Cucurbita species are valuable to study and preserve the genetic diversity of bottle gourd in Turkey. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Bottle gourd Cucurbits Genetic diversity Marker transferability Molecular markers
1. Introduction Bottle gourd (Lagenaria siceraria [Mol.] Standl.) is an edible, medicinal, and utilitarian plant species in the Cucurbitaceae family. Its estimated genome size is of ~334 Mb, distributed over 11 chromosomes (Decker-Walters et al., 2001). Phylogenetic analysis has showed a close genetic relationship of bottle gourd with other economically important cucurbit species such as cucumber, melon, and watermelon (Xu et al., 2011). Indeed, the initial partial sequence of bottle gourd genome revealed a 90% sequence similarity to the cucumber genome.
* Corresponding author. Tel.: þ90 432 2251701/2662; fax: þ90 432 2251104. E-mail address:
[email protected] (M. Yildiz). http://dx.doi.org/10.1016/j.bse.2015.01.006 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
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Africa is the presumed center of origin of bottle gourd (Pitrat et al., 1999), although wild progenitors have not been identified there (Xu et al., 2011). Even if Turkey is not the center of origin of bottle gourd, its landraces show wide phenotypic diversity in fruit size and shape (Yetisir et al., 2008). Recently, morphological diversity studies found larger diversity among domesticated bottle gourd than within the wild relatives, which has been attributed to human selection (Sakar, 2004; Morimoto et al., 2005; Koffi et al., 2009; Mladenovic et al., 2012). Levi et al. (2009) investigated the genetic relationship among landraces of L. siceraria from different countries and reported that these clustered into two major groups: one group included accessions from India, Mediterranean region, and Northeast Africa, and the other included accessions from Southern Africa, America, China, Indonesia, and Cyprus. Microsatellites, also known as simple sequence repeats (SSRs), consist of tandem repeats of simple nucleotide units (1e6 bp) that are widely spread throughout the genomes of plants and animals (Jarne and Lagoda, 1996). Another useful marker system, Sequence-Related Amplified Polymorphism (SRAP), developed by Li and Quiros (2001), has been demonstrated to have several advantages over other techniques of DNA fingerprinting, as it is simple, easy to carry out, and could be more useful for routine germplasm screening. Various molecular marker techniques have been successfully employed to study the genetic relationships among cucurbit species such as RAPD, SRAP, ISSR and SSR (Youn and Chung, 1998; Baranek et al., 2000; Gwanama et al., 2000; Ferriol et al., 2003; Inan et al., 2012; Gong et al., 2012). Unfortunately, few molecular resources are available for bottle gourd and its genetic relationship with other cucurbit species is unclear. Moreover, few reports investigated the level of genetic variation between bottle gourd accessions (Decker-Walters et al., 2001; Morimoto et al., 2006; Koffi et al., 2009; Xu et al., 2011; Srivastava et al., 2014). Therefore, it is desirable to develop new molecular tools to study genetic diversity and genome mapping of bottle gourd. In this regard, a recent work by Xu et al. (2011) reporting the initial partial sequence of bottle gourd, has taken an important first step towards the study of genetic diversity and genome structure of this species. However, besides these newly developed SSRs, the number of known SSR markers in bottle gourd is still too low for genomic studies. Genomic resources available for economically important species might be exploited in less studied, marginal crops. In the particular case of cucurbits, Stift et al. (2004) demonstrated that most of the 27 SSRs developed for Cucurbita pepo were transferable to Cucurbita moschata, Cucurbita maxima, and Cucurbita ecuadorensis. The transferability of SSR markers has aided to compare genomes within cucurbits (Dawei et al., 2011), and might be useful to clarify genome evolution of different species and genus. Turkey has always been a junction of cultures among Europe, Asia, and Africa. Besides, Turkey could have played an important role in spreading bottle gourd genetic resources from Africa to Asia or Europe. Despite the morphological diversity of bottle gourd present in Turkey, there is an evident lack of molecular markers to assess its genetic diversity. The number of SSR markers developed for bottle gourd is not enough for genome mapping and to develop highly saturated molecular maps, which in turn, would allow the identification of markers linked to traits of interest. Hence, we sought particularly to 1) evaluate the efficacy of 40 Cucurbita SSR markers to be used in bottle gourd accessions, 2) study the genetic relatedness among 30 Turkish bottle gourd accessions, and between the latter and 2 Luffa cylindrica and 29 Cucurbita accessions, and 3) provide information about the transferability of SSR markers to bottle gourd to molecular geneticists and breeders interested in developing linkage maps for this species. 2. Material and methods 2.1. Plant material Thirty bottle gourd landraces collected in 2010e2011 from 17 provinces of Turkey (Table 1) were used in this study. In addition, 11 accessions of C. maxima, 3 of C. moschata, 5 of C. pepo subsp. ovifera, 10 of C. pepo, and 2 of L. cylindrica were evaluated to compare genetic relationships with bottle gourd. Cucurbit germplasm was kindly provided by Yuzuncu Yil University, Cukurova University, Ondokuz Mayis University, and/or by growers and local seed suppliers. 2.2. Molecular analysis 2.2.1. DNA extraction Bulk DNA of eight individuals per accession was prepared from young leaves of three-week-old plants grown under greenhouse conditions. Total genomic DNA was isolated following the protocol by Doyle and Doyle (1990), with the minor modifications incorporated by Boiteux et al. (1999). Subsequently, DNA concentration was measured with NanoDrop 2000 (Thermo Scientific) and was adjusted into two sets of concentrations: 20 ng/ml and 5 ng/ml for SSR and SRAP applications, respectively for polymerase chain reaction (PCR). 2.2.2. SSR markers A total of 40 SSR markers, 16 derived from C. pepo and 24 from C. moschata, were selected based on their location on C. pepo genome (two SSRs for each linkage group; Gong et al., 2008). Briefly, PCR reactions were performed in a total volume of 15 ml, containing 7.15 ml water, 1.5 ml 10 DNA polymerase buffer, 1.2 ml dNTPs (2.5 mM each), 1 ml each primer at 5 mM, 0.15 ml Taq Polymerase at 10 U/ml (Fermentas) and 3 ml genomic DNA. The thermocycler was programmed as follows: initial cycle at 94 C for 5 min, 40 cycles at 94 C for 20 s, 48e65 C (depending on primer used) for 30 s, and 71 C for 1 min, followed by a final extension at 72 C for 5 min. The amplicons were size-fractionate for 5 h in a 3% (w/v) high-resolution agarose gel (Gene Pure
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Table 1 Cucurbit accessions used for genetic diversity analysis and marker transferability. Codea
Origin
Species
Codea
Origin
Species
L-01-1 L-01-2 L-06-1 L-15-1 L-19-1 L-21-1 L-30-1 L-30-2 L-30-3 L-30-4 L-33-1 L-33-2 L-33-3 L-35-1 L-39-1 L-39-2 L-39-3 L-39-4 L-54-1 L-54-2 L-54-3 L-56-1 L-61-1 L-63-1 L-63-2 L-63-3 L-65-1 L-65-2 L-72-1 L-80-1
Adana Adana Ankara Burdur Corum Diyarbakır Hakkari Hakkari Hakkari Hakkari Mersin Mersin Mersin _ Izmir
L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.
P-16-1 P-22-1 P-50-1 P-50-2 P-50-3 P-50-4 P-50-5 P-50-6 P-Aus-1 P-Aus-2 O-23-1 O-44-1 O-54-1 O-65-1 O-72-1 Mo-14-1 Mo-14-2 Mo-14-3 Mx-14-1 Mx-14-2 Mx-14-3 Mx-26-1 Mx-31-1 Mx-55-1 Mx-55-2 Mx-55-3 Mx-55-4 Mx-57-1 Mx-65-1 Lu-07-1 Lu-31-1
Bursa Edirne Nevs¸ehir Nevs¸ehir Nevs¸ehir Nevs¸ehir Nevs¸ehir Nevs¸ehir Austria Austria Elazig Malatya Sakarya Van Batman Bolu Bolu Bolu Bolu Bolu Samsun Eskis¸ehir _ Iskenderun Samsun Samsun Samsun Samsun Sinop Van Antalya Hatay
C. pepo L. C. pepo L. C. pepo L. C. pepo L. C. pepo L. C. pepo L. C. pepo L. C. pepo L. C. pepo L. C. pepo L. C. pepo ssp. ovifera C. pepo ssp. ovifera C. pepo ssp. ovifera C. pepo ssp. ovifera C. pepo ssp. ovifera C. moschata C. moschata C. moschata C. maxima C. maxima C. maxima C. maxima C. maxima C. maxima C. maxima C. maxima C. maxima C. maxima C. maxima L. cylindrica L. cylindrica
Kırıkkale Kırıkkale Kırıkkale Kırıkkale Sakarya Sakarya Sakarya Siirt Trabzon Sanlıurfa Sanlıurfa Sanlıurfa Van Van Batman Osmaniye
siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria siceraria
a Tripartite designation indicates genus or species/subspecies, the traffic plate number of the province of origin and numbered according to the order of collection, respectively. For genus: L Lagenaria, Lu Luffa; for species/subspecies: P pepo, O ovifera, Mo moschata, Mx maxima (e.g., L-65-1 means the first Lagenaria siceraria accession collected from Van province).
HiRes Agarose, ISC Bioexpress, Kaysville, UT) in 0.5 TAE buffer, and visualized with ethidium bromide. A 200 bp ladder (Fermentas) was used as molecular weight marker. 2.2.3. SRAP markers In this assay, 16 different primer combinations were evaluated using six forward and six reverse primers for amplification and polymorphism detection. The reaction mixture and protocols were similar to the ones described by Ferriol et al. (2003). Briefly, amplification was performed in 15 ml total volume reaction containing 7.15 ml water, 1.5 ml 10 DNA polymerase buffer, 1.2 ml dNTPs (2.5 mM each), 1 ml each primer at 5 mM, 0.15 ml Taq Polymerase at 10 U/ml (Fermentas), and 3 ml genomic DNA. The amplification protocol consisted of an initial denaturing step of 5 min at 94 C, followed by five cycles of three steps: 1 min of denaturing at 94 C, 1 min of annealing at 35 C, and 2 min of elongation at 72 C. In the following 30 cycles the annealing temperature was increased to 50 C, with a final extension step of 5 min at 72 C. The amplicons were size-fractionate during 4 h using 2% (w/v) high-resolution agarose gel in 0.5 TAE buffer, and visualized with ethidium bromide. A 200 bp ladder (Fermentas) was used as molecular weight marker. 2.3. Data analysis All amplicons were scored based on presence/absence to produce a binary matrix (1-presence/0-absence). Only clear and strong bands were recorded and used for further analysis. Polymorphism information content (PIC) of each SSR and SRAP marker was calculated with PowerMarker v3.25 software (Liu and Muse, 2005). Pairwise genetic similarity coefficient between individuals were calculated by shared allele distance (Chakraborty and Jin, 1993) using PowerMarker v3.25 software. The resulting distance matrix was subject to principal coordinate analysis (PCoA), as computed by GeneAlEx 6.5 (Peakall and Smouse, 2012). Accessions were clustered using the neighbor-joining method on PowerMarker v3.25, and visualized using the Interactive Tree of Life software (Letunic and Bork, 2011). 2.3.1. Population structure of Lagenaria siceraria The population structure and the inference of admixture ancestry were assessed with a model-based clustering method implemented in STRUCTURE 2.1 (Pritchard et al., 2000), using an admixture model with correlated allele frequencies. The K values were set from 1 to 10, with three independent runs for each value (50,000 burn-in periods and 150,000 Monte Carlo
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Markov Chain [MCMC]), and no a priori assumptions about the genetic grouping of the samples. The change in the log probability of data between successive K values was used to determine the real number of subpopulations. Independent runs for the correct number of subpopulations were matched by permutation in CLUMPP (Jakobsson and Rosenberg, 2007) to generate the optimum alignment over multiple runs. Subpopulations were assembled by grouping accessions with a membership probability higher than 0.70, while accessions with a lower membership probability were assigned to a mixed groups. 3. Results 3.1. Transferability of SSR markers across species Thirty-six (90%) working SSR markers (13 from C. pepo and 23 from C. moschata) were tested in L. siceraria (Table 2). The transferability of SSRs to L. siceraria varied with the source of origin of SSR markers; it was higher for SSRs originated from C. pepo (84.6%) than C. moschata (69.6%). The transferability of SSR markers from C. pepo and C. moschata to L. cylindrica was similar to L. siceraria (84.6 and 65.2%, respectively). 3.2. Diversity analysis A total of 36 SSR primers produced amplicons in the studied cucurbit species, and 27 of these were amplified in L. siceraria (Table 3). The number of amplicons per primer varied among species, ranging from 56 (L. cylindrica) to 142 (C. maxima). 60 amplicons were produced in L. siceraria, while 91, 117, and 86 were produced in C. pepo, C. pepo subsp. ovifera, and C. moschata, respectively. The diversity analysis for each species identified 59, 87, 96, 62, 148, and 31 amplicons in L. siceraria, C. pepo, C. pepo subsp. ovifera, C. moschata, C. maxima, and L. cylindrica, respectively. The most informative SSR primers were CMTp193 (PIC ¼ 0.48) for L. siceraria, CMTp125 (PIC ¼ 0.68) for C. pepo, CMTm84 (PIC ¼ 0.77) for C. pepo subsp. ovifera, and CMTp68 (PIC ¼ 0.82) for C. maxima. Five primers (CMTm68, CMTp247, CMTp68, CMTm209, and CMTp138) had a similar PIC value of 0.59 in C. moschata. In the case of L. cylindrica, CMTm252, CMTmC34, CMTm68, CMTm206, and CMTp138 were the only informative primers (PIC ¼ 0.38). The 16 SRAP primer combinations identified polymorphisms among cucurbits (Table 4). These primers generated 161 bands with an average of 10 bands per primer. The number of bands ranged from 18 in L. cylindrica to 80 in C. maxima, while 32, 39, 46, and 24 bands were observed in L. siceraria, C. pepo, C. pepo subsp. ovifera, and C. moschata, respectively. The most informative SRAP markers were as follows: Me2xEm1 (PIC ¼ 0.61) for L. siceraria, Me6xEm5 (PIC ¼ 0.61) for C. pepo, Me1xEm2 (PIC ¼ 0.77) for C. pepo subs. ovifera, and Me2xEm3 (PIC ¼ 0.59) for C. moschata. Three primers had a similar PIC value of 0.79 in C. maxima (Me6xEm6, Me5xEm2, and Me6xEm14), while Me1xEm2, Me2xEm3, and Me2xEm14 were the only informative markers for L. cylindrica (PIC ¼ 0.38). 3.3. Principal coordinate and cluster analysis Genetic distance obtained by the analysis of 453 fragments (292 bands from SSR and 161 bands from SRAP markers) ranged from 0.02 (L-54-3 vs. L-39-4) to 1.0 (several accessions), with a mean genetic distance of 0.71 between accessions. The mean genetic distance within the six cucurbit groups ranged from 0.17 (L. cylindrica) to 0.71 (C. maxima and C. pepo subsp. ovifera). The mean genetic distance was 0.18 among the 30 L. siceraria, 0.40 among the three C. moschata, and 0.47 among the 10 C. pepo accessions. Principal coordinate analysis clearly discriminated L. siceraria (group 1), L. cylindrica (group 2), and Cucurbita spp. (groups 3, 4, and 5) accessions (Fig. 1). The first two axes explained 79% of the total genetic variation. From the three groups that comprised Cucurbita ssp., one group (group 5) included all C. pepo and C. moschata accessions, together with four C. maxima accessions (Mx14-1, Mx14-2, Mx55-4, and Mx65-1). Another group (group 4) included four C. pepo subsp. ovifera and two C. maxima accessions (Mx14-3 and Mx31-1), while group 3 included one C. pepo subsp. ovifera (O-72-1), and five C. maxima accessions (Mx26-1, Mx55-1, Mx55-2, Mx55-3, and Mx57-1).
Table 2 Transferability of SSR markers to six cucurbit species: Lagenaria siceraria, Cucurbita pepo, Cucurbita pepo spp. ovifera, Cucurbita moschata, Cucurbita maxima, and Luffa cylindrica. Origin
Total working SSRs
L. siceraria
C. pepo
C. pepo subsp. ovifera
C. moschata
C. maxima
L. cylindrica
Total
Poly. SSRs
Total
Poly. SSRs
Total
Poly. SSRs
Total
Poly. SSRs
Total
Poly. SSRs
Total
Poly. SSRs
C. pepo
13
C. moschata
23
11 (84.6%) 16 (69.6%)
4 (36.4%) 8 (50%)
e
e
23 (100%)
9 (39.1%)
13 (100%) 23 (100%)
9 (69.2%) 13 (56.5%)
13 (100%) e
7 (53.8%) e
13 (100%) 23 (100%)
9 (69.2%) 14 (60.9%)
11 (84.6%) 15 (65.2%)
1 (9%) 4 (26.7%)
M. Yildiz et al. / Biochemical Systematics and Ecology 59 (2015) 45e53
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Table 3 Transferability of 36 SSR markers across the six cucurbit species. SSR
CMTp193 CMTp201 CMTm48 CMTm209 CMTp131 CMTmC67 CMTm120 CMTp46 CMTp174 CMTm130 CMTm252 CMTm84 CMTm144 CMTm66 CMTmC14 CMTp182 CMTm261 CMTm68 CMTp138 CMTm111 CMTm126 CMTm131 CMTp68 CMTm11 CMTm206 CMTmC34 CMTp158 CMTp125 CMTp247 CMTp210 CMTm224 CMTm187 CMTp132 CMTm219 CMTm83 CMTm207 Mean
LGa
LG1 LG1 LG2 LG2 LG3 LG3 LG4 LG5 LG5 LG6 LG6 LG7 LG7 LG8 LG8 LG9 LG9 LG10 LG10 LG11 LG11 LG12 LG13 LG13 LG14 LG14 LG15 LG16 LG16 LG17 LG17 LG18 LG19 LG19 LG20 LG20
C. pepo subsp. ovifera
C. moschata
Amp.b
L. siceraria No.c
PICd
C. pepo Amp.b
No.c
PICd
Amp.b
No.c
PICd
Amp.b
No.c
PICd
C. maxima Amp.b
No.c
PICd
L. cylindrica Amp.b
No.c
PICd
4 1 n.a 1 n.a n.a 1 1 3 n.a 1 2 1 n.a 1 2 n.a 6 2 3 n.a n.a n.a 5 2 2 3 2 1 2 3 2 1 3 4 1 1.67
4 1 n.a 1 n.a n.a 1 2 3 n.a 2 3 1 n.a 1 3 n.a 2 2 3 n.a n.a n.a 2 3 2 3 4 2 2 2 1 1 1 5 2 1.64
0.48 0.00 n.a 0.00 n.a n.a 0.00 0.06 0.41 n.a 0.06 0.32 0.00 n.a 0.00 0.32 n.a 0.24 0.33 0.41 n.a n.a n.a 0.36 0.26 0.16 0.31 0.28 0.12 0.36 0.36 0.00 0.00 0.00 0.52 0.12 0.15
3 4 1 3 3 1 1 1 3 2 1 2 3 2 2 3 1 5 3 3 1 2 3 5 2 2 1 6 2 3 5 2 1 4 3 2 2.53
2 4 2 3 5 2 1 1 2 2 1 2 3 4 2 3 1 3 3 1 2 2 4 1 2 1 1 5 2 3 4 2 1 3 4 3 2.41
0.36 0.61 0.27 0.41 0.64 0.36 0.00 0.00 0.33 0.16 0.00 0.16 0.47 0.67 0.36 0.55 0.00 0.47 0.31 0.00 0.16 0.16 0.61 0.00 0.16 0.00 0.00 0.68 0.36 0.41 0.58 0.16 0.00 0.49 0.45 0.41 0.30
7 6 2 3 3 1 2 2 5 1 4 3 3 1 3 3 2 7 2 2 3 1 3 6 2 2 2 7 1 4 5 2 3 6 5 3 3.25
3 3 2 3 4 2 3 2 3 1 3 5 4 2 2 2 2 3 2 2 3 2 3 2 2 2 3 4 2 2 3 2 3 4 3 3 2.67
0.50 0.50 0.36 0.50 0.67 0.27 0.50 0.27 0.50 0.00 0.50 0.77 0.67 0.27 0.27 0.27 0.36 0.50 0.27 0.27 0.50 0.27 0.50 0.27 0.27 0.27 0.50 0.67 0.36 0.27 0.50 0.36 0.50 0.67 0.50 0.50 0.42
3 4 1 3 2 1 1 1 3 1 2 1 2 1 2 3 1 6 3 3 1 2 3 6 1 2 1 5 2 3 5 1 1 4 3 2 2.39
2 2 1 3 2 1 1 1 2 1 2 1 2 2 1 2 1 3 3 1 1 2 3 2 1 1 1 2 3 2 2 1 1 2 2 2 1.72
0.35 0.35 0.00 0.59 0.35 0.00 0.00 0.00 0.35 0.00 0.35 0.00 0.35 0.35 0.00 0.35 0.00 0.59 0.59 0.00 0.00 0.35 0.59 0.35 0.00 0.00 0.00 0.35 0.59 0.35 0.35 0.00 0.00 0.35 0.35 0.35 0.24
6 8 2 3 4 2 2 2 6 2 4 3 3 2 3 5 2 12 3 3 2 1 4 8 1 3 4 8 2 5 6 2 3 5 9 2 3.94
6 7 2 5 3 3 3 3 7 3 4 4 4 4 3 5 2 7 4 3 2 2 7 5 1 4 4 6 3 6 6 3 3 6 6 2 4.11
0.78 0.77 0.25 0.70 0.55 0.55 0.49 0.49 0.80 0.39 0.58 0.55 0.58 0.65 0.57 0.67 0.36 0.72 0.62 0.55 0.37 0.37 0.82 0.70 0.00 0.58 0.64 0.75 0.39 0.70 0.76 0.55 0.48 0.70 0.73 0.36 0.57
3 n.a n.a n.a 4 n.a 1 1 4 1 3 1 1 n.a 1 1 n.a 6 5 1 n.a n.a 1 1 3 1 2 3 n.a 2 3 1 1 2 3 n.a 1.56
1 n.a n.a n.a 1 n.a 1 1 1 1 2 1 1 n.a 1 1 n.a 2 2 1 n.a n.a 1 1 2 2 1 1 n.a 1 1 1 1 1 1 n.a 0.86
0.00 n.a n.a n.a 0.00 n.a 0.00 0.00 0.00 0.00 0.38 0.00 0.00 n.a 0.00 0.00 n.a 0.38 0.38 0.00 n.a n.a 0.00 0.00 0.38 0.38 0.00 0.00 n.a 0.00 0.00 0.00 0.00 0.00 0.00 n.a 0.05
n.a. refers to no amplification. a Linkage group (LG) number according to SSR-based genetic linkage map of Cucurbita pepo (Gong et al., 2008). b Number of amplicons. c Number of alleles. d Polymorphism information content (PIC).
Neighbor-joining cluster analysis confirmed and clarified the results obtained by PCoA analysis (Fig. 2). One node held the 30 L. siceraria accessions, while another held the two L. cylindrica, and a third node held all the Cucurbita spp. The majority (27 out of 30) of L. siceraria accessions were separated into four clusters composed of 6, 11, 4, and 6 accessions, respectively. Two accessions (L-33-1 and L-72-1) were held by separate nodes, distant from the other L. siceraria accessions, while L-63-3 was held by one node in between the four previously described clusters. The genetic relationship among C. pepo, C. moschata, and C. maxima (group 5) was further clarified. Two C. moschata accessions had closer genetic relationships with two C. maxima accessions (Mo-14-3 with Mx55-4, Mo-14-1 with Mx-14-1), and these were placed among the C. pepo cluster. Within this cluster, one C. maxima accession (Mx-65-1) showed a larger genetic distance from the other accessions, being held by one separate node. Four C. pepo subsp. ovifera held by one node formed one cluster, while two C. maxima (Mx-14-3 and Mx-31-1) constituted a separate small cluster. Finally, another five C. maxima accessions assembled in one cluster, with one C. pepo subsp. ovifera accession (O-54-1) being held separately within this cluster. 3.4. Population structure L. siceraria The population structure analysis grouped the 30 L. siceraria accessions in two subpopulations. The estimated membership coefficients were higher than 0.65 for 21 accessions, which formed two subpopulations of 11 (subpopulation 1 hereafter) and 10 (subpopulation 2 hereafter) accessions. Nine accessions had an estimated membership coefficient lower than 0.65 and were assigned to a mixed group. The majority of accessions in subpopulation 1 were characterized by long neck-shaped fruit. In contrast, accessions in subpopulation 2 were characterized by dumbbell-shaped fruit. The total number of alleles varied between subpopulations, being subpopulation 1 the less genetically diverse, with 61 amplicons. Subpopulation 2 comprised 81 amplicons, while the mixed group included 72 amplicons. Likewise, the average genetic distance among accessions in
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Table 4 Transferability of 16 SRAP markers across the six cucurbit species. L. siceraria Primer Me1xEm2 Me2xEm1 Me2xEm2 Me2xEm3 Me2xEm6 Me2xEm14 Me3xEm2 Me6xEm5 Me6xEm6 Me7xEm1 Me7xEm2 Me7xEm5 Me7xEm6 Me1xEm14 Me5xEm2 Me6xEm14 Mean
C. pepo subsp. ovifera
C. moschata
Amp.a
No.b
PICc
C. pepo Amp.a
No.b
PICc
Amp.a
No.b
PICc
Amp.a
No.b
PICc
C. maxima Amp.a
No.b
PICc
L. cylindrica Amp.a
No.b
PICc
3 4 3 3 3 4 3 3 5 1 1 2 2 1 3 3 2.75
2 7 2 1 1 3 1 1 2 1 1 3 2 2 2 1 2
0.34 0.61 0.16 0.00 0.00 0.42 0.00 0.00 0.16 0.00 0.00 0.18 0.06 0.06 0.12 0.00 0.13
3 1 1 5 3 6 5 5 1 1 1 1 3 2 5 5 3
1 2 1 4 3 3 3 4 1 1 1 2 2 3 4 4 2.44
0.00 0.16 0.00 0.58 0.55 0.31 0.47 0.61 0.00 0.00 0.00 0.16 0.27 0.31 0.54 0.54 0.28
3 1 3 6 5 6 5 5 4 1 2 3 6 2 5 7 4
5 2 2 3 3 3 3 3 2 2 3 3 3 3 3 3 2.88
0.77 0.27 0.27 0.50 0.50 0.50 0.50 0.50 0.27 0.27 0.50 0.50 0.50 0.50 0.50 0.50 0.46
3 1 1 5 3 6 5 5 2 1 1 1 3 2 3 5 2.94
1 1 1 3 2 1 2 2 2 1 1 1 1 1 2 2 1.5
0.00 0.00 0.00 0.59 0.35 0.00 0.35 0.35 0.35 0.00 0.00 0.00 0.00 0.00 0.35 0.35 0.17
4 1 2 8 6 11 7 7 6 2 4 4 6 5 8 10 5.69
3 3 3 5 7 4 6 5 7 3 3 5 6 5 8 7 5
0.55 0.48 0.48 0.76 0.80 0.58 0.70 0.70 0.79 0.59 0.55 0.66 0.76 0.65 0.79 0.79 0.66
n.a 1 1 3 2 4 1 1 3 1 0 2 2 3 4 0 1.75
n.a 2 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1.12
n.a 0.38 0.00 0.38 0.00 0.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.07
n.a. refers to no amplification. a Number of amplicons. b Number of fragments. c Polymorphism information content (PIC).
subpopulation 1 was 0.10, 0.20 in subpopulation 2 and 0.19 in the mixed group. The place of origin was not associated with the observed population structure. 4. Discussion Bottle gourd is among the most widely dispersed and common plant species in the world. Since ancient times, it has been dispersed on different continents through trade routes (Mladenovic et al., 2012). In this study, genomic resources (SSR markers) from Cucurbita species showed high transferability to L. siceraria and L. cylindrica. For instance, the average PIC (0.15) L. siceraria was less than what obtained by Xu et al. (2011; PIC ¼ 0.39), who analyzed 44 Chinese bottle gourd landraces with 14 SSRs developed using bottle gourd genome sequence information. The higher value observed by Xu et al. (2011) might have resulted from the techniques employed to size-fractionate the amplicons in both experiments. Consequently, the average allele number per marker was also smaller in the present study (1.64 vs. 3.64 in Xu et al. (2011)). Today, extensive genomic resources (e.g., SSRs markers, SNP genotyping) are available in cucurbit crops such as cucumber, melon, squash, and watermelon. Our results indicate that these resources can be used to study other marginal crops like L. siceraria and L. cylindrica. The transferability of genomic resources depends on the phylogenetic relationship between germplasm. Here, the polymorphism detected in L. cylindrica was the lowest among cucurbit species evaluated. However, the transferability to L. siceraria and L. cylindrica was lower with C. moschata SSR markers. Previous phylogenetic studies among cucurbit species have
Fig. 1. Principal coordinate analysis (PCoA) of 61 cucurbit accessions based on shared allele distance. Different colored symbols represent cucurbit species: Lagenaria siceraria (30 accessions), Cucurbita pepo (10), Cucurbita pepo spp. ovifera (5), Cucurbita moschata (3), Cucurbita maxima (11), and Luffa cylindrica (2).
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Fig. 2. Neighbor-joining analysis of 61 cucurbit accessions based on shared allele distance. Blue braches refer to Lagenaria siceraria, brown to Cucurbita pepo, light blue to Cucurbita pepo spp. ovifera, green to Cucurbita moschata, purple to Cucurbita maxima, and red to Luffa cylindrica. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
shown significant genetic distance between C. moschata and C. pepo (Wilson et al., 1992). In this regard, watermelon is the closest species to L. siceraria (Schaefer et al., 2009) and thus its genomic resources should show high transferability to L. siceraria. For instance, bottle gourd SSR markers developed by Xu et al. (2011) showed a higher transferability to watermelon than to L. cylindrica and C. pepo (41% vs. 20% and 11% amplification rate, respectively). Here, transferability between C. pepo and L. siceraria was high (84.6%). This higher SSR transferability could be because Xu et al. (2011) designed SSRs from a random genomic sequence, while SSR markers employed in this study were strategically selected based on their position on the Cucurbita genome. Genetic sequence homology among cucurbit species varies across the genome, and chromosome rearrangement has been documented using synteny analysis (Ren et al., 2009; Diaz et al., 2011). Therefore, the transferability value obtained in our study should be more precise than the one obtained by Xu et al. (2011). SRAP markers are widely used in genetic studies of cucurbits and could be a valuable genetic tool for the study of marginal crops. Although the genetic diversity observed with SRAP was similar to that with SSR, the Cucurbita germplasm used here was selectively chosen and does not represent the actual genetic diversity of the genus. Further genetic studies with larger samples are required to establish an advantage of SRAP over SSR markers. Principal coordinate analysis separated accessions into three genera: 1) Lagenaria, 2) Luffa, and 3) Cucurbita. The Cucurbita was the most genetically diverse since it included four species spread over three groups, which were defined according to the use and origin of the accessions. This close genetic relationship between C. maxima and C. pepo subsp. ovifera could be associated with the domestication process of the two species. An also close genetic relationship was observed among C. pepo, C. moschata, and four C. maxima accessions. This group combined the majority of edible squash, including seed pumpkins. It comprised four naked (hull-less) seed C. pepo genotypes, two seed pumpkin genotypes selected by Abak et al. (1990), two naked seed pumpkin accessions from Austria, and one zucchini genotype. Our results suggest that the genetic relationship among cucurbits can be defined by their use rather than place of origin, due to human selection pressure. The L. siceraria germplasm had a low genetic diversity. However, population structure analysis grouped genotypes into two subpopulations. The long-necked and dumbbell-shaped fruit genotypes were grouped into subpopulations 1 and 2, respectively. We did not find an association with place of origin although accessions were collected from different regions of Turkey. Similar results have been documented for Chinese bottle gourd, with accessions clustering by fruit shape rather than geographic origin (Xu et al., 2011). Moreover, Mladenovic et al. (2012) found that genetic diversity patterns revealed by morphological characterization of Serbian bottle gourd germplasm were not associated with geographic origin. Although Yetisir et al. (2008) detected some clustering based on geographical origin of bottle gourds collected in the Mediterranean
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region of Turkey, these were also largely morphologically diverse in traits such as fruit size and shape. Thus, bottle gourd use and selection must have been based on fruit shape and size. Results obtained in this study indicate that germplasm collection programs should be based on morphological variation rather than location. In fact, it might be possible to obtain the majority of genetic variation in only a few places where bottle gourd is extensively used for multiple purposes. The present study revealed a narrow genetic relationship among the studied L. siceraria accessions, and an adequate transferability of genomic resources from major cucurbit crops. This high transferability of genomic resources (SSR markers) encourages its use in marginal crops such as L. siceraria and L. cylindrica to improve germplasm management and/or as a tool for marker-assisted selection in breeding programs. 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