Introduction
Genetic similarity data allows breeders to make informed decisions for selection of parents. To identify genetic diversity, several DNA marker systems have been used in potato such as RAPD (random amplified polymorphic DNA) (Demeke et al., Reference Demeke, Kawchuk and Lynch1993; Sosinski and Douches, Reference Sosinski and Douches1996; McGregor et al., Reference McGregor, Lambert, Greyling, Louw and Warnich2000), amplified fragment length polymorphism (AFLP) (Milbourne et al., Reference Milbourne, Meyer, Bradshaw, Baird, Bonar, Provan, Powell and Waugh1997; Rouppe van der Voort et al., Reference Rouppe van der Voort, van Eck, Draaistra, van Zandvoort, Jacobsen and Bakker1998; McGregor et al., Reference McGregor, Lambert, Greyling, Louw and Warnich2000; van Treuren et al., Reference van Treuren, Magda, Hoekstra and van Hintum2004; Akkale et al., Reference Akkale, Yildirim, Yildirim, Kaya, Öztürk and Tanyolaç2010), simple sequence repeat (SSR) (Kawchuk et al., Reference Kawchuk, Lunch, Thomas, Penner, Sillito and Kulcsar1996; Provan et al., Reference Provan, Powell and Waugh1996; McGregor et al., Reference McGregor, Lambert, Greyling, Louw and Warnich2000; Ghislain et al., Reference Ghislain, Spooner, Rodríguez, Villamón, Núñez, Vásquez, Waugh and Bonierbale2004; Moisan-Thiery et al., Reference Moisan-Thiery, Marhadour, Kerlan, Dessenne, Perramant, Gokelaere and Le Hingrat2005; Kandemir et al., Reference Kandemir, Yılmaz, Karan and Borazan2010a; Côté et al., Reference Côté, Leduc and Reid2013) inter-simple sequence repeat (ISSR) (Alhani and Wilkinson, Reference Alhani and Wilkinson1998; Prevost and Wilkinson, Reference Prevost and Wilkinson1999) and inter-retrotransposons amplified polymorphisms (IRAP) (Nováková et al., Reference Nováková, Šimáčková, Bárta and Čurn2009). Among them, SSR and AFLP marker systems are very powerful methods for distinguishing potato genotypes. However, they require expensive devices, technical expertise or fluorescently labelled primers for SSR analysis and high-resolution agarose or polyacrylamide gel. In addition, AFLP marker system is more difficult and time-consuming technique than other systems. Despite that, retrotransposon marker system is reproducible, easy to apply, cheap and requires basic molecular laboratory facilities. Kalendar et al. (Reference Kalendar, Antonius, Smykal and Schulman2010) developed inter-primer-binding sites (iPBS) retrotransposon marker system for both plant and animal kingdoms, without the requirement for prior sequence knowledge. Due to high copy numbers in the genome and distribution over a wide area of the chromosomes, retrotransposons are considered as excellent molecular marker systems (Schulman et al., Reference Schulman, Flavell and Ellis2004; Kalendar and Schulman, Reference Kalendar and Schulman2006; Kalendar et al., Reference Kalendar, Antonius, Smykal and Schulman2010).
Retrotransposons copy themselves and relocate to another region of a genome. Therefore, they cause mutations, increasing of genome size and genetic diversity. Plant genomes consist of large numbers of repetitive DNA elements, in addition, retrotransposons have the highest proportion among repetitive DNA elements. For example, 28.5% of Arabidopsis genome (Arabidopsis Genome Initiative, 2000), 14.7% of rice genome (Wang et al., Reference Wang, Shi, Hao, Ge and Luo2005), 45–60% of cotton genome (Hawkins et al., Reference Hawkins, Kim, Nason, Wing and Wendel2006) are composed of retrotransposons. Similarly, long terminal repeat (LTR) retrotransposons are predominate transposable elements in potato and represent 29.4% (214.1 Mb) of potato genome (The Potato Genome Sequencing Consortium, 2011). To the best of our knowledge, only Nováková et al. (Reference Nováková, Šimáčková, Bárta and Čurn2009) used IRAP primers as a retrotransposon marker system to distinguish 20 potato varieties, but iPBS marker system has not been previously used for potato.
The advantages of iPBS marker system are: (1) ability to screen large regions of plant genomes; (2) primers can be used for any organism; (3) easy to perform; and (4) comparatively cheap due to requirement of basic laboratory facilities. Besides, iPBS primers have previously been shown to be a powerful marker system for fingerprinting and genetic similarity studies in plants (Gailīte et al., Reference Gailīte, Ievinsh and Ruņģis2011; Raddová et al., Reference Raddová, Ptáčková, Čechová and Ondrášek2012; Andeden et al., Reference Andeden, Baloch, Derya, Kilian and Özkan2013; Fang-Yong and Ji-Hong, Reference Fang-Yong and Ji-Hong2014; Guo et al., Reference Guo, Guo, Hou and Zhang2014; Baloch et al., Reference Baloch, Derya, Andeden, Alsaleh, Cömertpay, Kilian and Özkan2015a, Reference Baloch, Alsaleh, de Miera, Hatipoğlu, Çiftçi, Karaköy, Yıldız and Özkanb). Because of these advantages, in the present study, iPBS marker system was used for fingerprinting potato genotypes and identification of genetic similarity within potato genotypes located in Turkey.
Materials and methods
Plant materials and DNA extraction
In the study, 151 potato accession located in Turkey were investigated. The collection (Table S1) contains 76 potato commercial varieties and 75 breeding lines developed in different potato-breeding programmes of Turkey. Approximately 0.2 g tissue for extraction was homogenized using a TissueLyser II (Qiagen) disrupter. Genomic DNA was extracted from sprouts or young leaves of genotypes using the standard CTAB protocol modified from Doyle (Reference Doyle, Hewitt, Johnson and Young1991). DNA concentration was measured by using a Biospec-nano UV–vis Spectrophotometer (Shimadzu).
iPBS markers and PCR conditions
Initially, 16 randomly selected potato genotypes were assessed using 45 iPBS retrotransposon markers developed by Kalendar et al. (Reference Kalendar, Antonius, Smykal and Schulman2010), in order to find convenient primers for producing sharp and clear band profile and to identify polymorphic ones for potato (Table 1). Seventeen of these primers were selected for fingerprinting the whole set of accessions. The PCR was performed in a 25 µl reaction mixture containing 25 ng DNA, 1× DreamTaq PCR buffer, 1 µM of primer for 12–13 nt primers or 0.6 µM for 18 nt primers, 0.2 mM dNTPs, 1 unit Taq DNA polymerase (DreamTaq, Thermoscientific). After an initial denaturation at 95°C for 3 min, the PCR was performed as follows: amplification for 30 cycles with denaturation at 95°C for 15 s, annealing at 50–55°C (dependent on primer, Table 1) for 60 s, and extension at 72°C for 2 min. After amplification, a final extension step of 72°C for 7 min was performed.
Description of 45 iPBS primers with their name, sequences and annealing temperature (T a)
a Primers written bold were used for fingerprinting 151 potato genotypes.
The PCR products were analyzed by 1.8% agarose gel using 0.5× TBE buffer at 6 V/cm for 2 h. To identify band size on the gel, a DNA ladder, GeneRuler DNA Ladder Mix (Thermo Scientific), were loaded into the first and the last wells on the gel. Agarose gels were stained using ethidium bromide and bands were visualized by Gel Doc™ XR+ gel imaging system (Bio-Rad) (Fig. 1). All PCR and electrophoresis analysis were repeated at least twice and only sharp and clear bands were scored. Two replicate DNA samples from Agria variety provided by two separate sources were used to assess the reproducibility of band profiles using 17 polymorphic iPBS primers. These two different Agria DNA samples were named as Agria-1 and Agria-2 in the study.
iPBS fingerprints of some potato genotypes by using 2229 iPBS primer, iPBS 2232 primer, 2277 iPBS primer and 2390 iPBS primer. M, DNA size ladder between 100 and 10,000 bp loaded in the first and last lanes on each gel.
Data analysis
A data matrix was constructed based on presence (1) or absence (0) of iPBS bands. Missing data were scored as ‘?’. Bands with size between 3000 and 100 bp were scored manually by three people using a 60″ (152 cm) widescreen plasma TV (LG 60PH670S) and scores were independently checked at least twice by three people. Genetic similarity among potato genotypes were estimated according to Jaccard's coefficient (Jaccard, Reference Jaccard1908) using PAST 3.14 software. Jaccard's coefficient is commonly used with dominant marker data where allele frequencies cannot be calculated (Reif et al., Reference Reif, Melchinger and Frisch2005). The Jaccard's coefficient (J) between two genotypes was calculated according to formula described by Hinze et al. (Reference Hinze, Fang, Gore, Scheffler, Yu, Frelichowski and Percy2015) as:
$$J = \displaystyle{a \over {a + b + c}},$$
where a is the number of bands common to both genotype, b is the number of bands only present in genotype 1, and c is the number of bands only present in genotype 2. Since iPBS marker system is dominant marker, polymorphism information content (PIC) was calculated according to formula described by Hinze et al. (Reference Hinze, Fang, Gore, Scheffler, Yu, Frelichowski and Percy2015) as:
$${\rm PIC}_b = {\rm 1} - (\,p^{\rm 2} + q^{\rm 2} ),$$
where p is the frequency of the band presence and q the frequency of band absence of the bth band of the iPBS primer and PIC b is the PIC of band b. PIC for dominant markers is a maximum of 0.5 when p = q = 0.5. Estimates of PIC are calculated for each band, and mean PIC value of an iPBS primer was calculated from over all bands in an iPBS primer. A neighbour-joining tree was generated by genetic similarity based on Jaccard's coefficient using PAST 3.14 software.
Results
For initial screening, 45 iPBS primers were tested on 16 potato genotypes. Among these primers, 17 iPBS retrotransposon primers produced strong, reproducible and polymorphic bands and these 17 primers were used for genotyping all 151 potato genotypes. After scoring, a high quantity of missing data was observed for Lady Rosetta, Compass, Elgar, Concordia, Atlantic and Brooke varieties. Therefore, the data of these six varieties were not used for genetic similarity analysis and clustering analysis. The 17 iPBS primers produced 290 scorable bands and among them 224 bands were polymorphic (Table 2). Band sizes produced by all primers ranged from 100 to 10,000 bp (Fig. 1). However, only bands with size between 100 and 3000 bp were scored.
iPBS marker results based on 151 potato genotypes
PIC, average polymorphism information content.
The number of scored bands per primer ranged from 10 to 26 with an average of 17.1 bands per primer (Table 2). The number of polymorphic bands per primer was between 6 and 21 with an average of 13.2 bands per primer (Table 2). The polymorphism percentage per primer ranged from 46.2% (2375) to 100.0% (2229) with an average of 77.2% per primer (Table 2). The mean PIC values of iPBS primers varied from 0.12 to 0.31 with an average of 0.23 per primer. iPBS primers of 2229, 2252, 2272 and 2374 showed highest PIC values with 0.31, 0.29, 0.29 and 0.29, respectively.
Genetic similarity based on Jaccard's coefficient of potato genotypes ranged from 0.61 to 0.93 with an average of 0.73 (Supplementary material). The lowest genetic similarity (0.61) was observed between PRI-11 and 0904-5, and between 08-212 and Volare whereas, the highest genetic similarity (0.93) was observed between PRI-03 and PRI-04 breeding lines (Supplementary data). The genetic similarity index between Agria-1 and Agria-2 that are the same genotypes provided by two different sources was calculated as 0.90. Similarly, high genetic similarity indexes were observed between the potato lines of PRI-01, PRI-02, PRI-03 and PRI-04 in range of 0.79–0.93 (Supplementary data). These four potato lines were created by applying a mutagen to Marfona variety in the mutation breeding programme at Potato Research Institute, Turkey. In addition, the lines of 0909–11 and 0909–12 from breeding programme of Nigde University are the progenies of Agria × Granola crossing. While the genetic similarity index was 0.80 between 0909–11 line and 0909–12 line, it was 0.80 between 0909–11 line and male parent, Agria and 0.81 between 0909–12 line and Agria (Supplementary data). A dendrogram was generated based on genetic similarity values of Jaccard's coefficient by neighbour-joining method (Fig. 2). The neighbour-joining tree clearly split 145 potato genotypes into nine groups. While all varieties were separated into different groups, breeding lines from Potato Research Institute were clustered together. Identification of genetic similarity using genetic fingerprinting data is a useful tool for potato breeders to make informed decisions for selection of parents to be used in crossing programmes. To create a high genetic variation in potato-breeding population, potato breeders can benefit from the result of the study by choosing parents having high genetic distance from each other.
Dendrogram of 145 potato genotypes using neighbour-joining method and Jaccard's index.
Discussion
Since production and marketing of varieties is very important for the seed trade, accurate identification of potato varieties is essential for global seed marketing. Identification of potato varieties based on morphological characteristics may be prone to error; therefore, molecular identification is required for variety verification. Although the morphological characteristics of potato genotypes located in Turkey are known, not enough information is available about their genotypic characteristics. Since there is a limited knowledge on DNA profiles of potato genotypes grown in Turkey, molecular techniques cannot be used to identify potato varieties. In addition, using genetic fingerprinting data allows breeders to make informed decisions for selection of parents to be used in breeding programmes. In order to facilitate accurate identification of potato varieties in Turkey and a breeder's decision for selection of highly diverse parents, molecular fingerprinting was performed in the study.
For genetic fingerprinting of potato genotypes located in Turkey, iPBS retrotransposon marker system was used in the study. To our knowledge, only Nováková et al. (Reference Nováková, Šimáčková, Bárta and Čurn2009) used a retrotransposon marker system (Inter-Retrotransposon Amplification Polymorphism) to distinguish potato varieties, but iPBS retrotransposon marker system has not previously been used for potato. One of the limiting factors for using retrotransposons as molecular markers is requirement for prior sequence knowledge of LTR retrotransposons. However, the iPBS retrotransposon marker system overcomes this problem by using the reverse transcriptase PBS of retrotransposons. Primers used in iPBS marker system were developed by utilizing universally conserved sequences of PBS that are adjacent to the 5′ LTR retrotransposons (Kalendar et al., Reference Kalendar, Antonius, Smykal and Schulman2010). Therefore, iPBS marker system has no requirement for prior knowledge of a genome sequence as in other retrotransposon marker systems, and it can be applicable to both the plant and animal kingdoms (Kalendar et al., Reference Kalendar, Antonius, Smykal and Schulman2010). Due to dispersal throughout the genome, high copy numbers in the genome and showing spread over a wide area of the chromosomes, retrotransposons were considered to have excellent potential as a source of molecular markers by Kalendar et al. (Reference Kalendar, Antonius, Smykal and Schulman2010). Although iPBS primers are universal markers for plant and animal kingdoms, they have been used to investigate genetic diversity in only a few plant species such as Saussurea esthonica (Gailīte et al., Reference Gailīte, Ievinsh and Ruņģis2011), Diospyros ssp. (Raddová et al., Reference Raddová, Ptáčková, Čechová and Ondrášek2012), Cicer species (Andeden et al., Reference Andeden, Baloch, Derya, Kilian and Özkan2013), Myrica rubra (Fang-Yong and Ji-Hong, Reference Fang-Yong and Ji-Hong2014), grape (Guo et al., Reference Guo, Guo, Hou and Zhang2014), Lens species (Baloch et al., Reference Baloch, Derya, Andeden, Alsaleh, Cömertpay, Kilian and Özkan2015a) and pea (Baloch et al., Reference Baloch, Alsaleh, de Miera, Hatipoğlu, Çiftçi, Karaköy, Yıldız and Özkan2015b). All of the earlier studies clearly demonstrated that iPBS retrotransposons were very efficient for genetic diversity and molecular studies in plants. However, this newly developed marker system has not been utilized for evaluation of genetic diversity in potato genetic resources. Therefore, we particularly sought to investigate the applicability of iPBS retrotransposon markers for differentiating the large collection of commercial cultivars and breeding lines of Solanum tuberosum germplasm commonly grown in different regions of Turkey. One of the main concerns for the molecular characterization and genetic diversity studies in plants is that selected marker systems must have high reproducibility and should be the source of high polymorphism, even in closely related genotypes. For this purpose, reproducibility of the iPBS retrotransposon markers in potato was tested with replicated polymerase chain reaction method. The results clearly showed that iPBS retrotransposon markers were highly reproducible in all tested cases. iPBS retrotransposons have several appealing features, such as high degree of polymorphism, technical simplicity, less time and minimum labor requirement, as well as reproducibility, which make this marker system of choice for cultivar fingerprinting and genetic diversity studies of large germplasm collections.
In this study, each iPBS primer produced the large number of bands and high polymorphism percentage. The results proved that iPBS retrotransposon marker system used was sufficient to distinguish a potato population. Similarly, Andeden et al. (Reference Andeden, Baloch, Derya, Kilian and Özkan2013) demonstrated the utility of a retrotransposon marker system for genetic diversity studies in Cicer species. They assessed genetic diversity and relationship of 71 accessions belonging to five wild Cicer species and one cultivated chickpea using iPBS retrotransposon and ISSR marker systems. The clustering results of their study from iPBS and ISSR markers systems were almost similar (Andeden et al., Reference Andeden, Baloch, Derya, Kilian and Özkan2013). Similarly, Baloch et al. (Reference Baloch, Derya, Andeden, Alsaleh, Cömertpay, Kilian and Özkan2015a) reported almost similar grouping of Lens accessions and species within clusters when iPBS and ISSR dendrograms were compared.
In Turkey, variety identification of potato is still based on morphological characters, such as tuber colour and shape, sprout appearance, leaf shape and size, flower features, etc. Most of the phenotypic traits for variety identification are polygenic in nature and affected by environmental conditions, growth and developmental stage of the plant. Although sprout appearance is considered identical for each potato cultivar, the presence or absence of light during post-harvest storage dramatically affects the morphology of emerging sprouts (Suttle, Reference Suttle, Vreugdenhil, Bradshaw, Gebhardt, Govers, Mackerron, Taylor and Ross2007). In addition, different developmental stages of potato are required according to various morphological traits for variety identification. This approach complicates accurate identification of potato varieties. In this case, potato variety identification among high numbers of varieties becomes time-consuming, less suitable for expeditious results and not effective for large samples of genetic resources. In Turkey, the potato industry needs an easy, rapid and trustable method for cultivar identification. To facilitate description and identification of cultivars, DNA markers are currently used. DNA fingerprinting is useful for breeders, seed companies and growers for protection of breeder's rights, checking line purity and identity of a variety. However, limited studies were carried out on genetic diversity and fingerprinting of potato genotypes located in Turkey (Akkale et al., Reference Akkale, Yildirim, Yildirim, Kaya, Öztürk and Tanyolaç2010; Kandemir et al., Reference Kandemir, Yılmaz, Karan and Borazan2010a, Reference Kandemir, Yılmaz, Karan and Borazanb). Akkale et al. (Reference Akkale, Yildirim, Yildirim, Kaya, Öztürk and Tanyolaç2010) investigated genetic diversity of 26 potato genotypes grown in Turkey using six AFLP primer pairs. As a result, six AFLP primer pairs produced 191 polymorphic bands, and 26 genotypes were clustered in six different subgroups. Another study in Turkey was on development of an SSR marker set for fingerprinting of major potato landraces and varieties grown in Central Anatolia (Kandemir et al., Reference Kandemir, Yılmaz, Karan and Borazan2010a). Five SSR markers (STM19, STM31, STM3012, STI32 and STI42) among sixteen markers adequately distinguished 15 potato genotypes investigated (Kandemir et al., Reference Kandemir, Yılmaz, Karan and Borazan2010a). The same study showed that three local varieties, Başçiftlik Beyazı, Aybastı Beyazı and Aleddiyan Beyazı, were found to be the same potato genotype even though they are named differently according to their growing area (Kandemir et al., Reference Kandemir, Yılmaz, Karan and Borazan2010a). Contrastingly, Kandemir et al. (Reference Kandemir, Yılmaz, Karan and Borazan2010b) determined 23 different potato genotypes in Başçiftlik Beyazı landrace of Turkey using 16 SSR markers. Compared to SSR and AFLP marker systems, iPBS marker system has advantages due to minimal laboratory equipment requirements, technical simplicity, quicker results, and using universal primers. However, the number of total scorable bands in the study was between 10 and 26 per primer, because of being dominant markers. Actually, the total band numbers were higher than scorable bands. In addition, a disadvantage of iPBS retrotransposon marker system is difficulty in consideration of faint bands as present or absent. Therefore, scoring of the bands of iPBS retrotransposon markers are relatively more difficult particularly, compared to SSR markers. Nevertheless, the results of our study showed that the iPBS marker system has potential to be used regularly for identification of potato varieties. These results allowed creation of a database; therefore, the data can be used for conservation or rapid identification of potato varieties registered in Turkey. The system, therefore, facilitates verification of varietal identity in cases in which one variety is marketed under the name of another variety. Although it is the first fingerprinting data for such a large potato population and has potential for use in varietal identification of potato genotypes in Turkey, the high number of total bands for each genotype that were produced from 17 primers may make confirmation of varieties relatively difficult compared with SSR systems. Moisan-Thiery et al. (Reference Moisan-Thiery, Marhadour, Kerlan, Dessenne, Perramant, Gokelaere and Le Hingrat2005) stated the reliability of fingerprint analyses for cultivar identification for any molecular marker system is limited when a cultivar is a sport or somatic mutation of a previously-released cultivar. In addition, the similar problem may appear for transgenic lines of a variety. However, identified genetic distance between potato genotypes in the study has more advantages to breeders for selection highly diverse parents to be used in a breeding programme.
Although potato has been grown in Turkey for roughly 150 years, very limited studies had been conducted for cultivar breeding. In 2005, however, a national project was initiated with cooperation of several research institutes, universities and private companies to develop national varieties and to increase seed potato production. This programme has yielded the registration of six national varieties up to now, and registration procedures of around ten variety candidates have also been started. Currently, several public and private institutes have ongoing breeding programmes in the country. Therefore, any knowledge relating to genetic diversity of available cultivars and breeding lines will contribute to increase the efficiency of these programmes. In this study, 17 polymorphic iPBS primers were identified for potato. In addition, pre-breeding data were produced by using iPBS retrotransposon marker system to help inform potato breeders. Potato breeding programmes to develop new potato varieties are generally based on the crossing different potato genotypes and selection of breeding lines with desired traits among the segregating progeny. To construct a breeding population, the use of parents with desirable traits and also having the greatest possible genetic distance to each other increases the possibility of generating high diversity. Therefore, breeders have more chance to select desired breeding lines among a breeding population. For this purpose, knowledge of genetic distance between parents as well as agronomic traits is one of the most important factors contributing to successful breeding. The study produced genetic distance data for potato genotypes located in Turkey. Therefore, potato breeders may take the data in account and results of the study may facilitate the decision of breeders for selection of highly diverse parents with traits of interest to construct highly segregated population in potato-breeding programmes. Further, a total of 151 potato genotypes in Turkey were molecularly characterized. Therefore, the DNA fingerprinting data produced from the study will be useful for variety description in the future. Finally, the study proved that iPBS marker system is a powerful and easy method for fingerprinting and distinguishing potato genotypes.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262117000041.
Acknowledgements
This work was funded by Scientific and Technological Research Council of Turkey (TÜBİTAK), Project No. 213O234. Gratitude is extended to Prof. Dr Sedat Serçe for excellent suggestions and scientific discussion on statistical analysis. We also thank Buse Leyla Cihangiroğlu for technical assistance during laboratory experiments. We greatly appreciate Dr Wayne Morris for his advice on improving the use of English in the manuscript.
