ГЕНЕТИКА РАСТЕНИИ
УДК 575.17
LARGE-SCALE DEVELOPMENT OF PIP AND SSR MARKERS AND THEIR COMPLEMENTARY APPLIED IN Nicotiana
© 2013 L. Huang4, H. Caoa, L. Yanga, Yu. Yu4, Yu. Wanga
a Tobacco Laboratory, College of Plant Protection, Shandong Agricultural University, Tai'an, Shandong, 271018China e-mail: lyang@sdau.edu.cn; chcsnd@163. com bCollege of Agronomy, Shandong Agricultural University, Tai'an, Shandong, 271018 China
e-mail: huang2002003@gmail.com Received Oktober 15, 2012
PIP (Potential Intron Polymorphism) and SSR (Simple Sequence Repeats) were used in many species, but large-scale development and combined use of these two markers have not been reported in tobacco. In this study, a total of 12,388 PIP and 76,848 SSR markers were designed and uploaded to a web-accessible database (http://yancao.sdau.edu.cn/tgb/). E-PCR analysis showed that PIP and SSR rarely overlapped and were strongly complementary in the tobacco genome. The density was 3.07 PIP and 1.72 SSR markers per 10 kb of the known sequences. A total of 153 and 166 alleles were detected by 22 PIP and 22 SSR markers in 64 Nicotiana accessions. SSR produced higher PIC (polymorphism information content) values and identified more alleles than PIP, whereas PIP could identify larger numbers of rare alleles. Mantel testing demonstrated a high correlation coefficient (r = 0.949, P < 0.001) between PIP and SSR. The UPGMA dendrogram created from the combined PIP and SSR markers was clearer and more reliable than the individual PIP or SSR dendrograms. It suggested that PIP and SSR can make up the deficiency of molecular markers not only in tobacco but other plant.
DOI: 10.7868/S0016675813070072
Tobacco (Nicotiana tabacum L.), an important species in the genus Nicotiana, is a raw material for the cigarette industry and is widely cultivated in many different countries [1]. It is well known that cultivated tobacco is a natural amphidiploid (2n = 24II) through the hybridization of wild progenitor species and N. sylvestris (n = 12) and N. tomentosiformis (n = 12) [2]. The recent taxonomic revision of Nicotiana produced a well defined group of species which was composed of 76 naturally occurring Nicotiana species within 13 sections [3]. The chromosome numbers of these 76 species belong to eleven types: 2n = 9II, 10II, 12II, 16II, 18II, 19II, 20II, 21II, 22II, 23II and 24II [4]. Species with chromosome numbers of 2n = 12II and 24II are euploid while the rest are aneuploid due to them losing one or more chromosome formations during the evolutionary process [4, 5]. Chromosome number 2n = 24II (33 species) was the most frequent type [6]. The highly polymorphic chromosome numbers have provided a large gene reservoir that can be used to study genetic diversity in tobacco.
Unlike other major crop species, research progress of tobacco genetic markers has been relatively slow [7]. Although many DNA markers, such as RAPD (Random Amplified Polymorphic DNA), ISSR (Inter Simple Sequence Repeat) and AFLP (Amplified Fragment Length Polymorphism), have been utilized in the genus Nicotiana for genetic diversity analysis [1,
8—10], the disadvantages of these markers, such as a small number, randomly amplification and poor reproducibility, have limited their wide application. In contrast, SSR (Simple Sequence Repeats) are ubiquitous throughout the eukaryotic genomes [11]. SSR has some positive attributes, such as having a high level of polymorphism, are co-dominant and are easily detected by PCR detection [12]. Since Bindler et al. [7] reported the first linkage map for tobacco, there has been much research into using SSR markers to analyze genetic relationships in Nicotiana [13—16]. With the increase in the numerous functional sequences and genome sequences for tobacco being submitted to the public database (PlantGDB, http://www.plant-gdb.org/ and TGI, tobacco genome initiative, http:// www.pngg.org/tgi/), large scale mining for SSR markers using these known sequences became possible. Recently, Bindler et al. [17] developed 5,119 SSR markers by exploiting TGI sequences but the number was still not large enough to be used in the study of tobacco.
ILP (Intron Length Polymorphism) has attracted more and more attention as a novel molecular marker, since it not only has similar advantages to SSR, but also some particular characteristics, such as directly reflecting variation within specific genes and subspecies [18]. Before ILP markers could be designed, the whole genome sequence and position of introns needed to be
identified, as happened with the successful use of ILP in the sequenced model plant, rice [19, 20]. Apart from a few model plants, most crop plants or economically important plants have been not sequenced yet. Fortunately, a database of expressed sequence tags (ESTs) has been constructed for plants [21], which meant that the positions of the introns could be annotated by a comparative genomics approach. So cross species amplification became possible when primers were designed in flanking exons in order to amplify introns by PCR [22]. Yang et al. [22] developed a database of PIP (Potential Intron Polymorphism) markers based on the cross species predicted position of in-trons. PIP markers have been used in several plants [23—25], but not in tobacco.
PIP and SSR have their own advantages. SSR markers have the highest expected heterozygosity compared with other markers [12]. The advantages of PIP markers include: subspecies specificity, neutrality (no phenotypic effect) and an ability to directly identify variation within genes. These advantages can complement SSR markers [18]. Thus, PIP markers, in combination with SSR markers, could be used to estimate genetic diversity. The combined use of these two marker systems in rice has been successful, but their use in tobacco has not been reported.
This study undertook the large-scale development of PIP and SSR markers and analyzed their distribution in the tobacco genome. In addition, a set of PIP and SSR markers were selected in order to undertake phylogenetic analysis and to analyze the prospects of the combined use of PIP and SSR in tobacco.
MATERIALS AND METHODS
Plant material and DNA isolation. A set of 64 accessions, including 12 wild species and 52 lines of cultivated tobacco (N. tabacum L.), were selected for this study (Table 1). Twelve wild Nicotiana species were chosen from three subgenera, including Petunioides, Rustica and Tabacum, and 52 cultivars, representing the five market classes of air-cured, burley, flue-cured, oriental and sun-cured tobacco, were also selected. Most materials were collected from China and America. These seeds were germinated and grown under greenhouse conditions. One leaf of young fresh material from one seedling per accession was used for DNA extraction. Genomic DNAs were extracted using the CTAB/NaCl method [26].
Development of PIP and SSR markers. The EST sequences of four tobacco species (N. tabacum, N. sylvestris, N. benthamiana and N. langsdoffi x N. sanderae) and the ORF (Open Read Frames) of N. tabacum were downloaded from PlantGDB and TGI, respectively.
A pipeline in Perl script was developed to design the PIP and SSR markers. The method of designing PIP markers was taken from the PIP database and used
120 base pair (bp) sequences cut from the tobacco EST sequences [22]. Primer pairs were designed for each side of the alternative splicing joint positions or the SSR loci using ePrimer3 [27]. The designed primers were tested using e-PCR (electronic PCR) [28] in all the tobacco sequences. Finally, suitable and reliable PIP and SSR markers were identified.
Selection of PIP and SSR markersy. All the PIP and SSR markers were tested by e-PCR, and then 44 markers (22 PIP and 22 SSR markers, Table 2) were selected out for real PCR. We selected those markers because of the 22 PIP markers could identify the different amplification products in two or more Nicotiana species, and the 22 SSR markers could amplify polymorphism in different Nicotiana species or the products had a polymorphic length of >5 bp in the same species from e-PCR.
PCR conditions and allele detection. PCR reactions were performed in 15 |L volumes containing 20— 25 ng DNA, 1.5 |L of 10x PCR buffer, 2.5 mmol L-1 MgCl2, 0.25 mmol L-1 dNTPs, 0.36 |imol L-1 forward primers, 0.36 |mol L-1 reverse primers and 1 U Taq DNA polymerase. Thermocycling conditions started with extension for 4 min initial denaturation at 94°C, followed by 30 cycles of 30 s at 94°C, 1 min at 55°C, 30 s at 72°C and a final extension at 72°C for 10 min. 6% non-denaturing PAGE (320 V, 2.5 h) was used to separate the PCR products and silver staining was used to visualize the DNA bands. The PIC (polymorphism information content) value was calculated as follows
[29]: PIC = 1 - wherep„ is the frequency of
the ith allele, and k is the total number of different alleles for the locus [30].
Construction of a phylogenetic tree. All distinct bands from the PCR products were scored as present (1) or absent (0). The binary matrix was used to generate a similarity coefficient matrix according to the Dice coefficient [31]. A dendrogram was constructed from the computed similarity matrix using the algorithm from UPGMA. To evaluate the robustness of the phylogenetic tree, Mantel testing [32] was used to compare co-phenetic matrices among the genetic similarities based on PIP, SSR and combined markers (in this paper, combined markers means PIP markers and SSR markers). Genetic similarity, dendrogram and co-phenetic correlations were calculated using NTSYS-pc version 2.1 [33]. The reliability of the dendrogram branches was checked using the Bootstrap method and 1000 replicates were checked using Free-Tree [34].
RESULTS AND DISCUSSION
PIP and SSR markers were widely distributed and covered the whole tobacco genome from e-PCR
Based on the EST sequences of four Nicotiana species and the original read sequences of N. tabacum, a
Table 1. Pedigree and origin o
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