Abstract
Simple sequence repeat (SSR) is a popular tool for individual fingerprinting. The long-core motif (e.g. tetra-, penta-, and hexa-nucleotide) simple sequence repeats (SSRs) are preferred because they make it easier to separate and distinguish neighbor alleles. In the present study, a new set of 8 tetra-nucleotide SSRs in potato (Solanum tuberosum) is reported. By using these 8 markers, 72 out of 76 cultivars obtained from Japan and the United States were clearly discriminated, while two pairs, both of which arose from natural variation, showed identical profiles. The combined probability of identity between two random cultivars for the set of 8 SSR markers was estimated to be 1.10 × 10−8, confirming the usefulness of the proposed SSR markers for fingerprinting analyses of potato.
Keywords: potato, Solanum tuberosum, cultivar identification, SSR markers
Introduction
Potato (Solanum tuberosum) is the fourth-largest food crop in the world, following maize, wheat, and rice. More than 4,500 potato varieties are cultivated in over 100 countries (Pieterse and Judd 2014). As the number of known varieties increases, it becomes difficult to identify them by morphological markers. Thus, reliable methods of correctly identifying cultivars are strongly needed to assess the genetic diversity of the potato germplasm.
SSR markers, or microsatellites, consist of tandemly repeated DNA sequences with a core unit of 1–6 base pairs (bp). They have many positive features useful for the genetic profiling of individuals, including abundance in plant genomes, multi-allelic co-dominant patterns, ease of use, and high variability in the number of core-motif repeats. In the long-core motif (e.g. tetra-, penta-, and hexa-nucleotide) SSRs, neighbor alleles are more easily separated from each other, while di-nucleotide SSRs are subject to a lower level of separation of neighbor alleles and a higher level of stuttering, which make the interpretation of electropherograms and the allele call less reliable (Cipriani et al. 2008). Long nucleotide repeats are widely adopted for genetic profiling in humans and animals (Butler et al. 2004, Butler 2006, Hammond et al. 1994, Hellmann et al. 2006, Ruitberg et al. 2001). Meanwhile, regarding plants, the use of long nucleotide repeats has been limited to the variety identification of a few crops: grape (Cipriani et al. 2008, 2010), Eucalyptus (Faria et al. 2011), olive (De la Rosa et al. 2013), peach (Dettori et al. 2015), and tea (Wang et al. 2016).
In potato, SSRs have been used to study the genetic relationships and distances between wild and cultivated potato (Ghislain et al. 2004, 2009, Milbourne et al. 1998). However, as with other crops, di- and tri-nucleotide SSRs have mainly been used, and few long-core motif SSRs have been reported (Ghislain et al. 2004, 2009, Milbourne et al. 1998).
In the present paper, we propose a new set of long-core motif SSR markers for potato with the aim of minimizing genotyping errors.
Materials and Methods
Plant materials and DNA extraction
Ten potato cultivars of in vitro cultures were obtained from the University of Idaho as representative cultivars in the United States. Potato tubers of Japanese cultivars were obtained from the Hokkaido Research Organization (HRO) Kitami Agricultural Experiment Station (9 cultivars), the Nagasaki Agricultural and Forestry Technical Development Center (8 cultivars), and the NARO Hokkaido Agricultural Research Center (49 cultivars) (Table 3). For each cultivar tested, DNA was extracted using the GM quicker 2 kit (Nippon Gene, Toyama, Japan) according to the supplier’s protocol.
Table 3.
Cultivar | Source | 4026/4027 | 8242 | 12002 | 16410 | 31924 | 35584 | 43016 | 46514 |
---|---|---|---|---|---|---|---|---|---|
Ainoaka | Nagasaki | AB | CF | BCD | BE | BD | CF | D | ABE |
Aiyutaka | Nagasaki | ABF | ABF | BD | BE | BD | C | G | AF |
Alturas | Idaho | C | BC | CE | DEK | BDE | B | DFG | AE |
Astarte | NARO | BD | BCF | D | DE | ABD | BE | D | AE |
Atlantic | NARO | BDG | ACD | CDEF | BK | BD | BCE | DFG | A |
Beniakari | NARO | BG | BF | DFG | BEK | DE | CEF | D | AC |
Benimaru | NARO | BDG | ABD | CDFG | EHIK | AB | BE | CE | AE |
Cal white | Idaho | DG | ABC | BCF | CDK | BE | BF | D | E |
Chelsea (Jenny) | NARO | B | BCG | ACD | DEFK | BCD | ABE | D | AE |
Cherie | NARO | BD | ABCD | CD | DEG | AD | B | DF | E |
Clearwater | Idaho | A | B | BCF | DK | BE | B | D | EG |
Cynthia | NARO | AB | BF | CD | DEHK | BD | B | CD | ABE |
Dansyakuimo (Irish Cobber) | NARO | B | AB | CF | BCIK | AB | BC | D | E |
Dejima | Nagasaki | ABF | ABF | BCD | BE | B | BC | Null | AFG |
Destroyer | NARO | B | BDF | CD | AEJK | CF | AB | BCDF | ACE |
Early Starch | NARO | B | CF | CD | BE | ABD | BE | D | ACG |
Eniwa | NARO | G | ABF | CDF | DEKL | BDE | BC | D | ACE |
Hanashibetsu | Hokkaido | BCD | AF | DFG | BE | BCD | BE | AD | AE |
Haruka | NARO | BC | BF | BCDG | BEG | BD | BCEF | DEG | AEF |
Hikaru | NARO | BCFG | BDEF | DE | BEK | ABCE | BCF | D | ACEF |
Hokkai 50 | NARO | B | BF | CDF | BCIK | AB | BC | D | AE |
Hokkai 98 (Inca Rouge) | NARO | B | CG | F | EK | F | B | D | E |
Hokkaikogane | NARO | BG | AB | CDF | EH | ABE | BC | D | AC |
Hugenmaru | Nagasaki | ABF | AF | BCD | BE | AB | B | G | AE |
Inca no hitomi | NARO | D | FG | FG | K | F | B | D | E |
Inca no mezame | NARO | B | CG | F | EK | F | B | D | E |
Inca Purple | NARO | BCG | BCF | CDF | EK | AB | B | AC | ACE |
Inca Red | NARO | B | BF | DF | EK | AC | BC | D | ABE |
Kitaakari | NARO | BF | ABF | CDF | BCEK | BE | BC | D | AEG |
Kitahime | NARO | BC | F | CG | EFGK | BCE | BEF | DE | E |
Kitamurasaki | NARO | BCG | B | CD | BEK | ADE | EF | D | ACE |
Kitamusashi | NARO | FG | BDF | BD | DE | BD | BE | E | ACEF |
Koganemaru | NARO | BF | AB | CD | BEK | BDE | ABE | DG | ACE |
Konahubuki | Hokkaido | BG | ABD | CD | CE | BD | B | D | AG |
Konayuki | Hokkaido | BG | AB | CDF | EHIK | AB | E | CD | AE |
Konayutaka | Hokkaido | BDF | ABE | DFG | BEK | BE | BC | D | AE |
Matilda | NARO | BD | CDG | DG | BEI | CD | ABE | D | EG |
May Queen | NARO | BDG | CDF | CFG | BEGI | BE | BE | EF | ABE |
Nishiyutaka | Nagasaki | B | BCF | CD | BEK | B | BE | Null | AEG |
Norin 1 | NARO | B | AB | CDF | BEHK | AB | BCE | D | AE |
Norking Russet | NARO | BF | BC | BCD | DE | BCE | BF | Null | AEG |
Northern Ruby | NARO | CG | BCF | CD | CEK | ABD | BF | DE | AE |
Okhotsk Chip | Hokkaido | BG | CDF | BCF | EK | BE | BEF | DF | AF |
Oojiro | NARO | B | AD | DF | BHIK | A | BCE | D | E |
Piruka | NARO | BF | ABC | BCD | BEK | BD | CE | CDG | ACE |
Prevalent | NARO | CD | CDG | BD | DE | BCD | AB | CE | E |
Ranger Russet | Idaho | B | CF | BDG | DEK | CD | BC | Null | AE |
Ranran Chip | NARO | BDF | BCF | BCD | BEK | BD | BCF | DG | ABE |
Red Andes | NARO | B | ABG | F | BDF | BDF | BE | D | ADE |
Red Moon | NARO | B | BDF | CD | AEJK | CF | AB | BCDF | ACE |
Rira Chip | Hokkaido | BCG | ACF | BCD | BEK | BE | BC | DG | AE |
Russet Bannock | Idaho | ABC | BCF | CF | DK | BDE | BC | D | EG |
Russet Burbank | Idaho | B | BDG | BCFG | DGK | BC | BEF | DE | AE |
Russet Norkotah | Idaho | BD | BCDG | CDFG | BDK | BCD | BC | D | AEF |
Saikai 31 (Dragon Red) | Nagasaki | ACE | AF | BCD | BEL | BD | BCF | D | ABE |
Sakurahubuki | NARO | EG | AB | CD | CEKL | BD | BC | D | AEG |
Sanjumaru | Nagasaki | AC | BDF | BC | BE | B | D | DG | AEF |
Sanyenimo (Vermont Gold Coin) | NARO | BG | ABDG | BCF | BCGK | B | BE | Null | AE |
Sayaakane | Hokkaido | BD | CF | CDF | E | BCD | BCE | D | AE |
Sayaka | NARO | BC | BF | CG | BFG | BD | BEF | DE | AE |
Setoyutaka | Nagasaki | DF | ABF | BDF | BEHK | ABD | BCE | CD | AEF |
Shadow Queen | NARO | CG | BCF | CD | BEK | ABCD | BEF | DE | AC |
Shepody | Idaho | ABG | CDFG | BCDG | BDG | BE | BE | Null | ABEG |
Shigetsu | NARO | BF | ABF | CD | BEK | ABD | BC | DG | ACE |
Snow March | Hokkaido | BDG | ABCF | CDE | BEK | B | BCE | DG | AE |
Snowden | NARO | BD | BCF | CDF | BEK | B | BC | D | ABEG |
Star Ruby | NARO | BDG | BCF | BCDF | BDK | BCE | BF | D | ACE |
Tawaramurasaki | NARO | BF | ACF | BDF | BE | D | ABE | C | AC |
Tokachikogane | NARO | BF | BF | BCD | BEK | ABD | BCE | DG | ACE |
Toya | NARO | BF | AC | BDF | BEK | BDE | BCE | G | A |
Toyoshiro | NARO | BG | ABD | CDF | EL | BDE | BC | D | AE |
Umatilla Russet | Idaho | BG | CG | BC | DK | BDE | B | D | CE |
Waseshiro | NARO | F | CDF | CDG | EK | ABE | BE | C | AE |
Western Russet | Idaho | BD | CF | BDG | DEK | BD | B | D | ABCE |
Yukirasya | NARO | BF | BCF | C | EK | D | B | D | ACE |
Yukitsubura | Hokkaido | BC | C | CDF | BDG | DE | BC | E | AE |
Symbols of the peaks are described in Table 2. Null indicates a cultivar in which no peaks are obtained with the corresponding primer pair.
PCR and DNA fragment analysis
Fifty-six SSR markers with a tetra-nucleotide motif from Spud DB (Hirsch et al. 2014) were initially selected. Using 4 Japanese and 4 US major cultivars, a preliminary test of PCR amplification was performed. After the screening, 8 markers were selected for efficient discrimination of cultivars.
Octaplex PCR reactions were carried in a 5 μL reaction mixture with 2.5 ng genomic DNA, 0.1 U of KOD -Multi & Epi- (Toyobo, Osaka, Japan) and appropriate concentrations of the primer pairs shown in Table 1. The forward primers were labeled with any of 6-FAM, HEX, NED, and PET fluorescent dyes. The PCR reactions were carried out with the following thermal profile: one cycle at 94°C for 2 min followed by 30 cycles at 98°C for 10 sec, 63°C for 30 sec, and 68°C for 30 sec. Electrophoresis was performed in a Genetic Analyzer 310 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR products were analyzed using GeneMapper v3.7 software (Thermo Fisher Scientific). For each locus, peaks were assigned letters in alphabetical order from the smallest to the largest (Table 2). The number of peaks and the number of profiles per marker were evaluated based on amplification of the 76 test cultivars. Discrimination power (DP) was calculated as DP = 1 − ∑Pi2 where Pi is the frequency of the ith profile.
Table 1.
Marker ID* | Chr. | Motif | Forward (5′ to 3′) | Reverse (5′ to 3′) | Conc. (μM) | peak range (bp) |
---|---|---|---|---|---|---|
4026/4027 | 1 | (CTAT)n(CTAG)n | NED-AACTTGCGGGAATAAGTGACG | ACTATACACACGTGCCCTGAAACTAG | 0.09 | 265–346 |
8242 | 2 | (CTTT)n | FAM-CGTCTTGGATGTCTTAGTTGTGG | GCAAAACCAGAAAGGCTAACAAAC | 0.08 | 191–218 |
12002 | 3 | (ACAT)n | NED-CCATGAACCTGAAGTTTTTCTGC | TGGATATCTTGTGCCTACAAGCTAG | 0.10 | 209–235 |
16410 | 4 | (ATAC)n | FAM-GTATGTTTGAGTAAAATCCTCCACCA′ | TTCTCTGCCCCCTTTTAATTTG | 0.16 | 258–354 |
31924 | 8 | (ATAC)n | VIC-CGAAGACACCAAATCGCTCAG | GAAACGCCATTAACATTTTACATCG | 0.07 | 136–250 |
35584 | 9 | (GAAA)n | VIC-AGTAAGTCAAACTCAACTCCAAGGTG | GTTCTAGATTATCTCACTCATGCCTTTC | 0.08 | 84–111 |
43016 | 11 | (ATCC)n | PET-CAAGCTGCATGAAAGCCATC | TTTGCCTAAAAGTTTGTAGTGTGAGG | 0.07 | 184–227 |
46514 | 12 | (TATC)n | PET-TGCTTTTTGTTTCCTTTTGTGTG | GGAATGAAACTAAGCCTTGCTCTG | 0.12 | 130–172 |
Marker IDs are the same as in Spud DB (http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml).
Table 2.
Marker ID | No. of peaks | No. of profiles | Discrimination power | Averaged peak size (bp) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| |||||||||||||||
A | B | C | D | E | F | G | H | I | J | K | L | ||||
4026/4027 | 7 | 27 | 0.912 | 265.0 | 307.4 | 312.9 | 319.7 | 339.3 | 343.0 | 345.5 | |||||
8242 | 7 | 31 | 0.942 | 190.6 | 193.5 | 194.5 | 198.4 | 206.3 | 214.2 | 218.2 | |||||
12002 | 7 | 28 | 0.920 | 208.9 | 212.9 | 216.9 | 217.8 | 224.5 | 230.7 | 234.6 | |||||
16410 | 12 | 36 | 0.927 | 257.6 | 266.9 | 271.0 | 279.0 | 281.0 | 310.3 | 325.2 | 335.5 | 339.5 | 346.3 | 349.9 | 353.6 |
31924 | 6 | 22 | 0.918 | 135.6 | 213.5 | 218.2 | 222.2 | 230.3 | 249.9 | ||||||
35584 | 6 | 17 | 0.874 | 84.0 | 91.9 | 95.9 | 99.8 | 103.7 | 111.2 | ||||||
43016 | 7 | 16 | 0.725 | 184.3 | 188.0 | 192.0 | 199.5 | 203.5 | 215.0 | 226.9 | |||||
46514 | 7 | 18 | 0.869 | 129.9 | 131.2 | 152.5 | 156.9 | 161.1 | 165.1 | 172.0 |
For each locus, peaks were assigned letters in alphabetical order from the smallest to the largest.
Results and Discussion
A total of 1,729 tetra-nucleotide SSRs were annotated by Spud DB (Hirsch et al. 2014). Among them, 56 SSRs were selected based on a high number of repeats, and were tested according to the following criteria: (1) two or more peaks detected in a preliminary screening with 8 cultivars, (2) no null peak, (3) at most one marker in each chromosome to ensure independence of individual markers. As a result, 8 SSRs were further selected and an 8-plex PCR condition was designed (Table 1).
Based on the analysis of the 76 test cultivars, the number of peaks ranged from 6 to 12 (average 7.4), and the number of profiles ranged from 16 to 36 (average 24.4). For each locus, the discrimination power (PD) ranged from 0.725 to 0.942, averaging 0.886 (Table 2). The probability of finding two random individuals with identical profiles at all 8 loci was an estimated 1.10 × 10−8, which provided enough discriminant power to identify the tested cultivars.
Among all peaks, the differences of peak sizes between B and C of marker 8242, C and D of 12002, and A and B of 46514 were within two bases, suggesting that these small differences of peak sizes may have been caused by insertion or deletion of nucleotide except for difference of SSR motif replication. Because one base difference of peaks is sometimes difficult to discriminate in peak callings, we propose to use reference cultivars in a practical discrimination experiment or to consider results of other markers profile when these peaks are used in cultivar discrimination.
The profiles of the 76 cultivars generated from the set of 8 SSR markers are shown in Table 3. By using 8 markers, 72 cultivars were distinguished from each other, except for two combinations of cultivars: the combination of “Red Moon” and “Destroyer”, and that of “Inca no mezame” and “Hokkai 98 (Inca Rouge)”. Both of the latter cultivars are known as skin color mutants derived from the former ones, suggesting that these two cultivar pairs respectively have the same genomic organization other than the corresponding gene for skin color.
Cultivar identification of potato has been reported previously, and the markers described by Ghislain et al. (2004, 2009) have been used widely. Since these markers are mainly di- and tri-nucleotide SSRs, the lower separation of neighboring alleles and the relatively high level of stutter bands are inevitable. In fact, Reid et al. (2011) reported that one allele of STM3023 (di-nucleotide SSRs) is located at the stutter position for the other allele, resulting in a complication of the allele call. Additionally, simplex PCR and the various annealing temperatures of the primers are time-consuming and labor-intensive.
The set of tetra-nucleotide SSRs described here has no or extremely little stuttering, resulting in good reproducibility and reliability of allele calling. The 8-plex PCR conditions designed in this study allow simple and rapid analysis of cultivars. These markers will be helpful for the rapid identification of potato cultivars, and consequently for protecting plant breeders’ rights.
Acknowledgments
The authors would like to thank the University of Idaho, HRO Kitami Agricultural Experiment Station, Nagasaki Agricultural and Forestry Technical Development Center, and NARO Hokkaido Agricultural Research Center for providing us with potato materials.
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