Solanum tuberosum - Springer Link

1996

325 CHARACTERIZATION OF SOLANUM TUBEROSUM SIMPLE SEQUENCE REPEATS AND APPLICATION T O POTATO CULT1VAR IDENTIFICATION L. M. Kawchuk ~, D. R. Lynch~,J. Thomas ~, B. Penner ~, D. Sillito 1, and F. Kulcsaf Abstract

With the continued introduction of new potato cultivars, accurate identification is becoming difficult but is essential for maintaining cultivar integrity and Plant Breeders' Rights. Hypervariable DNA sequences, referred to as simple sequence repeats (SSRs) or microsatellites, have been reported to be an excellent source of genetic markers. To determine the abundance, distribution, and composition of SSRs within Solanum tuberosum, 252 sequences were searched for tetranucleotide and smaller SSRs with a minimum length of 20 nucleotides and a m a x i m u m discrepancy of two nucleotides. In total, 40 unique SSRs were observed in the 252 S. tuberosum sequences examined and occurred at a frequency of one SSR every 8.1 kb. To assess the ability of site-specific amplified SSRs to identify potato cultivars, a simple (TCAC)m and compound (TCAC) m 9 (CTT) n SSR 5' to the starch synthase gene and a c o m p o u n d (C) 9 (CT) * , P q (AT)r 9 (G) s SSR 5 to the sequence encoding mature proteinase inhibitor I, were e x a m i n e d a n d shown to p r o d u c e u n i q u e DNA profiles for 73 of 95 tetraploid cultivars. In total, 24 alleles were observed at these loci and the accurately sized amplified DNA products can be used to establish a database for cultivar identification. Site-specific amplified alleles were somatically stable and have been conserved in clonal variants of Russet Burbank independently maintained for almost seven decades, a characteristic essential for cultivar identification. As genetic markers, the abundant, informative, and easily examined site-specific amplified alleles of SSRs are ideal for quickly and accurately determining cultivar identity of S. tuberosum ssp. tuberosum. Introduction Enforcement of regulations and proprietary fights requires fast and accurate identification of plant cultivars. For potato, however, identification is

'Agriculture and Agri-Food Canada, Research Center, P.O. Box 3000, Lethbridge, Alberta, Canada

T1J 4B1. ~Deparunent of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4. SBrooks Diagnostics Ltd., P.O. Box 1701, Brooks, Alberta, Canada T1R 1C5. Accepted for publication July 12, 1996. ADDITIONAL KEYWORDS: Microsatellites, allelic stability, PCR.

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extremely challenging with over 1,000 cultivars registered in North America and Europe (2, 29). Although genetic markers are ideal for identifying vegetatively propagated potato cultivars, existing procedures have several limitations. Isozymes are available only for a limited n u m b e r of loci and are influenced by plant development (6), restriction fragment length polymorphisms (RFLPs) require relatively large samples and take several days to examine (9), and random amplified polymorphic DNA (RAPD) analysis lacks the desired stringency (5). As a result, these procedures have been used only on a limited basis for the routine identification of potato cultivars. We examined the stability and suitability of using site-specific amplified DNA profiles as genetic markers for the identification of tetraploid potato cultivars. Simple sequence repeats (SSRs) are short sequences of one to five nucleotides repeated in tandem that are uniformly distributed in eukaryotic genomes and are frequently polymorphic, with a variable number of repetitive elements (11, 31). Simple sequence repeats have been observed in most organisms examined including mammals (15, 16, 18, 27, 30, 36), birds (19), "insects (8), fish (22), and plants (38). Oligonucleotides containing SSRs have been used routinely as restriction fragment length polymorphic (RFLP) probes and amplified using the polymerase chain reaction (PCR) to identify useful genetic markers. Simple sequence repeats have been examined in barley (38), canola (13), chickpea (38), grapes (32), rice (40), soybean (1, 20), tomato (25), and trees (4, 37). Veilleux, et al. (34) recently used SSRs to characterize the genetic composition of anther-derived plants of a diploid potato clone. The abundance, random distribution, and Mendelian inheritance of SSRs within plants suggests that they are ideal genetic markers for determining genetic diversity. However, application of these markers for cultivar identification requires that they are conserved for an extended length of time. A search of Solanum tuberosum ~,. DNA sequences was conducted to identify all tetranucleotide and smaller SSRs within the EMBL and GenBank databases. We report here the relative abundance, lengths, and composition of the SSRs and demonstrate the stability of SSRs over almost 70 years within vegetatively propagated S. tuberosum ssp. tuberosum. Oligonucleotide primers were used to amplify a simple (TCAC)m and compound (TCAC)m 9 (CTT)n S. tuberosum SSR 5' to the starch synthase gene and a compound (C) 9 (CT) 9 (AT)r 9 (G)s SSR 5' to the sequence encoding mature proteinase i;aPhibitorI.qThe allelic heterozygosity of 95 potato cultivars was determined at these loci to create a database of alleles for cultivar identification. Materials and Methods

Sequence ldentification

Searches for SSRs within S. tuberosum were performed using IntelliGenetics PC/GENE (Mountain View, CA) version 6.8. The SELECT program was

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used to identify all S. tuberosum sequences within the EMBL (release 36) and Genbank (release 79.0) databases. All m o n o n u c l e o t i d e t h r o u g h tetranucleotide SSR sequences were subsequently identified using the QSEARCH program set for a minimum length of 20 nucleotides and a maximum discrepancy of zero, one, or two nucleotides (Table 1). Each SSR is reported once and additional submissions excluded even though they may be polymorphic. Sequences selected because of their polyadenylation sequence were not reported.

DNA Preparation Potato genomic DNA was extracted from leaves and tubers of field and greenhouse propagated cultivars using a previously described procedure (7). Ninety-five randomly selected commercial tetraploid potato cultivars and seven clonal variants of Russet Burbank were examined (Fig. 1). Using published sequences (3, 14, 23, 33), oligonucleotide primers were synthesized to conserved sequences surrounding the (TCAC)m and (TCAC)m " (CTT)n SSRs 5' to the potato starch synthase gene and the (C)~ 9 (CT)q 9 (AT) r 9 (G) s SSR 5' to the sequence encoding mature proteinase in~aibitor I (Table 2). To facilitate analysis using an automated DNA sequencer, the Applied Biosystems (AB, Foster City, CA) fluorescent dye phosphoramidite 6-carboxyfluorescein (6-FAM) was incorporated into the primer 5' to the SSRs.

Simple Sequence Repeat Characterization Polymerase chain reaction (PCR) conditions were similar to those of Saiki, et al. (26) with a reaction volume of 100/A containing 5 ng of genomic DNA template, 0.2/zM of each deoxyribonucleotide primer, 200/tM of each dNTP, 2 mM MgC12, 50 mM KC1, 10 mM Tris-C1 pH 8.3, 0.01% gelatin, and 1 U Taq DNA polymerase (GIBCO BRL, Gaithersburg, MD). Samples were overlaid with mineral oil and PCR was performed using a Perkin-Elmer 480 thermocycler programmed for 4 min at 95 C followed by 26 cycles of I min at 95 C, 1 min at 55 C, and 2 min at 72 C with 10 min at 72 C during the final cycle. Amplified DNA products were separated in vertical non-denaturing 12% polyacrylamide gels, 20 cm in length, with TBE (89 mM Tris-borate, 2 mM EDTA at pH 8.0) for 3 h at 500 V using a Bio-Rad (Richmond, CA) Protean apparatus cooled to 6 C. Alternatively, aliquots of the amplified samples were e x a m i n e d by electrophoresis t h r o u g h horizontal n o n - d e n a t u r i n g 3% Metaphor (FMC BioProducts, Rockland, ME) agarose gels, 10 cm in length, for 4 h at 5 V / c m or 1 h at 15 V / c m with circulating TBE buffer (100 ml h -~) cooled to 15 C. Non-denaturing gels were subsequently stained with 0.5/~g/ml of ethidium bromide for 30 min. In a third alternative, amplified DNA products labelled with 6-FAM were combined with 6 fmol of size standard GENESCAN1000 (ABI), consisting of A/uI-restricted plasmid pBR322 DNA labelled with 6-carboxy-X-rhodamine (ROX), and denatured for 2 min at 90 C before being loaded onto a 24 cm well-to-read, 6% denaturing polyacrylamide gel. Gels were

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TABLE 1.--Accession designation for Solanum tuberosum SSRs composed of

tetranucleotide and smaller simple sequence repeats (SSRs) with a minimum length of 20 nucleotides and a maximum of zero, one, or two discrepancies. SSR

Perfect Match

O n e Discrepancy

Two Discrepancies

Total

A

L01401 X04078 X15494 Zl1680 Zl1741 Zl1882

X13497 X60397 X67511

9

C

M17108 "c

M17108 ~ X15494

3

M15186 X04077" $51460 ~ )(04118 X55752 X63103 X69760

14

AT

A01611 X04077" X55748 X67511 Z12611 ~ Z12824

CT

X04753 Z1261F

X13497

3

AAG

X58453"

L22576 I~ X73684 b

3

AAT

X13497

Z 13992

3

AGT

X66826

1

AATA

X13497

1

M 18881

2

ATI'A

X04077

CACT

X52416 c

Total

12

$51460"

X67511

1 9

"Sequences contain two distinct loci with the same SSR. ~Simple sequence repeat occurs within a coding sequence. cSimple sequence repeats e x a m i n e d for allelic diversity.

19

40

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electrophoresed for 14 h at 30 W on an ABI 373A DNA sequencer with the photomultiplier tube (PMT) adjusted so that the blue line was at 850 for the start o f each run. Product sizes were determined by ABI GENESCAN 672 software (version 1.2.2-1) using the Southern method. In total, n ( n - 1)/2 or 4,465 pairwise comparisons were made o f the SSR DNA products obtained from the 95 cultivars. Qualitative scores of all amplified DNA were used to generate similarity coefficients and cluster analysis was performed using the unweighted pair-gToup m e t h o d with arithmetical averages (24). The level of polymorphism of each locus was calculated by using the genetic diversity index, 1 - Zp/~,where Pi is the frequency of the i ~ SSR allele (28). Results

Occurrence of specific SSRs within the 252 reported DNA sequences from S. tuberosum and the type of SSR are indicated in Table 1. Complementary sequence and permutations of each SSR have been considered synonymous, providing a total of 48 possible repetitive elements that include 2 mononucleotides, 4 dinucleotides, 10 trinucleotides, and 32 tetranucleotides. In total, 40 SSRs, composed of 10 unique repetitive elements, with a minimum length of 20 nucleotides and a maximum 2 nucleotide discrepancy were detected in the 252 reported S. tuberosum sequences. Dinucleotide repeats were most comm o n (42%) followed by mononucleotide (30%), trinucleotide (18%), and tetranucleotide (10%) motifs. Maximum resolution and n u m b e r of amplified products were obtained using denaturing polyacrylamide gel electrophoresis and software capable of determining the size of amplified products. However, electrophoresis of the amplified products through Metaphor agarose or non-denaturing polyacrylamide gels was sufficient to detect many of the polymorphisms. Minor amplified products previously attributed to heteroduplexes formed during PCR (21) were occasionally observed with non-denaturing electrophoresis. Results were identical for DNA samples obtained from potato tubers and leaf tissue. Between zero and four amplified DNA products were observed at the various loci of any individual cultivar and a total of 851 amplified DNA products was observed in the 95 cultivars examined (Fig. 1). The proteinase inhibitor I SSR primers amplified DNA of two distinct sizes apparently from two loci. In total, 10 alleles were detected at the (TCAC)m locus, 7 alleles at the (TCAC) m * (CTI')n locus, 4 alleles at one o f the (C)p 9 (CT)~ 9 (AT)r 9 (G) s loci, and 3 alleles at the other (C) p 9 (CT) q 9 (AT) r 9 (G) s locu~s9Diversity values of 0.74, 0.77, 0.64, and 0.62 were calculated for the (TCAC) m, (TCAC)m 9 (CTr)n, and the two (C)p 9 (CT), 9 (AT)r 9 (G)s SSRs, respectively. Amplified alleles from the (T~AC) m, (TCAC)m 9 (CTT)., and (C)p 9 (CT)q 9 (AT)r 9 (G)s SSRs were capable of distinguishing 73 of 95 tetraploid cultivars. Twenty cultivars were distinguished using the (TCAC)m SSR, another 15 cultivars were distinguished using the (TCAC)m 9 (CTI')n SSR, and an addi-

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tional 38 cultivars were distinguished using the (C)p 9 (CT)q 9 (AT)r 9 (G)s SSRs (Fig. 1). No polymorphism was detected among the seven Russet Burbank clonal variants examined. Discussion

O u r results demonstrate that amplification of a SSR with site-specific primers results in reproducible polymorphic alleles that can be used for potato cultivar identification. This procedure is very discriminating having produced unique DNA profiles for 73 of 95 tetraploid potato cultivars examined using oligonucleotide primers specific for the SSRs located 5' to the starch synthase and proteinase inhibitor I genes in potato. Results indicate that a database of conserved SSR alleles capable of distinguishing commercial potato cultivars can be established to meet producer and regulatory needs. The 40 tetranucleotide and smaller SSRs identified within reported S. tuberosum DNA sequences were distributed throughout the genome. This corresponds to a SSR every 27.8 kb for a perfect match, similar to the rate of 1 per 18.2 kb in S. tuberosum previously calculated using 8 identified SSRs (35). Additional SSRs were detected within reported S. tuberosum DNA sequences by increasing the maximum discrepancy to two nucleotides, thereby increasing the average frequency of finding a SSR to every 8.1 kb. Only two of the identified SSRs occurred within coding sequences (Table 1), in agreement with previous reports (20, 35) that all but a few trinucleotide SSRs occur within n o n c o d i n g plant sequences, probably a c o n s e q u e n c e of the restrictions imposed on hypervariability within a coding sequence. A higher frequency of SSRs is expected since many of the S. tuberosum database sequences are derived from eDNA and the SSRs reside mostly in untranscribed 3', 5', or intron sequences. Several procedures have been developed for the isolation of additional SSRs from genomic DNA (12, 36, 39). Each procedure utilizes oligonucleotide probes or primers designed to detect and facilitate the cloning of a specific family of SSRs and surrounding sequences. An alternative procedure that does not require the isolation of the SSR, but unfortunately resembles the RAPD procedure in that random genomic sequences are amplified under conditions less stringent than those of site-specific amplification, uses a single SSR oligonucleotide primer to randomly amplify complementary SSRs (10). Simple sequence repeats represent an unlimited source of polymorphic genetic markers with a high genetic diversity index. In a tetraploid cultivar, the number of DNA profiles that can be produced by site-specific amplification is n + n ( n - 1 ) / 2 + n ( n - 1 ) ( n - 2 ) / 6 + n ( n - 1 ) ( n - 2 ) ( n - 3 ) / 2 4 or (n 4 - 2n ~ + l l n 2 + 14n)/24 where n is the number of alleles at a specific locus. Site-specific amplification of the (TCAC)m SSR 5' to the starch synthase gene produced 10 alleles and is therefore capable in theory of distinguishing 385 tetraploid cultivars. Several SSRs can be examined simultaneously using a single amplification reac-

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FFrux FPTUWX CFJMOPTUWX CFJMOPSTWX FJOPTUWX FJOPSTUWX FJOPSTUWX FJOPSTWX DFJMOPSTUVWX - Elba LI Rouge FJOPQSTUVW FJOPSTW - RussetBurbank FJOPSTVW -Saco C~-q~n Mountain FJOPSTVX FJOPSTVX FJOPQSTVX F]OPSTUVX FJOPQSTUVX FJOPSVX , ~way FJOPSVX FJOPSVX I Rarimn FJOPSVX Allagash Russet CEFJLMOPSTVX I~nsll CFJMOPSTUVX Irit~ Collier CF|JMOPSTV Ocdrce CFIJMNOPVX On~ri,, CFIJMNOPSTVX BFIJMNOPVX Norchlp FUNOPTUWX AC Chal9 FIJNOPSTUWX I Katal~iin Trent FIJNOPSTUWX FUNOPSTUX FIJNOPSTUW FUNOPSTWX Kenn,d~ FIJNOPSWX Norcnief FIJNOPSWX FIJNPSTUVW Keswick Tcton CFUNOPSTUVW Bclchi FIPQTUVX Idi~ ~ d FUNOPTUVX Islander FINPTUVX Bellide FINPSTVX ] ChinoOk FINPSTVX Chipbclle FINPSTVX Fro~der Russr162 FINPSTVX i Sl~inaw Gold FINPSVX Wauseon FINPSVX F1NPSVX FIJNOPSVX , C~'iv.m Jemse 8 FUNOPSVX FIJNOPSVX I Yellow Finn FIJNOPSVX FUNOPSVX ' Wbi= Pomiac Red Pontiac FUNOPQSVX FUNOPSTVX . Snmvden BelRus FUNOPQSTVX FUNOPQSTUVX , Carula FIJNOPQSTUVX ' Red Gold FIJNPQSTUVX , Russian Blue FUNPQSTUVX W.rba FIJNPQSTUVX ' T r ~ Blue Sangrr FIJNPQSUVX RUxtCCNorkotah FUNPQSTV FJNP(~TVX = Coastal Russet .- Fide Russet FJP~S rVX Buete FIP VX Rosa FHJNOPSVW Centennial Russet FJPSTVX La Br162 FPSTVX Ersmosa FPSTUVX Nooksack FPSTV I UU: Russet FPSTV Nor n FPSTV FPSTUVW AC Ptarmigan EFJLOPSTUWX Conestoga EFJLOPSTW Pink Pearl EFJLOPSWX EFLPSTUW Gemchtp EFJLNPTUVW EFJLOPTUVX Shel~dy EIJLNQSUVX Chkftain EFJLPQSTVX t Guldru~ EFJLPQSTVX Lxmhi R u s ~ EFIJLNPQSTVX Viking EFIJLNPQSVX Norland EFILNPSVX EFLPSVX Sburchip Bintje CEULMNOSTV Yuk~m Gold EIJLNOPSTUV Ca=cade FHJMPQSVX WhiO: Rose FJPQSV Redsen CFJMPQSTW Alaska Frostlcss ACJKMORSVX J SVX ~GO~LMOSVX Urgcma Bison CEOQSVW

FIG. 1. Separation of 95 potato cuhivars as determined by UPGMA cluster analysis of similarity coefficients calculated from amplified DNA products produced by SSRs 5' to the starch synthase and proteinase inhibitor I genes. Amplified DNA products of the (TCAC)m SSR (A, B, C, D, E, F, G, H, I,J), the (TCAC)m 9 (CqT) n SSR (K, L, M, N, O, P, Q), and the (C)p 9 (CT)q 9 (AT)r 9 (G)s SSRs (R, S, T, U and V, W, X) are given for each cultivar. Identical DNA patterns were detected for Russet Burbank clonal variants BC, COLO, IDAHO D, IDAHO E, MAN, NB, and PE1.

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tion by c o m b i n i n g p r i m e r s specific for i n d e p e n d e n t loci (36). W h e n e x a m i n i n g m o r e t h a n o n e locus the possible n u m b e r o f different b a n d i n g patterns is y', where y is the n u m b e r o f amplified p a t t e r n s at a specific locus a n d x is the n u m b e r o f u n l i n k e d loci e x a m i n e d . A l t h o u g h SSRs r e p r e s e n t hypervariable areas o f the g e n o m e , they are sufficiently conserved in plants to b e i n h e r i t e d for at least a few g e n e r a t i o n s i n a M e n d e l i a n fashion (20). I n each o f the 95 tetraploid potato cultivars exami n e d , the simple a n d c o m p o u n d SSRs were somatically stable with identical alleles observed in leaf a n d t u b e r tissues. Identical allelic profiles in clonal selections, i n d e p e n d e n t l y m a i n t a i n e d since 1930 (17), d e m o n s t r a t e s the l o n g - t e r m stability o f these alleles in a vegetatively p r o p a g a t e d c r o p a n d e n s u r e s t h e i r c o n t i n u e d effectiveness for cultivar identification. Site-specific amplification of SSRs u n d e r s t r i n g e n t c o n d i t i o n s u s i n g e x t r e m e l y small samples provides results that are unlikely to be i n f l u e n c e d by c o n t a m i n a t i n g D N A from n o n s o l a n a c e o u s sources. I n g e n e r a l , site-specific a m p l i f i e d SSRs r e p r e s e n t a n u n l i m i t e d source of easily e x a m i n e d g e n e t i c m a r k e r s ideal for the fast a n d accurate identification of S. tuberosum ssp. tuberosum cultivars.

Acknowledgements We t h a n k F o o d P r o d u c t i o n a n d I n s p e c t i o n , A g r i c u l t u r e a n d Agri-Food C a n a d a , V a n c o u v e r , British C o l u m b i a for p r o v i d i n g m a n y o f the cultivars e x a m i n e d . This research was s u p p o r t e d by the Alberta Agricultural Research Institute Project 950643. LRC c o n t r i b u t i o n 3879533.

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