High SSR diversity but little differentiation between accessions of Nordic timothy (Phleum pratense L.)

High SSR di e si y bu li le di e en ia ion be ween accessions o No dic imo hy (Phleum p a ense L.) P. TanhuanPää and O. Manninen Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, Jokioinen, Finland Tanhuanpää, P. and Manninen, O. 2012. high SSR di e si y bu li le di e en ia ion be ween accessions o no dic imo hy (Phleum p a ense L.). – He edi as 149: 114–127. Lund, Sweden. eiSSn 1601-5223. Recei ed 20 Sep embe 2011. accep ed 29 May 2012. a la ge collec ion o genebank accessions o he hexaploid ou c ossing o age g ass species imo hy (Phleum p a ense L.) was o he i s ime analysed o SSR di e si y on indi idual, popula ion and egional le el. Timo hy is he mos impo an o age g ass species in he no dic coun ies. eigh y-eigh imo hy accessions om no dic coun ies and eigh accessions a ound eu ope we e analysed wi h ecen ly de eloped simple sequence epea (SSR) ma ke s. Timo hy p o ed o be e y polymo phic: he 13 selec ed SSRs ampli ied a o al o 499 polymo phic alleles, he numbe o alleles pe SSR locus a ying om 15 o 74. Taking all SSR alleles oge he , he obse ed numbe in each accession anged om 95 o 203. Le els o di e si y we e ound o be signi ican ly di e en be ween coun ies, ege a ion zones and di e en cul i a ypes. howe e , he di e en ia ion be ween accessions was low: mos o he a ia ion (94%) in he s udied imo hy ma e ial was due o a ia ion wi hin accessions and only 5% was be ween accessions and 1% be ween coun ies. Lack o geog aphical di e en ia ion may e lec he ou c ossing and hexaploid na u e o imo hy. Ou esul s showed ha neu al SSR ma ke s a e sui able o demons a ing le els o di e si y bu no alone adequa e o esol e popula- ion s uc u e in imo hy. no dic imo hy ma e ial seems o be di e se enough o b eeding pu poses and no decline in he le el o di e si y was obse ed in a ie ies compa ed o wild imo hy popula ions. Challenges in analysing SSR ma ke da a in a hexaploid ou c osse we e discussed. Pi jo Tanhuanpää, Plan Genomics, Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, FI-31600 Jokioinen, Finland. e-mail: [email p o ec ed] he edi as 149: 114–127 (2012) © 2012 The au ho s. This is an Open access a icle. DOi: 10.1111/j.1601-5223.2012.02244.x Timo hy (Phleum p a ense L.) is a cool-season pe ennial g ass species dis ibu ed na u ally h oughou eu ope and pa s o no h a ica and asia. Wild popula ions o P. p a ense ep esen a polyploid se ies om diploids o oc oploids. The cul i a ed o m o imo hy is hexaploid. The uni o mi y o he molecula p o ile in ag icul u al P. p a ense sugges s ha he o ma ion o his hexaploid is p obably pos -glacial (S e w a e al. 2011). The genomic composi ion o hexaploid imo hy has no been ully esol ed ye , bu he e is some e idence ha he genome con ains ou doses o be olonii genome and wo doses o hae icum. Bo h o hese genomes de i e om he same p ogeni o and a e no e y di e en ia ed which explains ha bo h hexasomic and e adisomic inhe i ance has been epo ed in imo hy (S e w a e al. 2011). Timo hy is cul i a ed o hay, silage and pas u e ac oss he no he n hemisphe e. in no dic coun ies imo hy is he mos impo an o age g ass species due o adap a ion o he cool and ela i ely humid no he n clima e. The main goal in imo hy b eeding o his egion is o combine high yield, good win e su i al, and high eeding quali y. Timo hy b eeding elies on b oad gene ic a ia ion and u ilisa ion o he e osis, which can be achie ed by combin- ing gene ically dis an indi iduals wi h good combining abili y in a syn he ic a ie y. The e o e, plan b eede s ha e o be su e ha hey ha e su icien gene ic a ia ion a ailable o hei b eeding p og ammes. Gene ic di e si y wi hin a plan species e lec s bo h he li e his o y ai s and dis ibu ion o he species. Pe ennial, ou c ossing species a e known o ha e highe gene ic di e si y and less di e en ia ion among popula- ions han annual sel pollina o s (Ha m i c k and Go d 1996). Timo hy is a pe ennial wind pollina ing species whe e hexaploidy is expec ed o u he ise he le el o di e si y. The abundan cen e model (B o w n 1984) p e- sumes educed neu al gene ic di e si y wi hin pe iphe al compa ed o mo e cen al popula ions (ec k e e al. 2008). al hough he dis ibu ion o hexaploid imo hy co e s mos o eu ope (co n e 1998), a he no he n ma gin, namely no he n bo eal and alpine ege a ion zones, ha sh win e condi ions may limi su i al o imo hy. This may be e lec ed in he le els o di e si y. P e ious s udies ha e also shown ha gene ic di e si y o plan popula ions may ei he inc ease o dec ease wi h inc easing al i ude (Ya n e al. 2009). no dGen, he no dic Gene ic Resou ce Cen e , has a collec ion o 716 accessions o imo hy, o igina ing mos ly om no dic coun ies. Six y- ou o hese a e cul i a s, o he s ep esen na u al popula ions o old land aces. no dic coun ies ep esen a wide geog aphical egion he edi as 149 (2012) SSR di e si y in No dic imo hy 115 wi h a ying g ow h condi ions om sou he n nemo al zone in Denma k o no he n alpine ege a ion zone in no he n no way. in addi ion o la i ude, g ow h condi- ions a e also a ec ed by longi ude since condi ions in wes e n no way a e ma i ime and in eas e n Finland mo e con inen al. Th ee hund ed and se en y- h ee imo hy accessions in he no dGen collec ion ha e been p e iously cha ac e ised o mo phological and ag o- nomic ai s in Finland, no way, iceland and Sweden du ing 1995–1996 (<www.no dgen.o g/index.php/skand/ con en / iew/ ull/344>). Cha ac e isa ions we e mos ly made on coa se, ela i e scale and a ia ion wi hin each accession was no aken in o accoun . This da a gi es an o e all pic u e o he pheno ypic a ia ion p esen in he collec ion. howe e , i doesn’ ully desc ibe he le els o gene ic di e si y, he gene ic s uc u e o a ia ion be ween and among popula ions no he gene ic dis ances be ween indi iduals o popula ions. The e a e many molecula ma ke sys ems a ailable o di e si y analyses, om which we chose simple sequence epea s (SSRs) o s udying di e si y in imo hy. P ime s o 355 SSR loci in imo hy ha e been de eloped (ca i e al. 2003), and some o he loci ha e been loca ed on a diploid imo hy map (ca i e al. 2009). SSRs a e mos ly codominan ly inhe i ed, e y polymo phic, and wi h he use o di e en luo escen labels, can be mul i- plexed in PCR. The in o ma ion con en pe locus is big- ge in SSRs compa ed o dominan ma ke s because homo- and he e ozygo es a e de ec ed. howe e , in polyploid species in e p e a ion o exac ma ke geno- ypes is no s aigh o wa d and SSR alleles a e usually analysed as p esence/absence ma ke s. This s udy is pa o a wide no dic collabo a i e esea ch p ojec , whe e he a ia ion o no dGen imo hy collec ion was e alua ed bo h on pheno ypic and geno ypic le el. he e we epo he esul s o he assess- men o gene ic di e si y using SSR ma ke s. Ou aim was o s udy whe he geog aphical loca ion ( ege a ion zone, la i ude, longi ude, al i ude) a ec s he le el o gene ic di e si y. in addi ion, we s udied i gene ic ma ke s could ind a popula ion s uc u e in he no dic imo hy ma e ial and hus help inding he e o ic g oups among he collec- ion o imo hy o be used in a ie y b eeding. MaTeRiaL anD MeThODS Plan ma e ial eigh y-eigh imo hy accessions om no dic coun ies (Table 1, Fig. 1) we e selec ed om no dGen collec ion based on geog aphical dis ibu ion and p e ious pheno- yping da a o ep esen as wide geog aphical and ai a ia ion as possible, and 15–20 andomly selec ed indi iduals pe accession we e analysed. Mos o he accessions, namely 59, we e classi ied as wild accessions, 17 as land aces and 11 as a ie ies o b eede s ma e ial. accessions we e di ided o six g oups acco ding o coun- y o o igin: no way (26), Finland (25), Sweden (25), Denma k (10), iceland (2), and exo ic (8) including all o igins ou side no dic coun ies. exo ic accessions we e ob ained om di e en genebank collec ions. accessions wi h known geog aphical coo dina es we e di ided o six ege a ion zones (mo e n 1999): 1  nemo al (11), 2  bo eonemo al (15), 3  sou he n bo eal (14), 4  middle bo eal (22), 5  no he n bo eal (11), and 6  alpine (2). Dnas we e ex ac ed using he me hod o i n k e e al. (1993) wi h he ollowing modi ica ions: lyophilised lea es we e c ushed wi h a Fas P ep FP120 Cell Dis up e (BiO 101, The mo Sa an , Wal ham, Ma, uSa), in 1 ml CTaB (hexadecyl ime hyl-ammonium b omide) bu e supplied wi h 70 u o ibonuclease a (Omega Bio- ek, no c oss, Ga, uSa) and 0.05 mg o p o einase K (Finnzymes, espoo, Finland). ex ac ions we e i s done wi h phenol/chlo o o m/isoamyl alcohol (25:24:1) and hen wi h chlo o o m. Dna concen a ions we e mea- su ed using he GeneQuan ii Rna/Dna Calcula o (Pha macia Bio ech L d., Camb idge, uK). SSR analyses SSRs de eloped o imo hy (ca i e al. 2003) we e used o assessing di e si y in he selec ed accessions. a he beginning o he s udy, 35 imo hy SSRs we e selec ed using he ollowing c i e ia: s ong ampli ica ion (ca i e al. 2003), p e e ably SSRs wi h inucleo ide epea s (SSRs con aining inucleo ide o highe o de epea s ha e less s u e ing: Ho l o n 2001), and some SSRs which ha e been localised on one posi ion on he diploid imo hy map (ca i e al. 2009). The SSRs we e op imised and es ed o hei polymo phism, mul i- plexing possibili ies, and easiness o in e p e a ion. One p ime o each p ime pai was labelled wi h a luo escen dye, FaM (5-ca boxy luo escein), heX (hexachlo o- 6-ca boxy luo escein) o TeT (6-ca boxy e achlo o- luo escein) o enable sepa a ion and isualisa ion o ampli ica ion p oduc s wi h a MegaBaCe 500 Sequence (Ge heal hca e, Buckinghamshi e, uK) using MegaBaCe eT400-R Size S anda d. Thi een bes SSRs (Table 2) we e selec ed o inal analyses and we e ampli ied using wo di e en PCR p og ams in a PTC-220 Dna engine Dyad Pel ie The mal Cycle (MJ Resea ch, Wal ham, Ma, uSa) o a Bio-Rad Dna engine Te ad 2 The mal Cycle (Bio-Rad, he cules, Ca, uSa). The i s i e SSRs in Table 2 we e ampli ied wi h i e cycles o 15 s a 94°C, 15 s a 65°C, and 30 s a 72°C, ollowed by 30 simila cycles excep ha he anneal- ing empe a u e was 60°C. The p og am s a ed wi h an ini ial dena u a ion s ep o 5 min a 94°C and was 116 P. Tanhuanpää & O. Manninen he edi as 149 (2012) Table 1. Nine y-six accessions o Phleum p a ense ssp. p a ense analysed in he s udy wi h 499 SSR ma ke s (each SSR allele ea ed as a sepa a e ma ke ). numbe code accession no. Genebank name Coun y Cul i a ype1La i ude Longi ude al i ude2 Veg. zone3 no. o ind. Obse ed no. o ma ke s no. o p i a e ma ke s aa4ai5PWD6 1 nGB10828 no dgen Va88108 Denma k W 1 19 120 0 113.1 28.6 31.0 2 nGB10829 no dgen Va88112 Denma k W 1 19 141 2 131.9 32.9 38.7 3 nGB10830 no dgen Va88119 Denma k W 1 19 124 0 116.7 28.8 30.7 4 nGB10831 no dgen hF88266 Denma k W 1 19 148 2 137.9 28.4 32.5 5 nGB15461 no dgen Vildbje g aC0103 Denma k W 56°11′ n 8°49′58″ e 40 1 19 188 0 171.5 33.3 44.0 6 nGB16650 no dgen ejsing Denma k W 56°31′21″ n 8°47′11″ e 1 19 142 0 132.9 31.2 36.2 7 nGB1672 no dgen BiLBO Denma k CV 19 162 1 149.7 31.7 40.1 8 nGB1675 no dgen POTa Denma k CV 20 119 0 110.1 30.0 32.5 9 nGB4053 no dgen SR SaLTuM Mh0202 Denma k W 57°14′ n 9°46′ e 6 1 19 145 0 135.2 30.6 36.2 10 nGB4548 no dgen nR FaRuP Mh0202 Denma k W 55°21′ n 8°41′ e 2 1 20 154 0 139.4 30.3 35.1 11 nGB132 no dgen LiPinLahTi Me0901 SeP a Finland L 63°28′ n 29°18′ e 100 4 19 186 0 167.5 31.1 39.1 12 nGB9285 no dgen OTTO Finland CV 18 188 1 175.9 35.9 46.8 13 nGB14394 no dgen KäRKÖLä hM0102 Finland W 60°55′16″ n 25°17′27″ e 3 19 175 0 162.2 32.7 42.6 14 nGB14399 no dgen MahLaMäKi Mh0104 Finland W 61°22′00″ n 22°56′19″ e 70 3 20 179 0 162.5 32.9 41.7 15 nGB14403 no dgen näReKuMPu Mh0103 Finland L 61°57′14″ n 28°26′05″ e 3 20 203 2 180.8 35.0 46.3 16 nGB14404 no dgen PaaTTinen Mh0201 Finland L 60°35′11″ n 22°22′16″ e 2 20 176 0 158.4 33.6 40.5 17 nGB14415 no dgen huOLiLa Mh0204 Finland L 60°41′00″ n 21°45′06″ e 2 20 141 0 128.0 30.6 35.0 18 nGB14417 no dgen MeDVaSTÖ Mh0101 Finland W 60°06′02″ n 24°37′36″ e 25 2 18 121 0 115.2 28.4 32.5 19 nGB14419 no dgen KiiKaOJa Mh0201 Finland W 61°30′33″ n 22°32′10″ e 3 20 194 1 174.4 32.3 42.3 20 nGB747 no dgen nuVVuS aK0401 Finland W 69°50′ n 26°19′ e 160 6 20 144 1 131.4 30.3 34.7 21 nGB748 no dgen uTSJOKi aK0602 Finland W 69°55′ n 27°03′ e 70 6 20 169 1 153.6 33.7 39.8 22 nGB754 no dgen haLOSenRanTa eh0101 Finland W 66°41′ n 27°30′ e 145 4 19 183 0 168.4 34.7 43.9 23 nGB757 no dgen PeKKaLa eh0703 Finland W 66°21′ n 26°52′ e 145 4 19 189 0 173.5 36.2 44.7 24 nGB1095 no dgen LaiTaSaaRi Me0201 Finland L 64°51′ n 25°56′ e 4 20 192 1 170.2 34.2 43.1 25 nGB1096 no dgen TuOMiOJa Me0201 Finland L 64°36′ n 25°02′ e 4 18 191 1 177.5 33.6 44.0 26 nGB1107 no dgen JYRinKi Me0101 Finland L 63°55′ n 24°26′ e 4 19 188 0 171.5 33.6 43.5 27 nGB1111 no dgen MäLäSKä Me0101 Finland L 64°24′ n 26°19′ e 4 20 194 1 172.7 33.6 44.3 28 nGB151 no dgen VaRiSLahTi Me0102 Finland L 62°42′ n 28°42′ e 115 3 18 190 0 176.5 33.9 41.1 29 nGB1115 no dgen KiLPau Me0101 Finland L 64°20′ n 25°07′e4 19 194 0 178.8 35.1 44.6 30 nGB1119 no dgen KaTeRMa Me0401 Finland L 64°03′n 29°09′ e 4 19 189 0 170.9 31.9 40.5 (Con inued) he edi as 149 (2012) SSR di e si y in No dic imo hy 117 Table 1. (Con inued). numbe code accession no. Genebank name Coun y Cul i a ype1La i ude Longi ude al i ude2 Veg. zone3 no. o ind. Obse ed no. o ma ke s no. o p i a e ma ke s aa4ai5PWD6 31 nGB1122 no dgen nääDänMaa Me0202 Finland L 62°30′ n 28°14′ e 3 19 188 0 174.7 35.1 45.5 32 nGB2791 no dgen nORRGÅRD aP0101 Finland L 63°32′ n 22°31′ e 3 19 186 2 169.8 33.3 42.5 33 nGB2798 no dgen LÅnGÅMinne aP0201 Finland L 62°54′ n 21°43′ e 3 20 186 1 164.9 33.6 41.2 34 nGB2836 no dgen LanKaMaa aP0202 Finland L 62°23′ n 26°14′ e 3 19 184 0 166.8 32.1 41.5 35 nGB4066 no dgen TaMMiSTO Finland CV 19 180 1 164.5 32.7 38.6 36 nGB4140 no dgen KORPa iceland L 19 193 1 176.2 33.3 43.9 37 nGB4141 no dgen aDDa iceland CV 19 117 0 111.2 31.3 34.6 38 nGB7557 no dgen KLeVeLanD 01-5-43-2 no way W 63°13′ n 11°03′ e 300 4 20 178 0 160.9 33.7 41.7 39 nGB7559 no dgen SVenDGÅRD 01-5-43-4 no way W 63°13′ n 11°03′ e 100 4 15 152 0 152.0 33.9 42.9 40 nGB7573 no dgen ØVRe heRSTaD 01-5-44-3 no way W 63°52′ n 11°16′ e 200 3 20 164 1 146.6 32.2 39.2 41 nGB7577 no dgen VOLDen 01-5-44-8 no way W 63°22′ n 9°56′ e 20 3 17 179 2 170.4 33.8 42.8 42 nGB7592 no dgen SKJØLSViK 01-5-46-5 no way W 62°57′ n 7°48′ e 20 3 19 182 0 168.4 35.3 44.9 43 nGB7597 no dgen MÅna 01-6-48-13 no way W 62°06′ n 10°37′ e 600 5 20 155 0 141.4 32.8 40.3 44 nGB7709 no dgen nORDSKOT 01-2-13-6 no way W 67°50′ n 14°50′ e 15 4 19 186 1 169.9 32.6 42.7 45 nGB10785 no dgen SanDBu 01-6-49-4 no way W 61°52′ n 9°07′ e 420 5 19 95 0 86.8 32.6 28.9 46 nGB13647 no dgen LeVeLD no way P 18 106 1 102.6 32.6 34.1 47 nGB17194 no dgen i jo d 1-1-2-2 no way W 70°27′42″ n 27°06′30″ e 5 20 166 0 149.5 31.4 37.2 48 nGB17198 no dgen Ka asjok 1-1-3-2 no way W 69°28′31″ n 25°30′23″ e 5 18 179 1 166.4 32.4 42.5 49 nGB2169 no dgen BODin no way CV 19 167 0 153.4 30.3 38.8 50 nGB2170 no dgen VÅTi7702 no way B 17 119 0 115.0 34.0 35.2 51 nGB2180 no dgen GRinDSTaD no way CV 18 185 1 169.2 31.2 39.5 52 nGB2917 no dgen KLOMSeT 01-6-54-6 no way W 59°28′ n 8°36′ e 130 3 19 164 0 152.3 35.1 42.5 53 nGB2918 no dgen huSeTeR 01-9-70-1 no way W 59°40′ n 11°23′ e 135 2 20 164 0 150.2 32.1 39.0 54 nGB2922 no dgen SØRhuS 01-6-48-2 no way W 62°06′ n 10°40′ e 520 5 18 178 0 166.5 34.4 43.3 55 nGB2927 no dgen ØSTeRØYa 01-9-71-1 no way W 59°05′ n 10°12′ e 10 2 20 182 1 164.0 34.2 42.7 56 nGB2930 no dgen GRauTeKnaPP 01-6-54-7 no way W 59°10′ n 8°49′ e 95 2 20 194 0 174.6 34.4 44.7 57 nGB4226 no dgen haTLeSTaD 01-7-56-3 no way W 61°21′ n 6°06′ e 5 17 158 0 151.6 34.9 42.2 58 nGB4227 no dgen hÅRKLau 01-7-56-4 no way W 61°25′ n 6°15′ e 5 19 155 1 146.8 35.6 43.6 59 nGB4231 no dgen GJeRDÅKeR 01-7-58-1 no way W 60°40′ n 6°30′ e 100 5 19 156 1 143.6 31.3 38.4 118 P. Tanhuanpää & O. Manninen he edi as 149 (2012) 60 nGB4508 no dgen eneBO 01-6-48-5 no way W 61°15′ n 12°19′ e 550 4 20 161 0 149.2 33.2 38.9 61 nGB4523 no dgen FOSS 01-9-71-3 no way W 59°25′ n 11°21′ e 100 2 19 188 1 170.8 35.1 45.3 62 nGB7548 no dgen naMSVaTn 01-5-40-1 no way W 64°58′ n 13°34′ e 500 5 18 150 0 140.8 29.8 35.4 63 nGB7551 no dgen SOLeM 01-5-42-1 no way W 63°45′ n 9°45′ e 20 3 17 168 0 161.2 34.5 44.2 64 nGB722 no dgen KuOSSenJaRKa JP0404 Sweden W 66°41′ n 19°45′ e 260 5 19 115 0 108.8 29.5 32.7 65 nGB728 no dgen PJeSKeR Ph0405 Sweden W 65°32′ n 19°42′ e 350 4 18 182 0 171.2 33.9 43.4 66 nGB11428 no dgen JOnaThan Sweden CV 16 139 0 138.0 34.2 39.8 67 nGB11430 no dgen aRGuS Sweden CV 19 154 1 141.0 31.7 37.0 68 nGB13226 no dgen RaGnaR Sweden CV 17 111 0 107.9 32.8 34.3 69 nGB14224 no dgen SÖnDRaRP iB0101 Sweden W 57°36′02″ n 14°26′39″ e 268 2 20 175 1 158.4 33.6 42.2 70 nGB14236 no dgen LÖVhuLT iB0103 Sweden W 57°39′11″ n 14°45′23″ e 269 2 19 170 1 156.6 31.2 40.0 71 nGB731 no dgen RÖRMYRBeRG JP0204 Sweden W 64°40′ n 19°09′ e 350 4 20 182 2 163.2 31.9 41.1 72 nGB16958 no dgen LYa LJunGheD FO0201 Sweden W 56°24′13″ n 12°53′42″ e 175 1 18 152 0 142.3 30.7 37.7 73 nGB16975 no dgen nORRa KYLSäTeR FO0103 Sweden W 58°36′04″ n 11°59′51″ e 117 2 19 176 1 161.8 33.5 42.2 74 nGB733 no dgen SÖDRa SunDeRBYn Me0101 Sweden L 65°40′ n 21°52′ e 20 4 15 185 1 185.0 36.2 46.6 75 nGB16977 no dgen RYR, STORa BeRGeT FO0101 Sweden W 58°48′16″ n 12°29’25″ e 47 2 19 179 1 165.0 33.5 43.1 76 nGB16981 no dgen BRäCKeTORP FO0501 Sweden W 59°02′48″ n 12°29′26″e166 2 16 153 0 149.8 33.8 41.7 77 nGB17061 no dgen STORa ROThuLT haJ0201 Sweden W 57°55′53″ n 15°40′33″ e 180 2 20 187 0 168.5 33.4 43.3 78 nGB1306 no dgen BRaTTÅKeR GB0101 Sweden W 64°18′ n 19°33′ e 344 4 18 184 2 173.6 32.9 41.4 79 nGB1310 no dgen STORhäGGSJÖ GB0104 Sweden W 63°59′ n 20°00′ e 100 4 19 138 0 130.9 34.6 41.9 80 nGB1320 no dgen SKaRPMYRBeRG PR0601 Sweden W 64°37′ n 16°18′ e 400 5 20 194 0 173.4 33.5 42.5 81 nGB1327 no dgen haMMaRn PR0401 Sweden W 63°48′n 20°29′ e 10 4 19 162 0 151.4 33.6 41.2 82 nGB1330 no dgen äLGSJÖ Sh0302 Sweden W 64°13′ n 17°29′ e 390 4 20 185 0 166.0 34.7 43.3 83 nGB1331 no dgen VäSTanSJÖ Sh0102 Sweden W 63°45′ n 18°59′ e 170 4 19 170 0 158.4 32.8 42.1 84 nGB1332 no dgen KLuBBSJÖ Sh0301 Sweden W 63°51′ n 19°07′ e 250 4 18 185 1 172.4 34.0 43.1 85 nGB1537 no dgen eSKeLheM TL0104 Sweden W 57°29′ n 18°10′ e 2 18 139 1 131.7 30.7 36.1 86 nGB2530 no dgen RäMne GJ0301 Sweden W 59°00′ n 12°04′ e 110 2 19 165 0 152.8 31.9 39.6 87 nGB4349 no dgen BeneSTaD JK1506 Sweden W 55°31′ n 13°54′ e 40 1 20 171 1 153.4 32.4 40.6 88 nGB4350 no dgen BOaRP SB2106 Sweden W 55°56′ n 13°47′ e 180 1 18 175 1 163.9 34.3 42.6 89 Pi381926 GRin F ance P 19 131 0 122.3 31.1 34.6 90 Pi406317 GRin Russia P 19 165 1 151.7 31.8 38.5 (Con inued) he edi as 149 (2012) SSR di e si y in No dic imo hy 119 Table 1. (Con inued). numbe code accession no. Genebank name Coun y Cul i a ype1La i ude Longi ude al i ude2 Veg. zone3 no. o ind. Obse ed no. o ma ke s no. o p i a e ma ke s aa4ai5PWD6 91 ihaR151908 ihaR Ge many P 19 150 0 137.4 31.4 34.8 92 Pi210426 GRin G eece P 18 146 2 138.9 31.5 38.3 93 Pi325461 GRin Russia P 19 170 8 157.8 31.7 39.8 94 Pi204480 GRin Tu key P 19 158 3 144.3 31.6 37.6 95 14G2400116 RiCP Czech Republic P 19 186 2 170.5 34.2 44.0 96 RCaT040682 RCaT hunga y W 20 157 6 143.5 30.5 39.1 1CV  ad anced cul i a , L  adi ional cul i a , land ace, B  b eeding, esea ch ma e ial, gene ic s ock, P  pending, unknown cul i a ype, W  wild popula ion, weedy. 2me e s abo e sea le el. 3 ege a ion zones, acco ding o Moen 1999. 4co ec ed numbe o all ma ke s in each accession. 5mean numbe o all alleles obse ed in each indi idual. 6mean numbe o pai wise di e ences (PWD) (euclidean dis ances) be ween indi iduals in each accession. Fig. 1. Geog aphic loca ion o 71 imo hy accessions. numbe codes a e p esen ed in Table 1. ollowed by a inal ex ension s ep o 7 min a 65°C. The ollowing eigh SSRs in Table 2 we e ampli ied wi h he PCR p og am desc ibed in ca i e al. (2003). The PCR ampli ica ion eac ions in 10 ml con ained 0.25 u o FiRePol Dna polyme ase i (Solis BioDyne Ou, Ta u, es onia), he bu e B wi h 2.5 mM MgCl2 supplied by he enzyme manu ac u e , 100 mM each dnTP, 10 ng o Dna, and 125–500 nM each p ime . Sui able SSR combi- na ions we e ound wi h he Fas PCR so wa e (ka l e n d a e al. 2009), and he 13 SSRs we e mul iplexed in i e PCR ( hose ampli ied oge he a e g ouped in Table 2). Da a analyses allele pheno ypes o he plan s we e isually sco ed using a bina y code (1/0) o he p esence o absence o allele peaks wi hou knowing he doses o he alleles. When calcula ing gene ic dis ances each SSR allele was hus ea ed as a sepa a e ma ke locus. howe e , o he POPDiST p og am ( o m i u k e al. 2009), he allele pheno ype was eco ded locuswise i.e. he allelic con en o an indi idual a each o he 13 SSR loci was desc ibed. Gene ic di e si y o an accession was desc ibed in h ee di e en ways: 1) co ec ed numbe o all alleles ( ma ke s) in each accession (aa), whe e he obse ed numbe o alleles was co ec ed o a sample size n  15 120 P. Tanhuanpää & O. Manninen he edi as 149 (2012) Table 2. SSRs used in he di e si y analysis o imo hy accessions. SSR Repea mo i Repea class Fluo escen label exp. size (bp)1 allele size ange (bp) no. o alleles no. o alleles/ accession Mean no. o alleles/ indi idual Mos common allele2 no. o p i a e alleles3 Miss. in . (%) Linkage g oup4 a03a07 (TG)33 pe ec heX 140 94–207 50 4–17 2.10 0.64 5 2.5 unknown C02C08 (aaG)13 pe ec FaM 241 212–278 24 4–19 2.69 0.61 0 2.7 LG6 C01B11 (TTC)16 pe ec TeT 194 153–249 31 9–21 3.91 0.76 5 0.8 unknown C02h01 (TTC)17 pe ec FaM 146 95–201 53 8–26 2.93 0.44 8 2.4 unknown D01e04 (Caa)8(Taa)10 compound TeT 158 95–288 56 8–23 2.82 0.67 11 1.9 LG1 B03F07 (TC)14 pe ec heX 130 112–172 32 2–18 1.44 0.29 2 15.1 unknown C01e11 (TTC)11 pe ec FaM 123 98–142 15 2–10 1.12 0.53 0 18.8 LG5 a09h08 (TG)16 pe ec TeT 255 230–267 15 4–9 3.55 0.86 5 1.1 unknown D01G10 (TGa)7(CGa)4舰 … (TGa)16 compound and in e up ed FaM 231 208–336 74 7–24 2.51 0.44 9 2.4 LG2 B03a09 (Ga)19 pe ec TeT 226 193–247 40 8–22 2.62 0.28 3 0.3 LG4 a03e06 (TTG)28 pe ec heX 238 166–290 49 3–22 2.46 0.91 10 0.5 unknown D01h08 (aaT)13 pe ec FaM 147 116–170 20 4–15 1.88 0.28 1 7.1 LG6 a10a10 (Ca)31 pe ec TeT 232 161–243 40 6–22 2.68 0.68 7 3.2 LG3 1acco ding o Cai e al. 2003. 2Occu ence o he mos common allele. 3alleles p esen in only one accession. 4Re e s o he diploid imo hy map (Cai e al. 2009). he edi as 149 (2012) SSR di e si y in No dic imo hy 121 di e en dis ance indices: euclidean dis ance om a lequin, nei’s dis ance (ne i 1972) om Genalex, and Tomiuk and Loeschke dis ance om Popdis ) wi h geo- g aphic dis ance (km) was es ed using a Man el- es (ma n e l 1967) in he so wa e Genalex. Man el es was also used o compa e di e en gene ic dis ance indices. ReSuLTS Di e si y in SSR loci Thi een SSRs (Table 2) we e selec ed o assess gene ic di e si y in imo hy accessions. O hese, i e included a dinucleo ide mo i and eigh a inucleo ide mo i , o which wo we e compound ones. in some cases he allelic se ies (allele sizes i he assump ion o inc emen s o wo o h ee nucleo ides) was pe ec (C02C08, B03F07, C01e11, D01h08) bu usually some alleles we e missing. Gene ally, he allele sizes ollowed nea ly he inc emen o wo o h ee bases. howe e , a ew ex a alleles ha did no i he allele se ies exis ed in all SSRs excep C01e11. in some SSRs (C02h01, D01G10, B03a09, a03e06), he e seemed o be ano he allele se ies di e ing om he common one wi h one base pai (Fig. 2). The exis ence o his o he se ies mos p obably was he ou come o an indel mu a ion in he SSR amplicon. as a consequence, he size o all he alleles a isen he ea e had shi ed wi h one base pai . The 13 selec ed SSRs ampli ied a o al o 499 polymo - phic alleles, he numbe o alleles pe SSR locus a ying om 15 (C01e11 and a09h08) o 74 (D01G10) (Table 2). The a e age epea leng h o alleles in SSR loci co ela ed posi i ely (  0.69) wi h he o al numbe o alleles in he loci. Mos o he alleles we e qui e a e, ca. 40% occu ed in no mo e han 1% o all he indi iduals, o wi h housand imes o esampling wi hou eplacemen , 2) mean numbe o all alleles obse ed in each indi id- ual (ai) and 3) mean numbe o pai wise di e ences (PWD) (euclidean dis ances) be ween indi iduals in each accession, which was coun ed wi h he p og am aRLeQuin e . 2.000 (Sc H n e i d e e al. 2000). Di e - ences in he le el o di e si y be ween di e en g oups like coun ies, ege a ion zones (mo e n 1999), o cul i a ype we e analysed by anOVa P oc GLM (SaS en e p ise Guide 4.3). Co ela ions we e coun ed be ween di e si y and la i ude, longi ude and al i ude (P oc CORR, SaS en e p ise Guide 4.3) o hose accessions whe e in o ma ion o collec ion si e map coo dina es o ele a ion was a ailable. Gene ic di e gence be ween accessions o g oups was analysed by analysis o molecula a iance (aMOVa) (ex c o i e e al. 1992) using he p og am Genalex 6.4 (Pe a k a l l and Sm o u S e 2006). Signi icance o he esul s was es ed by pe mu ing he Dna ma ke da a 999 imes. a neighbo -joining (nJ, Sa i o u and ne i 1987) dend o- g am was cons uc ed using he p og am MeGa e . 4 ( a m u a e al. 2007). The gene ic dis ances be ween accessions o he dend og am we e calcula ed wi h he p og am POPDiST ( o m i u k e al. 2009), whe e he es ima ion o gene ic dis ances is based on g ouping o allele pheno ypes (dis ance measu e o o m i u k e al. 1998), in which case he deg ee o ploidy is o no impo - ance. as a as we know, POPDiST is he only p og am o di e si y s udies ha can handle codominan ma ke s in polyploids. P incipal coo dina es analysis (PCa) based on nei’s gene ic dis ances be ween accessions was pe - o med using he so wa e Genalex 6.4 (Pe a k a l l and Sm o u S e 2006). Map coo dina es we e a ailabe o 71 accessions. Co ela ion be ween gene ic dis ance (desc ibed wi h Allele size (bp) 192.8 201.2 203.1 204.1 205.0 206.0 206.9 207.9 209.0 209.7 210.7 211.7 212.7 213.7 214.5 215.6 216.4 217.5 218.4 219.4 220.2 221.3 222.2 223.2 224.1 225.1 226.3 227.1 228.1 229.0 230.9 232.9 233.7 234.8 236.8 238.8 240.6 242.5 244.2 246.5 No. o indi iduals 0 100 200 300 400 500 se ies 2 se ies 1 217.5 se ies 1 se ies 2 Fig. 2. an example o he wo allelic se ies in SSR locus B03a09. 122 P. Tanhuanpää & O. Manninen he edi as 149 (2012) we e mo e di e se han Danish accessions when aa was compa ed (Tukey’s es , p  0.05). Fo PWD, Denma k showed less di e si y han Finland, no way o Sweden (Tukey’s es , p  0.05). Danish accessions we e also less di e se han Finnish, Swedish, no wegian o exo ic based on ai. accessions o igina ing om sou he n bo eal o middle bo eal ege a ion zone we e mo e di e se han hose om nemo al o alpine ege a ion zone when aa o PWD we e compa ed (Tukey’s es , p  0.05). Vege a ion zones explained 33% o he a ia ion in di e si y le els be ween imo hy accessions (Table 3). Signi ican di e - ences be ween ege a ion zones we e also obse ed o ai bu hey we e e y small and explained only a mino ac ion o a ia ion be ween indi iduals (Table 3). acces- sions wi h he cul i a ype L, meaning land ace o adi- ional, locally cul i a ed accession, had a highe aa when compa ed o cul i a s o wild accessions. Land aces also we e mo e di e se han cul i a s based on PWD o ai (Tukey’s es , p  0.05). no co ela ion was obse ed be ween la i ude o al i- ude and he di e si y indices. howe e , a weak co ela- ion was obse ed be ween longi ude and aa (  0.29, p  0.013). Gene ic di e gence be ween accessions and g oups aMOVa was pe o med in o de o di ide he o al gene ic a ia ion in o h ee componen s: a ia ion wi hin accessions, among accessions and among g oups. acco d- ing o aMOVa analysis, mos o he a ia ion (94%) in he s udied imo hy ma e ial was due o a ia ion wi hin accessions and only 5% was be ween accessions and 1% be ween coun ies (Table 5). no gene ic di e - gence was obse ed be ween ege a ion zones o cul i a ypes (aMOVa, p  0.05). no clea clus e ing o accessions based on coun ies o any o he g ouping was seen in ei he PCa (Fig. 3) o nJ dend og am (Supplemen a y ma e ial appendix a1 Fig. a1). in PCa, he i s wo axes explained 42.4% o he a ia ion among he 96 accessions. Mos o he acces- sions clus e ed oge he apa om a couple o excep ions. Gene ic dis ance ma ices coun ed wi h di e en ways 10% o he accessions. This usually caused a high occu ence o he mos common allele in a SSR locus (Table 2). The mean numbe o alleles in an indi idual a ied om 1.12 o 3.91 depending on he SSR locus. Ve y ew indi iduals (1.2%) ca ied six alleles in any SSR locus. The mos he e ozygous SSRs we e C01B11 and a09h08: 66% and 54% o he indi iduals in he s udy, espec i ely, con ained ou alleles o mo e in hese loci. On he o he hand, he alleles in he SSR loci B03F07 and C01e11 o en occu ed alone (in 48% and 67% o he indi iduals, espec i ely). This is pe haps spu ious, due o he exis ence o null alleles, which is also e lec ed by he la ge numbe o appa en missing in o ma ion in hese loci (15.1% and 18.8%, espec i ely, Table 2). The i e SSRs wi h a dinucleo ide epea ampli ied an a e age o 35.4 alleles compa ed o 40.3 alleles ampli- ied by he eigh SSRs wi h a inucleo ide epea (Table 2). The a e age obse ed numbe o alleles/acces- sion was 11.7 and 13.4, and o p i a e alleles 3.8 and 4.9 in SSRs wi h di- and inucleo ide epea s, espec i ely ( esul s no shown). howe e , none o hese di e ences we e s a is ically signi ican ( - es , p  0.05). Gene ic di e si y wi hin accessions Taking all he 499 SSR alleles ( indi idual ma ke s) oge he , he obse ed numbe in each accession anged om 95 (nGB10785) o 203 (nGB14403) (Table 1). Mos o he ma ke s we e polymo phic i.e. e y ew exis ed in all indi iduals o an accession. The numbe o p i a e alleles i.e. alleles ha did no exis in any o he accession was gene ally low bu accessions Pi325461 ( om Russia) and RCaT040682 ( om hunga y) included eigh and six p i a e alleles, espec i ely. ai anged om 28.4 (nGB14417) o 36.2 (nGB733 and nGB757) (Table 1). Gene ic di e si y wi hin accessions measu ed as PWD a ied om 28.9 (nGB10785) o 46.8 (nGB9285) (Table 1). Le els o di e si y we e ound o be signi ican ly di e en be ween coun ies, ege a ion zones and di e - en cul i a ypes (anOVa, Table 3, 4). Finnish accessions Table 3. ANOVA able showing F- alues, signi icance le els P and R2 o compa isons o di e en g oups o hei le els o SSR di e si y. He e AI ep esen s he numbe o alleles on indi idual le el. To al numbe o alleles (aa) numbe o pai wise di e ences (PWD) numbe o alleles pe indi idual (ai) Di e si y index d F p R2F p R2F p R2 G ouping accession 95 3.52 < 0.001 0.16 Coun y o o igin 5 4.15 0.002 0.19 4.40 0.001 0.20 14.15 < 0.001 0.04 Vege a ion zone 5 6.86 < 0.001 0.33 6.78 < 0.001 0.33 13.29 < 0.001 0.04 Cul i a ype 2 8.58 < 0.001 0.17 4.46 0.014 0.10 6.05 0.002 0.01