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