RESEARCH ARTICLE
Gene ic di e si y, popula ion s uc u e and linkage
disequilib ium in No dic sp ing ba ley (Ho deum ulga e
L. subsp. ulga e)
The e
´se Beng sson .The PPP Ba ley Conso ium .Ou i Manninen .
Ahmed Jahoo .Jihad O abi
Recei ed: 29 Sep embe 2016 / Accep ed: 24 Janua y 2017
ÓThe Au ho (s) 2017. This a icle is published wi h open access a Sp inge link.com
Abs ac Gene ic di e si y, popula ion s uc u e and
genome-wide linkage disequilib ium (LD) was es i-
ma ed in No dic sp ing ba ley (Ho deum ulga e L.
subsp. ulga e) by geno yping 180 b eeding lines wi h
48 SSR ma ke s and 7842 high-con idence SNPs using
he Illumina In inium 9K assay. In o al 6208 SNPs
we e polymo phic and selec ed o u he s a is ical
analysis. A Man el es e ealed a s ong posi i e
co ela ion wi h a Pea son’s co ela ion coe icien
( ) o 0.86, be ween he es ima es o gene ic dis ances
based on SSR and SNP da a. Popula ion s uc u e
analysis iden i ied wo g oups wi h a clea ances y
and one g oup wi h an admixed ances y. The g oups
we e p ima ily sepa a ed based on ow- ype and
geog aphical o igin. A e age LD o he whole
popula ion decayed below a c i ical le el o
2
=0.20 wi hin a ange o 0–4 cM. To a oid
con ounding e ec s o he s ong popula ion s uc u e,
LD decay o he di e en g oups was analysed
sepa a ely and anged om 0 o 12 cM. A slowe LD
decay was ound wi hin he wo- owed lines compa ed
o he six- owed lines and he wo- owed lines
o igina ing om he no he n pa , which could be
he esul o s ong selec ion o mal ing quali y and
yield in he sou he n pa . No la ge di e ence in
gene ic di e si y was obse ed be ween popula ion
sub-g oups, bu di e ences a ce ain ch omosomal
egions we e e iden .
Keywo ds Ho deum ulga e L. Linkage
disequilib ium Mic osa elli es Molecula ma ke s 
Plan b eeding SNP a ay
In oduc ion
Cul i a ed ba ley (Ho deum ulga e L. subsp. ul-
ga e) is one o he mos impo an c ops in he No dic
coun ies co e ing a o al a ea o 1.56 million ha in
2014 (h p:// aos a 3. ao.o g). No dic ba ley b eeding
began mo e han 100 yea s ago, s a ing by selec ions
in he land ace gene pool o he p esen mode n eli e
cul i a s de eloped by c osses be ween pu e lines o
ad anced ma e ial (Kolodinska B an es am e al.
2004; Fischbeck 1992). Exo ic sou ces a e used in
mode n b eeding, bu hen p ima ily as dono s o
single esis ance genes agains majo diseases
Elec onic supplemen a y ma e ial The online e sion o
his a icle (doi:10.1007/s10722-017-0493-5) con ains supple-
men a y ma e ial, which is a ailable o au ho ized use s.
T. Beng sson (&)A. Jahoo
Depa men o Plan B eeding, Swedish Uni e si y o
Ag icul u al Sciences, Box 101, 230 53 Alna p, Sweden
e-mail: [email p o ec ed]
O. Manninen
Bo eal Plan B eeding L d, Mylly ie 10, 31600 Jokioinen,
Finland
A. Jahoo J. O abi
No dic Seed A/S, Ko nma ken 1, 8464 Gal en, Denma k
123
Gene Resou C op E ol
DOI 10.1007/s10722-017-0493-5
(Melchinge e al. 1994; Weibull e al. 2003). Today’s
cul i a ion o gene ically uni o m cul i a s is aising
conce ns abou loss o gene ic di e si y. A p e ious
s udy o he gene ic di e si y in sp ing ba ley ge m-
plasm in he No dic and Bal ic egion epo ed a sig-
ni ican dec ease o gene ic di e si y in he sp ing
ba ley om sou he n pa s o he in es iga ed egion in
he middle o he wen ie h cen u y, bu no in he
sp ing ba ley om he no he n pa s (Kolodinska
B an es am e al. 2007). Likewise, no signs o gene ic
e osion we e obse ed in a ecen s udy o gene ic
di e si y o ba leys om he no he n Eu opean a ea
o e a hund ed yea s o ba ley b eeding (Rajala e al.
2016). This highligh s he impo ance o knowledge
ega ding he le el o gene ic di e si y in b eeding
ma e ial, since i enables he de ec ion o any changes
in di e si y ha migh lead o gene ic e osion.
Se e al ypes o molecula ma ke s such as ampli-
ied agmen leng h polymo phism (AFLP), andom
ampli ied polymo phic DNA (RAPD), simple
sequence epea s (SSR), di e si y a ay echnology
(DA T), and single nucleo ide polymo phism (SNP)
ha e been used o s udy gene ic di e si y and s uc u e
in c ops (Kesawa and Das Kuma 2009). SSRs ha e
he ad an age o be abundan , highly polymo phic and
mul i-allelic, and he e o e o en p o ide mo e in o -
ma ion compa ed o biallelic ma ke s such as SNPs.
On he o he hand, he new iSelec geno yping
pla o m, based on he Illumina In inium assay, allows
he simul aneous es ing o 7842 gene-de i ed SNPs
(Comad an e al. 2012). The gene ic polymo phism o
he SNP and SSR ma ke sys ems a e gene a ed
h ough di e en mechanisms, hus hey could gi e
di e en iews o he s uc u e o a popula ion.
Analysing he gene ic s uc u e wi hin a popula ion
is a c i ical s ep o he way o unde s and and e eal he
complexi y wi hin his popula ion (P i cha d e al.
2000). Fac o s such as human o en i onmen ally
d i en selec ion, gene ic d i , ma ing sys em and
g ow h habi can ha e an e ec on he popula ion
s uc u e (Buckle and Tho nsbe y 2002; Flin -Ga cia
e al. 2003). S udies o wo ldwide (Malyshe a-O o
e al. 2006), Eu opean (Ros oks e al. 2006), Ame ican
(Hamblin e al. 2010) and No dic (Rajala e al. 2016)
ba ley ge mplasm ha e shown ha cul i a ed ba ley
has a clea le el o popula ion s uc u e wi h majo
subpopula ions caused by di e ences in ea ype, i.e.
wo- ow and six- ow, and seasonal g ow h habi , i.e.
win e and sp ing (Hamblin e al. 2010; Malyshe a-
O o e al. 2006; Ros oks e al. 2006). In addi ion o
hese majo subpopula ions, i has been shown ha
Ame ican ba ley accessions u he can be di ided
in o mino sub-popula ions co esponding o he
b eeding p og ams, which migh be alloca ed o
limi ed exchange o ma e ial be ween he b eeding
p og ams and/o a esul o local adap a ion (Hamblin
e al. 2010).
Ano he impo an ac o o conside is linkage
disequilib ium (LD), which is he non- andom asso-
cia ion o alleles be ween wo loci and shows he
co ela ion be ween gene ic polymo phisms, e.g.
SNPs, and hei his o y o mu a ions and ecombina-
ion (Flin -Ga cia e al. 2003). LD is impo an since
he a e o i s decay in a gi en species de e mines he
numbe and densi y o he molecula ma ke s needed
o pe o m GWAS (Ra alski 2002). In many sel -
pollina ed species such as ba ley whe e LD ex ends
o e long ch omosomal dis ances (Malyshe a-O o
e al. 2006), ewe ma ke s a e needed o co e he
whole genome, whe eas a highe ma ke densi y is
needed when LD decays e y apidly in species such
as maize whe e i declines o nominal le els wi hin
1.5 kb (Reming on e al. 2001).
The aim o he Public P i a e Pa ne ship (PPP) o
p e-b eeding in ba ley, pa ly unded by he No dic
Council o Minis e s (NMR), is o lay a ounda ion o
ba ley b eeding o disease esis ance and yield
s abili y o mee cu en and u u e challenges in he
No dic egion. This collabo a ion is be ween i e
b eeding companies and h ee go e nmen al o gani-
za ions in he No dic egion. One o he goals wi h his
p og am is o iden i y ma ke s linked wi h ai s o
in e es ia genome-wide associa ion s udies
(GWAS). The main objec i e wi h he p esen s udy
is o de e mine he popula ion s uc u e and he LD
decay in a No dic ba ley panel, in o de o es ima e he
ela ionships among indi iduals.
Ma e ials and me hods
Plan ma e ials
A o al o 134 and 46 sp ing ba ley Ho deum ulga e
L. subsp. ulga e b eeding lines and cul i a s, espec-
i ely, we e included in his s udy. The selec ed sp ing
ba ley b eeding lines and cul i a s a e he ea e
e e ed o as lines. Equal numbe o lines was selec ed
Gene Resou C op E ol
123
by b eede s om each o Bo eal Plan B eeding
(Finland), G amino B eeding AS (No way), Ag icul-
u al Uni e si y o Iceland (AUI Iceland), Lan ma
¨n-
nen Lan b uk (LSW Sweden), No dic Seed and Seje
Plan e o aedling I/S (Denma k). The lines we e cho-
sen o ep esen he a ailable gene ic a ia ion in
cu en eli e No dic ba ley ge mplasm. Ou o he 180
lines, ele en lines we e emo ed om u he analyses
since hey we e duplica es, o due o incomple e
geno yping. Ou o he emaining 169 lines, 124 we e
wo- owed and 45 six- owed.
DNA ex ac ion
DNA was ex ac ed om 2-week-old seedlings, using
a CTAB (Ce yl T ime hyl Ammonium B omide)
me hod as desc ibed ea lie by (O abi e al. 2014).
The DNA was p ecipi a ed wi h isop opanol, washed
wo imes wi h 75% e hanol, ai -d ied and inally
dilu ed in TE bu e (pH 8.0).
Mic osa elli e geno yping
All lines we e geno yped using 48 mic osa elli e
ma ke s e enly dis ibu ed o e all ch omosomes.
PCR ampli ica ions we e pe o med on a GeneAmp
Ò
PCR Sys em 2700 he mal cycle (Applied Biosys-
ems, Fos e Ci y, CA, USA) using a single uni e sal
ouchdown PCR p og am as p e iously desc ibed in
(O abi e al. 2014). The o wa d p ime s we e 50-
labeled wi h luo escen dyes o achie e he maximum
mul iplex capaci y o he ABI 3130xl sequence .
Di ec and M13-labelling we e used o he
mic osa elli e agmen s, wi h 6-ca boxy luo escein
(6-FAM, blue) o hexachlo o-6-ca boxy luo escein
(HEX o VIC, g een) and 50- luo escein phospho-
amidi e (NED, yellow) o he di ec labelling. Fo he
M-13 labelling, 6-FAM (blue), VIC (g een) and NED
(yellow) we e used. Fo agmen de ec ion he ABI
3130xl DNA analyze (Applied Biosys ems, Fos e
Ci y, CA, USA) was used and he agmen analysis
and geno yping we e pe o med using he GeneMa -
ke geno yping so wa e p og am, e sion 1.85 (So
gene ics, S a e College, PA, USA).
SNP geno yping
All lines we e geno yped wi h he ba ley iSelec SNP
chip based on he Illumina In inium 9K assay. The
geno yping o he lines was ou sou ced o T ai Gene -
ics. The chip consis s o 7842 high-con idence SNPs
de i ed om exp essed genes (Comad an e al. 2012).
Da a analysis
Gene di e si y and ma ke allele equency
Gene ic dis ances be ween geno ypes, gene ic di e -
si y, allele equency and p i a e alleles (alleles
p esen only in one g oup) we e calcula ed using an
in-house p og am w i en in VBA (Visual Basic o
Applica ions) and implemen ed in Mic oso Excel
2007 (Mic oso , Redmond, WA, USA). The p og am
u ilises R language so wa e .2.14.2 (R De elopmen
Co e Team 2012), which includes he Mode n Applied
S a is ics wi h S-plus (MASS) package (Ripley 2002).
The a e age numbe o alleles pe locus pe g oup
ep esen s how polymo phic a gi en ma ke was
wi hin each g oup, and his alue was calcula ed o
each SNP ma ke . The a e age numbe o alleles pe
ma ke is be ween 1 and 2, whe e ma ke s wi h a
numbe o 1 we e conside ed monomo phic.
Modi ied Roge ’s dis ances (MRD) based on
(W igh 1978) we e calcula ed o he SSR da a based
on he ollowing equa ion:
MRD ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
2mXm
i¼1Xai
k¼1ðpij qijÞ2
whe e p
ij
and q
ij
a e he allele equencies o he j h
allele a he i h ma ke o he wo ba ley lines in
conside a ion; a
i
is he numbe o alleles a he i h
ma ke ; and mis he numbe o SSR loci. The gene ic
dis ances (D
SM
) based on he SNP da a we e de e -
mined by es ima ing a simple ma ching coe icien
(S
SM
) (Rei e al. 2005):
DSM ¼1SSM;SSM ¼ ij þyij
ij þwij þxij þyij
whe e
ij
e e s o he allele in common be ween wo
lines, iand j;w
ij
is he numbe o alleles p esen in i
and absen in j;x
ij
is he numbe o alleles p esen in j
and absen in i; and y
ij
is he numbe o alleles absen in
bo h iand j. Co ela ions be ween he MRD (SSR) and
D
SM
(SNP) ma ices we e calcula ed using Man el es
(Man el 1967). The polymo phic in o ma ion con en
(PIC) o he indi idual ma ke s was calcula ed as
explained by Bo s ein e al. (1980):
Gene Resou C op E ol
123
PIC ¼1X
n
i¼1
p2
iX
n1
i¼1X
n
j¼iþ1
2p2
ip2
j
whe e p
i
is he equency o he i h allele, and nis he
numbe o alleles pe ma ke .
To show gene ic di e si y and di e en ia ion along
he ba ley ch omosomes Shannon’s di e si y indices
we e calcula ed using GenAlEx . 6.5.0.1 (Peakall and
Smouse 2006,2012) based on he SNP da a.
Popula ion s uc u e analysis
To de e mine popula ion s uc u e o he ba ley lines
using SNP ma ke s, he so wa e package STRUC-
TURE .2.3.4 based on a Bayesian clus e ing
app oach, was used (P i cha d e al. 2000). STRUC-
TURE was un 10 imes o each hypo he ical numbe
o subpopula ions (K) be ween 1 and 12 wi h he
ploidy le el se as 2. The Ma ko chain Mon e Ca lo
(MCMC) was se o 9999 bu n-in phases ollowed by
9999 i e a ions. S uc u e Ha es e .0.6.94 (Ea l and
on Hold 2012), was used o es ima e he mos likely
numbe o g oups (K) using he Del aK me hod
(E anno e al. 2005). Popula ion s uc u e based on he
SSR ma ke s was calcula ed as desc ibed abo e wi h
he same se ings bu by using he p e iously desc ibed
VBA p og am in Excel.
Analysis o Molecula Va iance (AMOVA), Nei’s
unbiased gene ic dis ance and P incipal Coo dina es
Analysis (PCoA) we e calcula ed using GenAlEx .
6.5.0.1.
Linkage disequilib ium analysis
The TASSEL 3.0 so wa e (h p.//www.
maizegene ics.ne ) was used o calcula e he LD (al-
lele equency co ela ion,
2
) es ima es be ween he
SNP ma ke pai s using he ull ma ix op ion. Only
in a-ch omosomal compa isons we e included and
ma ke s wi h mino allele equency (MAF) below
0.05 we e excluded. Thus 4884 ou o he o al 6280
polymo phic ma ke s we e subjec ed o analysis. To
es ima e he LD decay, he in a-ch omosomal
2
alues we e plo ed agains he gene ic dis ance wi h a
second-deg ee smoo hed loess cu e i ed using he
p og am R (R De elopmen Co e Team 2012) and a
baseline based on he c i ical alue o
2
was d awn.
The c i ical alue o
2
, as an e idence o linkage, was
calcula ed based on he me hod desc ibed in B e-
seghello and So ells (2006), by squa e oo ans-
o ming he
2
- alues and aking he 95 h pe cen ile o
unlinked
2
- alues. In he analysis ma ke s loca ed
mo e han 50 cM apa we e conside ed unlinked.
Resul s
Popula ion s uc u e in No dic sp ing ba ley
The STRUCTURE analysis indica ed ha he ba ley
panel could be di ided in o wo g oups wi h common
ances y ([0.7) K1
SSR
(n =109) and K2
SSR
(n =50)
and one g oup (n =10) wi h an admixed ances y
( 0.7), based on he SSR da a. AMOVA analysis
e ealed ha he K1
SSR
,K2
SSR
and admixed
SSR
g oup
we e signi ican ly sepa a ed (p 0.001) and
explained 35% o o al molecula a iance (Online
Resou ce 1). This is simila o he esul ob ained om
he PCoA analysis, whe e he i s wo p incipal
coo dina es combined explained 32.1% (21.7 and
10.4%) o he a ia ion (Fig. 1a). The subdi ision o
he popula ion along he i s p incipal coo dina e
(PC1) co esponded o he sepa a ion o he ba ley
popula ion in o K1
SSR
and K2
SSR
. The K1
SSR
and
K2
SSR
g oup co esponded o wo- owed lines and six-
owed lines, espec i ely, wi h he excep ion o six
wo- owed lines ound in K2
SSR
. In he small
admixed
SSR
g oup, nine wo- owed lines and one
six- owed line om mainly he no he n pa s we e
ound.
Also o he SNP da a, wo g oups wi h a common
ances y ([0.7) (K1
SNP
:n =109; K2
SNP
:n =47) and
one g oup (n =13) wi h an admixed ances y ( 0.7)
we e in e ed by he STRUCTURE analysis. Th ee
clus e s we e obse ed in he PCoA analysis, whe e
he i s and second p incipal componen s explained
24.6 and 5.9% o he a ia ion, espec i ely, o 30.5%
combined (Fig. 1b). AMOVA analysis e ealed ha
he K1
SNP
,K2
SNP
and admixed
SNP
g oups we e
signi ican ly sepa a ed (p alue 0.001) and explained
42% o he o al molecula a iance (Online Resou ce
1). The wo- owed lines we e dis ibu ed be ween
K1
SNP
and he admixed
SNP
g oup and he six- owed
lines we e ound in g oup K2
SNP
, wi h he excep ion o
wo wo- owed lines ha we e ound in he la e
g oup. The lines wi h an admixed ances y in he
admixed
SNP
g oup we e wo- owed and we e, jus as
Gene Resou C op E ol
123
he lines in he admixed
SSR
g oup, mainly om he
no he n pa s. Mo e de ailed in o ma ion ega ding
he o igin o he lines and he in e ed popula ion
s uc u e g oups can be ound in Online Resou ce 2.
Gene ic di e si y in No dic sp ing ba ley
Ma ke sys em compa ison
The ba ley lines we e geno yped using 48 SSR
ma ke s. The same lines we e also geno yped using
he iSelec 9K SNP ba ley chip esul ing in a o al o
6208 polymo phic SNP ma ke s. The SSR ma ke s
p oduced 234 sco able loci. The numbe o alleles pe
ma ke anged om 1 o 15 wi h an a e age o 4.9
alleles pe SSR ma ke . The a e age polymo phic
in o ma ion con en (PIC) alue was 0.46 o he SSR
ma ke s and 0.28 o he SNP ma ke s. The gene ic
di e si y index was highe o he SSR ma ke s
(0.514) han o he SNP ma ke s (0.359).
Man el es showed a s ong co ela ion be ween he
SSR-based Modi ied Roge ’s gene ic dis ances and he
SNP-based simple ma ching coe icien , wi h a Pea -
son’s alue o
2
=0.86 (p alue 0.0000).
Gene ic di e si y based on ea ow ype
Allelic ichness pa ame e s in he o m o a e age
numbe o alleles pe locus, numbe and p opo ion o
p i a e alleles and gene ic di e si y o each ma ke
ype based on ea ow ype a e p esen ed in Table 1.
The e we e no majo di e ences in he a e age
numbe o alleles pe locus ound in he wo- owed
lines (SSR, 4.0; SNP: 1.9) compa ed o he six- owed
lines (SSR: 3.5, SNP: 1.8).). The gene ic di e si y was
highe in he wo- owed lines (SSR: 0.431; SNP:
0.305) compa ed o he six- owed lines (SSR: 0.386;
SNP: 0.225). The wo- owed lines had also a highe
numbe o p i a e alleles (SSR: 64; SNP: 1145)
compa ed o he six- owed lines (SSR: 41; SNP: 447).
AMOVA analysis e ealed ha 35 and 40% o he
molecula a iance be ween he lines based on he
SSRs and SNPs, espec i ely, could be explained by
he ow- ypes (da a no shown).
Gene ic di e si y based on popula ion s uc u e
Allelic ichness pa ame e s in he o m o a e age
numbe o alleles pe locus, numbe and p opo ion o
p i a e alleles and gene ic di e si y o each ma ke
ype based on he in e ed popula ion s uc u e g oups
a e p esen ed in Table 2. Fo he SSRs he highes
a e age numbe o alleles pe locus and gene di e si y
was ound in he admixed
SSR
g oup (3.7; 0.404).
Howe e , his g oup also had he lowes p opo ion o
p i a e alleles (0.30), whe eas he highes p opo ion
o p i a e alleles was ound wi hin he six- owed lines
in g oup K2
SSR
(0.86).
No majo di e ences in he a e age numbe o
alleles pe locus we e ound be ween he h ee
subg oups, based on he SNP da a. The highes
p opo ion o p i a e alleles (10.2) was, no di e en
om he esul s ob ained wi h he SSRs, ound wi hin
he six- owed lines in K2
SNP
whe eas he lowes
Fig. 1 a Associa ions be ween s uc u e g oups e ealed by
p incipal coo dina e analysis o he No dic sp ing ba ley
collec ion based on he SSR da a. bAssocia ions be ween
s uc u e g oups e ealed by p incipal coo dina e analysis o he
No dic sp ing ba ley collec ion based on he SNP da a
Gene Resou C op E ol
123

p opo ion o p i a e alleles (0.54) we e ound in he
admixed
SNP
g oup. The highes gene di e si y (0.279)
was ound in K1
SNP
, whe eas he lowes (0.198) was
ound in he admixed
SNP
g oup.
Ma ices showing ela ionships be ween he s uc-
u e g oups we e gene a ed based on Nei’s unbiased
gene ic dis ance o SSR and SNP da a (Table 3). In
he SSR ma ix, he smalles dis ance (0.272) was
ound be ween he K1
SSR
and admixed
SSR
g oup (bo h
mainly wo- owed lines om he sou he n and no h-
e n pa s, espec i ely). The la ges dis ance (0.566)
was ound be ween g oups K2
SSR
(mainly six- owed
lines) and admixed
SSR
(mainly wo- owed lines om
he sou he n and no he n pa s, espec i ely). Simila
esul s we e seen in he SNP ma ix, whe e he
smalles dis ance (0.217) was ound be ween g oups
K1
SNP
and admixed
SNP
(mainly wo- owed lines om
he sou he n and no he n pa s, espec i ely) and he
la ges (0.338) be ween K2
SNP
(mainly six- owed
lines) and admixed
SNP
(mainly wo- owed lines om
he no he n pa s).
When compa ing gene ic di e si y along he ba ley
ch omosomes, di e ences be ween he popula ion
s uc u e g oups we e e iden in se e al genomic
egions (Fig. 2). Bo h g oup K2
SNP
and admixed
SNP
we e low in di e si y on ch omosome 2H (a ound
83–113 cM), 3H (a ound 37–64 cM) and 5H (a ound
108–127 cM), whe eas he K1
SNP
lines we e e y
di e se in hese egions. A la ge di e ence in gene ic
di e si y we e also obse ed on ch omosome 6H
(a ound 55–58 cM), whe e he K1
SNP
and K2
SNP
lines
we e much mo e di e se compa ed o admixed
SNP
lines. Righ be o e his egion on ch omosome 6H
(a ound 49 cM), a egion wi h low di e si y was seen
o he K1
SNP
lines, bu he e he di e si y was
main ained o he wo o he g oups. On ch omosome
7H (a ound 68–88 cM) he lines in g oup K2
SNP
we e
e y di e se in con as o he low di e si y obse ed
o he K1
SNP
and admixed
SNP
lines in he same
egion.
Linkage disequilib ium in No dic sp ing ba ley
LD o he whole popula ion, he popula ion s uc u e
g oups and he ea ow- ypes we e calcula ed based on
he SNP ma ke s. The pe cen age o unlinked ma ke
pai s anged be ween 40 and 42% and no la ge
di e ences we e ound be ween he di e en g oups.
The numbe o in a-ch omosomal ma ke -pai s and
he numbe o unlinked pai s in he o al popula ion
and in he di e en g oups a e p esen ed in Table 4.A
o al o 1,795,852 in a-ch omosomal ma ke pai s
we e ound in he en i e popula ion. The mean
2
- alue
o he en i e popula ion, he ea ow- ypes and
popula ion s uc u e g oups we e calcula ed o he
whole genome and o he se en ch omosomes
sepa a ely (Table 5). The mean
2
- alue o he whole
genome o he en i e popula ion was ound o be 0.10.
The highes and lowes mean
2
- alue o he whole
genome was ound in he admixed
SNP
and K1
SNP
g oup (
2
: 0.19; 0.07), espec i ely. The in e al in
which he Loess cu e in e cep s he c i ical alue
(backg ound LD) was conside ed as he LD decay.
The backg ound LD and he LD decay o he en i e
popula ion, he ea ow- ypes and popula ion s uc u e
g oups we e calcula ed o he whole genome and o
he se en ch omosomes sepa a ely (Table 6, Online
Resou ce 3). A e age LD o he whole popula ion
decayed below he c i ical le el (
2
=0.20) wi hin a
ange o 0–4.0 cM. Howe e he LD decay o he
di e en ch omosomes, ea ow- ypes and popula ion
s uc u e g oups anged om 0 o 12, wi h he mos
ex ended LD decay ound o 5H in he six- owed lines
and in g oup K1
SNP
wi h an a e age
2
o 8–12
Table 1 Allele equency and gene ic di e si y in wo- and six- owed ba ley, based on SSR and SNP da a
Popula ion size (n) A e age numbe o
alleles pe locus
Numbe o
p i a e alleles
P opo ion o
p i a e alleles
Gene ic
di e si y (D)
S anda d
de ia ion (D)
SSR SNP SSR SNP SSR SNP SSR SNP SSR SNP
Two- ow 124 4 1.9 64 1145 0.5 9.2 0.431 0.305 0.063 0.100
Six- ow 45 3.5 1.8 41 447 0.9 9.9 0.386 0.225 0.123 0.233
To al 169 0.514 0.359 0.434 0.448
Gene Resou C op E ol
123
(Table 6). When compa ing he whole genome, he
six- owed lines, g oup K2
SNP
and he lines om he
no he n pa s in g oup admixed
SNP
had a mo e apid
LD decay wi h an a e age
2
be ween 0 and 4
compa ed o he wo- owed lines and g oup K1
SNP
,
wi h lines om he sou he n pa s, whe e he a e age
LD decay we e mo e slow wi h an a e age
2
be ween
4–8 and 8–12, espec i ely.
Discussion
Compa ison o ma ke sys ems
I is o in e es o compa e i he in o ma ion ega ding
popula ion s uc u e and gene ic di e si y is a ec ed
by he ma ke sys em o choice, since he e a e
di e en mu a ional mechanisms behind SSR ( epli-
ca ion slippage) and SNP (poin mu a ion) ma ke s.
SSRs ha e been he mos commonly used o s udies
o gene ic di e si y, mainly due o hei abundance in
he genome, ep oducibili y and high le el o poly-
mo phism. Howe e , he inc eased a ailabili y o he
SNP ma ke s and he as and highly au oma ed
geno yping echnologies, ha e ecen ly mo ed he
a en ion o he use o he SNPs in s udies o gene ic
di e si y and popula ion s uc u e. This s udy showed
ha he a e age PIC and gene ic di e si y alues we e
highe o he SSRs compa ed o he SNPs. Howe e ,
hese alues ha e a maximum alue o 0.5 o bi-
allelic ma ke s such as SNPs when he ma ke s sco es
a e 50% (0) and 50% (1). Taking his in o
Table 2 P i a e alleles and gene ic di e si y in he s uc u e g oups based on SSR and SNP da a
G oup SSR da a G oup SNP da a
Popula ion
size
A e age
numbe
o alleles
pe locus
No. o
p i a e
alleles
P opo ion
o p i a e
alleles
Gene ic
Di e si y
(D)
S anda d
de ia ion
(D)
Popula ion
size
A e age
numbe
o alleles
pe locus
No o
p i a e
alleles
P opo ion
o p i a e
alleles
Gene ic
Di e si y
(D)
S anda d
de ia ion
(D)
K1
SSR
109 2.5 42 0.39 0.392 0.275 K1
SNP
109 1.9 639 5.86 0.279 0.153
K2
SSR
50 3.5 43 0.86 0.397 0.063 K2
SNP
47 1.8 481 10.23 0.235 0.208
Admixed
SSR
10 3.7 3 0.30 0.404 0.120 Admixed
SNP
13 1.6 7 0.54 0.198 0.495
To al 169 0.514 0.434 To al 169 0.359 0.448
Table 3 Nei’s unbiased gene ic dis ance be ween di e en
s uc u e g oups, based on he (a) SSR da a, (b) SNP da a
K1
SSR
K2
SSR
Admixed
SSR
(a)
K1
SSR
0.000
K2
SSR
0.292 0.000
Admixed
SSR
0.272 0.566 0.000
K1
SNP
K2
SNP
Admixed
SNP
(b)
K1
SNP
0.000
K2
SNP
0.250 0.000
Admixed
SNP
0.217 0.338 0.000
Gene Resou C op E ol
123
conside a ion, he SNPs would be jus as o e en mo e
in o ma i e han he SSRs. A highe numbe o p i a e
alleles we e ound wi h he SNPs compa ed o he
SSRs (Tables 1,2). Howe e , conside ing he di e -
en numbe o ma ke s, he SSRs ac ually had he
highes p i a e allele equency, which could be
expec ed since he SNPs a e bi-allelic and ha e a
lowe mu a ion a e compa ed o he SSRs (Ma inez-
A ias e al. 2001; Li e al. 1981; K uglyak e al. 1998).
I has ea lie been epo ed ha he e is s ong
co ela ion be ween hese wo ma ke sys ems in
ba ley (Va shney e al. 2008; Va shney e al. 2010).
Fig. 2 Shannon’s di e si y index calcula ed as olling means o e 20 adjacen loci. The s a and end posi ion o each ch omosome a e
indica ed wi h e ical lines a he bo om o he igu e
Table 4 Numbe o in a-
ch omosomal ma ke -pai s
in he o al popula ion and
in he di e en g oups
Unlinked ma ke -pai s
e e s o a ma ke -pai
dis ance [50 cM
Popula ion
size
To al
pai s
Unlinked
pai s
Unlinked
pai s (%)
To al popula ion 169 1,795,852 744,332 41
Two- ow lines 124 1,464,051 609,353 42
Six- ow lines 45 923,896 367,226 40
K1
SNP
109 1,166,044 482,154 41
K2
SNP
47 981,129 393,735 40
Admixed
SNP
13 663,021 262,178 40
Table 5 Mean
2
- alues o in a-ch omosomal ma ke -pai s in he whole genome and o each ch omosome
Ch omosome No. o
SNPs
To al
popula ion
Two- owed
lines (n =124)
Six- owed
lines (n =45)
K1
SNP
(n =109)
K2
SNP
(n =47)
Admixed
SNP
(n =13)
1H 437 0.12 0.05 0.11 0.05 0.11 0.15
2H 768 0.11 0.07 0.12 0.06 0.11 0.23
3H 724 0.07 0.08 0.10 0.08 0.10 0.17
4H 578 0.09 0.07 0.11 0.06 0.10 0.18
5H 1025 0.10 0.10 0.10 0.05 0.10 0.21
6H 726 0.09 0.09 0.14 0.10 0.13 0.17
7H 626 0.09 0.07 0.08 0.07 0.09 0.19
Whole genome 4884 0.10 0.08 0.11 0.07 0.10 0.19
Gene Resou C op E ol
123
This was also demons a ed he e wi h a s ong and
posi i e co ela ion ( =0.86) be ween he modi ied
Roge ’s dis ances based on he SSR da a and he
simple ma ching coe icien based on he SNP da a.
Also he PCoA and s uc u e analysis g ouped he
lines in a simila way wi h he wo ma ke sys ems.
Howe e , he PCoA clus e ing based on he SNP da a
showed clea e and mo e dis inc g oupings han he
SSR-based PCoA, bu no majo di e ence in he
amoun o molecula a iance explained was ound
be ween he wo ma ke sys ems. Nei he did he
AMOVA analyses o he s uc u e g oups e eal any
la ge di e ences be ween he wo ma ke sys ems
(Table S1). Howe e , supe imposing he esul s om
he popula ion s uc u e analysis on he esul s om
he PCoA p o ided a clea e image and highe
esolu ion o he popula ion s uc u e based on he
SNPs compa ed o he SSRs (Fig. 1a, b). Tha e eals
he abili y o SNPs o explain he popula ion s uc u e
a a mo e speci ic le el. This was also seen in a s udy
o gene ic di e si y and popula ion s uc u e o 375
ice a ie ies (Singh e al. 2013), whe e a compa ison
o he SSR and SNP ma ke sys ems e ealed ha a
he s uc u e le el he SNPs we e be e a desc ibing
gene ic ela edness whe eas a he di e si y le el he
SSRs showed a be e g ouping o samples. In con as ,
some s udies o gene ic di e si y and popula ion
s uc u e in maize epo a be e es ima e o popula-
ion s uc u e wi h SSRs compa ed o SNPs (Yang
e al. 2011; Hamblin e al. 2007). The di e en esul s
be ween hose epo s and his s udy migh be due o
he di e en numbe s o SNP ma ke s used ( 900 s.
6208) o due o he complexi y o he maize genome.
Acco ding o a heo e ical p edic ion by La al e al.
(2002), (k-1) imes mo e bi-allelic ma ke s a e
needed o achie e a compa able accu acy o he
gene ic dis ance as a se o SSRs wi h kalleles. Wi h
he a e age o abou 3 alleles pe SSR ma ke in his
s udy, he numbe o SNPs needed would be [(3 -1)
948] =96, which a e abou 65 imes less compa ed
o he 6208 used. Al oge he he esul s om his s udy
show ha he numbe s o SNPs used a e mo e han
enough o e ie e a compa able accu acy o gene ic
di e si y in ba ley as he se o SSRs used. In addi ion,
he SNPs seem o p o ide a highe esolu ion o he
gene ic ela edness han ob ained wi h he SSRs.
Di e si y and ela ionships wi hin No dic sp ing
ba ley
The a e age gene ic di e si y o he No dic sp ing
ba ley collec ion analysed he e was 0.514 and 0.359
based on he SSRs and SNPs, espec i ely. These a e
simila o he di e si y es ima es o No dic b eeding
lines and cul i a s eleased a e 1970 (0.601) epo ed
in an ea lie s udy based on SSRs (Kolodinska
B an es am e al. 2007). In addi ion a simila esul
based on SSRs was epo ed in accessions om
Eu ope (0.593), E i ea (0.573) and E hiopia (0.620),
whe eas he Ho deum ulga e subsp. spon aneum (K.
Koch) and H. ulga e accessions om he Wes Asia
No h A ica (WANA) egion had a highe di e si y
(0.826 and 0.762, espec i ely) (O abi e al. 2007).
Table 6 In e al o he es ima ed LD decay (cM) in he o al popula ion and o he di e en g oups
Ch omosome No. o
SNPs
To al
popula ion
Two- owed
lines (n =124)
Six- owed
lines (n =45)
K1
SNP
(n =109)
K2
SNP
(n =47)
Admixed
SNP
(n =13)
1H 437 0–3 6–9 3–6 6–9 3–6 3–6
2H 768 0–3 3–6 3–6 6–9 3–6 0–3
3H 724 7–11 7–11 4–7 7–11 4–7 4–7
4H 578 0–3 3–5 0–3 3–5 0–3 0–3
5H 1025 0–4 0–4 0–4 8–12 4–8 0–4
6H 726 0–3 5–8 0–3 5–8 0–3 0–3
7H 626 0–3 7–10 7–10 7–10 3–7 0–3
Whole genome 4884 0–4 4–8 0–4 8–12 0–4 0–4
Backg ound LD whole genome
a
0.20 0.16 0.19 0.10 0.19 0.31
a
The 95 h pe cen ile o unlinked (abo e 50 cM) squa e oo ans o med
2
alues
Gene Resou C op E ol
123