لخّصلي

Abstra ct The oc currence of f boron tox xicity becom mes a majo or constrain nt in cereal l production n in the world, thus cause significant t yield loss particularl y in wheat , one of th he most sus sceptible crops to o boron tox xicity.List of ei ghty Aegilo ops-wheat a accessions c collected fro om differen nt country a and used analysis of f the present t study Name of f The Species * *Genome Country Aegilops speltoides S S Aegilops Aegilops Aegilops Aegilops Aegilops Aegilops longissima sharonensis bicornis searsii kotschyi S1 S Ssh S Sb S Ss S US U cylindrostach hys U US Aegilops euvariabilis US U Aegilops geniculata sy yn. ovata U UM Name of f the accession ns TS-100, TS-02, TS-41 1, TS-43, Israel (7) TS-47, T TS-76, TS-01 Unknown (1 ) TS-117 Turkey (1) TS-118 Syria (1) TS-132 TL-01, T TL-02, TL-04 4, TL-05, Israel (8) TL-07, T TL-09, TL-17 7, TL-21 Jordan (1) TL-24 TH-17, T TH-02, TH-03 3, TH-04, TH -07, Israel (9) TH-10, T TH-11, TH-15 5, TH-01 Israel (2) TB-04, T TB-05 Egypt (3) TB-07, T TB-10, TB-12 2 Israel (4) TE-03, T TE-09, TE-21 , TE-27 Syria (1) TE-36 TKK-01 , TKK-03, TK KK-06, Israel (6) Tkk-11, TKK-27, TKK KK-21 Israel (4) TKC-01 , TKC-04, TK KC-06, TKC-0 08 Unknown (1 ) TKE-02 TKE-03 , TKE-12, TKE-19, Israel (9) TKE-24 , TKE-42 , TKE-46, TK KE-62, TKE-6 66, Unknown (1 ) TO-01 Israel (2) TO-07, T TO-13 TKE-22, 44 www.macroth hink.org/jab Journal of A J Applied Biote echnology ISSN 2 2327-0640 2015, Vol.Few primers s were take en from w wheat chrom mosomes, 5 5A, 5B, an nd 5D bec cause recen nt study (Schnur rbusch et al l., 2008) ide entified som me orthologo ous sequenc ce of B tole erant genes on these chromo osomes. The e total PCR reaction vo olume was 1 13 ul, comp posed of 2.0 0 ul genomi ic DNA, 1.5 ul 1 10X PCR bu uffer (Tris w with 15 mM M MgCl2, Co onc. 10x), 0 0.75 ul dNT TPs (Contain ns dCTP, dGTP, d dTTP and dATP all in n the conc. 2.2 Hyd droponic scr reening The hyd droponic sc creening wa as done in a a growth ch hamber with h light set to o 16 h day at 22 oC and 8 h h night at 15 oC. Hig gh quality s seeds were sterilized i in a solutio on of 1.3% sodium hypoch lorite (2.5% % bleach sol lution) plus s one drop o of Tween 20 0 (surfactan nt) in distille ed water 43 www.macroth hink.org/jab Journal of A J Applied Biote echnology ISSN 2 2327-0640 2015, Vol.Agrono omic manag gement of B B toxicity is more of se erious task t than dealing g with B de eficiency (Sutton et al., 200 07). Boron s stress is on ne of the ke ey limiting factors for crop produ uction in 42 www.macroth hink.org/jab Journal of A J Applied Biote echnology ISSN 2 2327-0640 2015, Vol. 3, No. 2 many ar reas of the world (Jeff feries et al., 2000). Mate erials and M Methods 2.1 Plan ls nt Materials Seventy y-nine geno otypes from m 12 wild species w within the A Aegilops-Tri iticum grou up were obtaine d from Dr. Moshe Feld dman, Depa artment of P Plant Scienc ces, The We eizmann Ins stitute of Science e, Rehovot 7 76100, Isra el. These g enotypes w were collecte ed from sev veral Medite erranean region s sources (Ta able 1).2.3 DNA NA extraction n Fresh le eaves from 13-days ol ld seedlings s were used d for DNA extraction f followed by y CTAB mini-pr rep method (IRRI, 199 97). The D DNA sampl es were an nalyzed both h qualitativ vely and quantita atively usin g a spectrop photometer and 0.8% a agarose gel electrophor resis. 2.4 SSR R marker ge notyping Thirty-f four selecte ed SSR prim mers (along with three Barley prim mers) were used for th he study.Sample es were subj jected to the e following g thermal pr rofile for am mplification n in a therm mocycler: after the e initial 7 m min at 95 ?C C, SSR mark ker amplific cation comp prised 10-15 5 touchdow wn cycles of 94 ?C C for 30 s, a annealing fo for 30 s, dec creasing the temperatur re by 0.5 ?C C per cycle u until the specifie ed annealing g temperatu ure was reac ched, and 7 72 ?C for 30 0s.T The first m matrix was p produced fro om the stan ndardized ro oot length d data using S SIMINT procedu ure based o on Average Taxonomic c Distance (i.e. DIST coefficient t in the pro ocedure). Standar rdization of f data was d done by STA TAND proce edure of NT TSYS. The second ma atrix was generat ed with hel p of SSR da ata using N Nei genetic d distance (Ne ei, 1972).Previou us studies w were inappro opriate in co ontext of the e number of f species inc cluded and the type of mole ecular mark ker used fo or the diver rsity studies s. On the o other hand w wild emme er wheat Triticum m dicoccoi ides and ry ye (Secale e cereale) are also i important g genetic sto ocks for improve ement of cu ultivated wh heat.Ae. speltoid des, Ae euv variablis an d Ae. sears sii also exp perience tole erance perf formance ag gainst B toxicity y. Due to t the inconsi stency, no reliable co onclusion c can be draw wn for any y of the genotyp pes from the e species Ae e. kotschyi a and Ae. bico ornis. Wheat is very suscept ible to B to oxicity and p production is hampere d in toxic e environment ts. Due to th he stock of diffe erent genes in an inte erbreeding p population used in br reeding pro ograms, the genetic diversit ty among w wheat cultiva ars has decr reased (We ei et al., 200 00, as cited in Li et al. ., 2006). M Moreover, mo ost of the pr revious stud dies (Konsta antinos et a al., 2010; Be elkadi et al., 201 1, Baghizad deh et al., 2 2011, Nagha avi et al., 2 2009) that w were devoted d to identif fying the genetic relationsh ip and div versity of A Aegilops sp pecies wer re based on n RAPD m markers.2.5 Stat tistical Anal lysis The alle ele size at e each microsa atellite locu us was meas sured in bas se pairs by u using Alpha aEaseFC 45 www.macroth hink.org/jab Journal of A J Applied Biote echnology ISSN 2 2327-0640 2015, Vol.Deve elopment of f tolerant va ariety is no w of utmos st importan nce since agronom mic manage ement of so oil boron is b becoming i ineffective t to mitigate t the toxicity y. Due to narrow genetic bas se of the ex xisting whe eat cultivars s, genetic v ariation for r this charac cter was poorly reported.Their der ivatives gen notypes TL L-09 and TL L-17 were found to ex xplained conside erable tolera ance to boro on. On other r hand, amo ong the SSR R markers us sed, Xgwm 192 was the mos st robust in n identifying g boron tol lerance poly ymorphism.B But, surpris singly these species gav ve better root len ngth under 3 3 mM B. Am mong the sp pecies evalu uated Ae. sh haronensis, Ae. longiss sima and Ae. kots schyi reveal led better to olerance ag gainst exces s B than th e other spec cies (Figure e 1). Resu ults 3.1 Mor rphological l Characteri ization 3.1.1 Ro oot Length Variation In the h hydroponic screening, variation in n root lengt th of the ge notypes of each specie es under differen nt concentra ations (0, 3 and 10 mM M) is presen nted in the Figure 1.3, No. 2 for 8 m minutes and then thorou ughly rinsed d. Seeds we ere placed o on filter pap per containin ng 0.2% of Pipra acil (Pipera acillin Sodiu um antibiot tic) solution n in labeled d petri dishe es for germ mination.The Aegilops g genera are s still neglect ted in B toleranc ce research, , and the ex xistence of s such a large e pool of ge enotypic var riation is pr romising and can n be used in n wheat bre eding progr rams for de eveloping B -tolerant cu ultivars.3.69 (Felsenstei in, 2009) w ith 1000 permuta ation and co o-phenetic c correlation c coefficient w was calcula ated by NTS SYSpc prog gram ver. 2.11 (R Rohlf, 2005 5) for inter rnal validat tion of the phylogene etic tree.?C until electrop phoresis...3.46

Abstra
ct
The oc
currence of
f boron tox
xicity becom
mes a majo
or constrain
nt in cereal
l production
n in the
world,
thus cause
significant
t yield loss
particularl
y in wheat
, one of th
he most sus
sceptible
crops to
o boron tox
xicity. Deve
elopment of
f tolerant va
ariety is no
w of utmos
st importan
nce since
agronom
mic manage
ement of so
oil boron is b
becoming i
ineffective t
to mitigate t
the toxicity
y. Due to
narrow
genetic bas
se of the ex
xisting whe
eat cultivars
s, genetic v
ariation for
r this charac
cter was
poorly
reported. T
The present
t study was
s devoted t
to identify
such varia
ation by us
sing two
differen
nt genetic b
background
d of wheat
including
Aegilops a
and Triticum
m, which c
could be
readily
used in wh
heat breedin
ng program.
. Morpholo
ogical and g
genetic scree
ening revea
aled that
two spe
ecies Ae. lo
ongissima a
and Ae. shar
ronensis ex
xpressed tol
lerance aga
ainst boron
toxicity.
The m
olecular m
marker analy
ysis such
as unweig
ghted pair
group met
thod and p
principal
coordin
nate analysis
s confirmed
d these two
species to
be more to
lerant to ex
xcessive bor
ron with
higher r
root length
. Their der
ivatives gen
notypes TL
L-09 and TL
L-17 were
found to ex
xplained
conside
erable tolera
ance to boro
on. On other
r hand, amo
ong the SSR
R markers us
sed, Xgwm
192 was
the mos
st robust in
n identifying
g boron tol
lerance poly
ymorphism.
. The diver
rsity and va
ariability
observe
ed in this wo
ork could op
pen new av
venue in dev
veloping B t
toxicity tole
erant wheat
variety.
Keywo
rds: Boron
n-toxicity, G
Genetic dive
ersity, SSR,
Aegilops,
Triticum, R
Root length,
, Cluster
analysis
s

1. Intro
   oduction
   Boron (
   (B) is an ess
   sential micr
   ronutrient fo
   or plant gro
   owth and de
   velopment.
   Crop produ
   uction is
   hamper
   red seriousl
   ly under b
   oth conditi
   ions of def
   ficient and
   excessive
   boron in t
   the soil.
   Agrono
   omic manag
   gement of B
   B toxicity is
   more of se
   erious task t
   than dealing
   g with B de
   eficiency
   (Sutton
   et al., 200
   07). Boron s
   stress is on
   ne of the ke
   ey limiting
   factors for
   crop produ
   uction in
   42
   www.macroth
   hink.org/jab
   Journal of A
   J
   Applied Biote
   echnology
   ISSN 2
   2327-0640
   2015, Vol.
   3, No. 2
   many ar
   reas of the
   world (Jeff
   feries et al.,
   2000). Agr
   ronomic ma
   anagement o
   of B toxic s
   soils is a
   robust
   practice (S
   Sutton et a
   al., 2007)
   and also,
   detoxificati
   ion of exc
   cess B from
   m soils.
   Develop
   pment of w
   wheat cultiva
   ars that are t
   tolerant to B
   B is the only
   y long-term
   m viable app
   proach to
   deal w
   with B toxi
   icity and c
   crop produc
   ction (Schn
   nurbusch e
   et al., 2010
   0). Wheat
   is very
   suscept
   ible to B to
   oxicity and p
   production
   is hampere
   d in toxic e
   environment
   ts. Due to th
   he stock
   of diffe
   erent genes
   in an inte
   erbreeding p
   population
   used in br
   reeding pro
   ograms, the
   genetic
   diversit
   ty among w
   wheat cultiva
   ars has decr
   reased (We
   ei et al., 200
   00, as cited
   in Li et al.
   ., 2006).
   Hence,
   Wheat imp
   provement p
   programs re
   equire the in
   nclusion of
   diverse gen
   netic backgr
   round as
   a source
   e of target g
   genes. Land
   draces, wee
   edy species
   and wild re
   elatives of w
   wheat are g
   generally
   the prin
   ncipal sourc
   ce of these
   e genes (Li
   et al., 200
   06). Aegilop
   ps, which i
   is one of t
   he most
   closely
   related gen
   nera of whe
   eat, possess
   ses tremend
   dous genetic
   c variability
   y and could
   d be the
   source
   of desirabl
   e B toleran
   nt genes. M
   Many other
   value-added
   d genes of
   interest ha
   ave been
   incorpo
   orated to cu
   ultivated wh
   heat from A
   Aegilops spe
   ecies (Schn
   neider et al.
   ., 2008). In
   case of
   wheat, m
   most of the
   e earlier stud
   dies (Schnu
   urbusch et a
   al., 2008, Sc
   chnurbusch
   h et al., 2007
   7, Torun
   et al., 2
   2006; Jeffer
   ries et al., 2
   2000, Paull
   l et al., 199
   92, 1991), f
   for B toxic
   city toleranc
   ce, were
   conduct
   ted on brea
   ad wheat an
   nd durum w
   wheat. The
   Aegilops g
   genera are s
   still neglect
   ted in B
   toleranc
   ce research,
   , and the ex
   xistence of s
   such a large
   e pool of ge
   enotypic var
   riation is pr
   romising
   and can
   n be used in
   n wheat bre
   eding progr
   rams for de
   eveloping B
   -tolerant cu
   ultivars. So
   far only
   one rep
   ort (Emon e
   et al., 2012)
   ) is availabl
   le which att
   tempted to c
   categorize th
   he Aegilops
   s species
   for B to
   olerance. M
   Moreover, mo
   ost of the pr
   revious stud
   dies (Konsta
   antinos et a
   al., 2010; Be
   elkadi et
   al., 201
   1, Baghizad
   deh et al., 2
   2011, Nagha
   avi et al., 2

2009. that w
      were devoted
      d to identif
      fying the
      genetic
      relationsh
      ip and div
      versity of A
      Aegilops sp
      pecies wer
      re based on
      n RAPD m
      markers.
      Previou
      us studies w
      were inappro
      opriate in co
      ontext of the
      e number of
      f species inc
      cluded and
      the type
      of mole
      ecular mark
      ker used fo
      or the diver
      rsity studies
      s. On the o
      other hand w
      wild emme
      er wheat
      Triticum
      m dicoccoi
      ides and ry
      ye (Secale
      e cereale)
      are also i
      important g
      genetic sto
      ocks for
      improve
      ement of cu
      ultivated wh
      heat. Due to
      o genetic sim
      milarities to
      o cultivated
      d wheat, the
      transfer
      of usefu
      ful genes fro
      om these sp
      pecies is po
      ossible. Thu
      us, the eval
      luation of g
      genetic dive
      ersity of
      Aegilop
      ps, Triticum
      m and rye co
      ould provid
      de valuable
      informatio
      n for genet
      tic improve
      ement of
      wheat.
      Therefore,
      in the prese
      ent study 80
      0 genotypes
      s comprised
      d of Aegilop
      ps, Triticum
      and rye
      species
      were subj
      ected to a
      diversity s
      study utiliz
      zing thirty-t
      two SSR m
      markers. Th
      he main
      objectiv
      ve of the stu
      udy was to
      explore the
      e genetic di
      iversity and
      d relationsh
      hip of the sp
      pecies at
      the DNA
      A level, and
      d identify S
      SR markers
      s linked to B
      Boron toxic
      ity toleranc
      ce.

2. Mate
   erials and M
   Methods
   2.1 Plan
   ls
   nt Materials
   Seventy
   y-nine geno
   otypes from
   m 12 wild
   species w
   within the A
   Aegilops-Tri
   iticum grou
   up were
   obtaine
   d from Dr.
   Moshe Feld
   dman, Depa
   artment of P
   Plant Scienc
   ces, The We
   eizmann Ins
   stitute of
   Science
   e, Rehovot 7
   76100, Isra
   el. These g
   enotypes w
   were collecte
   ed from sev
   veral Medite
   erranean
   region s
   sources (Ta
   able 1). One
   e rye (Secal
   le cereale ‘
   Blanco’) ob
   btained from
   m the USD
   DA-Sears
   Collecti
   ion; Univer
   rsity of Miss
   souri, USA
   was also in
   ncluded in th
   his characte
   erization.
   2.2 Hyd
   droponic scr
   reening
   The hyd
   droponic sc
   creening wa
   as done in a
   a growth ch
   hamber with
   h light set to
   o 16 h day
   at 22 ºC
   and 8 h
   h night at
   15 ºC. Hig
   gh quality s
   seeds were
   sterilized i
   in a solutio
   on of 1.3%
   sodium
   hypoch
   lorite (2.5%
   % bleach sol
   lution) plus
   s one drop o
   of Tween 20
   0 (surfactan
   nt) in distille
   ed water
   43
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   hink.org/jab
   Journal of A
   J
   Applied Biote
   echnology
   ISSN 2
   2327-0640
   2015, Vol.
   3, No. 2
   for 8 m
   minutes and
   then thorou
   ughly rinsed
   d. Seeds we
   ere placed o
   on filter pap
   per containin
   ng 0.2%
   of Pipra
   acil (Pipera
   acillin Sodiu
   um antibiot
   tic) solution
   n in labeled
   d petri dishe
   es for germ
   mination.
   Petri p
   lates were
   shifted to
   a refriger
   rator (4 °C
   C) for adjus
   sting seedli
   ing growth
   h during
   germina
   ation. The h
   hydroponic
   nutrient so
   olution cons
   sisted of 0.4
   4 mM CaCl
   l2; 0.65 mM
   M KNO3;
   0.25 mM
   M MgCl2·6
   6H2O; 0.1 m
   mM (NH4)2S
   SO4; and 0.
   04 mM NH
   H4NO3 in di
   ionized wat
   ter (Yau,
   2002). T
   The nutrien
   nt solutions
   were supple
   emented wi
   ith H3BO3 to
   o make it at
   t 3 mM and
   d 10 mM
   of B co
   oncentration
   ns. Four see
   edlings per
   accession w
   with three r
   eplications
   were grow
   n in 2 L
   of nutri
   ient solution
   n with 0, 3
   3 and 10 m
   mM B, respe
   ectively for
   10 days. A
   All genotyp
   pes were
   categor
   ized as tole
   rant or susc
   ceptible base
   ed on their
   seedling roo
   ot length.
   Table 1
   in SSR
   . List of ei
   ghty Aegilo
   ops-wheat a
   accessions c
   collected fro
   om differen
   nt country a
   and used
   analysis of
   f the present
   t study
   Name of
   f The Species

*Genome
Country
Aegilops
speltoides
S
S
Aegilops
Aegilops
Aegilops
Aegilops
Aegilops
Aegilops
longissima
sharonensis
bicornis
searsii
kotschyi
S1
S
Ssh
S
Sb
S
Ss
S
US
U
cylindrostach
hys U
US
Aegilops
euvariabilis
US
U
Aegilops
geniculata sy
yn. ovata U
UM
Name of
f the accession
ns
TS-100,
TS-02, TS-41
1, TS-43,
Israel (7)
TS-47, T
TS-76, TS-01
Unknown (1
) TS-117
Turkey (1)
TS-118
Syria (1)
TS-132
TL-01, T
TL-02, TL-04
4, TL-05,
Israel (8)
TL-07, T
TL-09, TL-17
7, TL-21
Jordan (1)
TL-24
TH-17, T
TH-02, TH-03
3, TH-04, TH
-07,
Israel (9)
TH-10, T
TH-11, TH-15
5, TH-01
Israel (2)
TB-04, T
TB-05
Egypt (3)
TB-07, T
TB-10, TB-12
2
Israel (4)
TE-03, T
TE-09, TE-21
, TE-27
Syria (1)
TE-36
TKK-01
, TKK-03, TK
KK-06,
Israel (6)
Tkk-11,
TKK-27, TKK
KK-21
Israel (4)
TKC-01
, TKC-04, TK
KC-06, TKC-0
08
Unknown (1
) TKE-02
TKE-03
, TKE-12,
TKE-19,
Israel (9)
TKE-24
,
TKE-42
, TKE-46, TK
KE-62, TKE-6
66,
Unknown (1
) TO-01
Israel (2)
TO-07, T
TO-13
TKE-22,
44
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Journal of A
J
Applied Biote
echnology
ISSN 2
2327-0640
2015, Vol.
3, No. 2
Aegilops
biuncialis
U
UM
Lebanon (1)
Israel (2)
TO-44
TN-03, T
TN-13
Aegilops
triuncialis
UC
U
Israel (3)
Israel (10)
Triticum
. dicoccoides
Secale ce
ereale
Iran (1)
Turkey (1)
Brazil (1)
TW-03,
TW-09, TW-1
11
TTD-54
, TTD
D-09,
TTD-15
,TTD-24,
TTD-25
, TTD-30,
TTD-04
TTD-64
TTD-68
Blanco r
rye
TTD-12,
TTD-47,
TTD-48,
*Slager
ren (1994).
2.3 DNA
NA extraction
n
Fresh le
eaves from
13-days ol
ld seedlings
s were used
d for DNA
extraction f
followed by
y CTAB
mini-pr
rep method
(IRRI, 199
97). The D
DNA sampl
es were an
nalyzed both
h qualitativ
vely and
quantita
atively usin
g a spectrop
photometer
and 0.8% a
agarose gel
electrophor
resis.
2.4 SSR
R marker ge
notyping
Thirty-f
four selecte
ed SSR prim
mers (along
with three
Barley prim
mers) were
used for th
he study.
The ma
ajority of th
he primer pa
airs were se
elected from
m chromoso
omes 7B an
nd 7D of wh
heat and
2H, 3H
H, and 4H of
f barley, wh
hich are beli
ieved to be
associated
with the bo
oron toleran
nce. Few
primers
s were take
en from w
wheat chrom
mosomes, 5
5A, 5B, an
nd 5D bec
cause recen
nt study
(Schnur
rbusch et al
l., 2008) ide
entified som
me orthologo
ous sequenc
ce of B tole
erant genes
on these
chromo
osomes. The
e total PCR
reaction vo
olume was 1
13 µl, comp
posed of 2.0
0 µl genomi
ic DNA,
1.5 µl 1
10X PCR bu
uffer (Tris w
with 15 mM
M MgCl2, Co
onc. 10×), 0
0.75 µl dNT
TPs (Contain
ns dCTP,
dGTP, d
dTTP and
dATP all in
n the conc.
of 10 mM
M), 1.0 µl fo
forward prim
mer, 1.0 µl
reverse
primer,
0.5 µl Taq
q DNA pol
lymerase (c
conc. 5 U/
µl) and 8.2
25 µl steril
le deionized
d water.
Sample
es were subj
jected to the
e following
g thermal pr
rofile for am
mplification
n in a therm
mocycler:
after the
e initial 7 m
min at 95 °C
C, SSR mark
ker amplific
cation comp
prised 10–15
5 touchdow
wn cycles
of 94 °C
C for 30 s, a
annealing fo
for 30 s, dec
creasing the
temperatur
re by 0.5 °C
C per cycle u
until the
specifie
ed annealing
g temperatu
ure was reac
ched, and 7
72 °C for 30
0s. This wa
as then follo
owed by
25–35 c
cycles of am
mplification
n with the sp
pecified ann
nealing tem
mperature, an
nd a final ex
xtension
at 72
°C for 10
0 min. Aft
er amplific
cation, the
PCR tube
e was stor
red at 4 °
°C until
electrop
phoresis. Vi
isualization
of amplific
cation prod
ducts was ac
ccomplishe
ed on a 3%
agarose
gel in 0
0.5 × TBE
buffer. The
e agarose ge
els were sta
ained with e
ethidium br
romide solu
ution for
20-25
min. The
stained a
agarose ge
el was illu
uminated b
by UV-tran
ns-illuminat
tor and
photogr
raphed for a
assessing th
e DNA prof
files.
2.5 Stat
tistical Anal
lysis
The alle
ele size at e
each microsa
atellite locu
us was meas
sured in bas
se pairs by u
using Alpha
aEaseFC
45
www.macroth
hink.org/jab
Journal of A
J
Applied Biote
echnology
ISSN 2
2327-0640
2015, Vol.
3, No. 2
4.0 so
ftware (htt
tp://www.al
lphainnotec
ch.com/softw
ware.htm:
Alpha Inn
notech Inc
c.). The
summar
ry statistics
s including
the number
r of alleles
per locus,
heterozygo
osity, gene d
diversity
and pol
lymorphism
m informatio
on content
(PIC) valu
ues were ca
alculated us
sing Power
Marker
version
3.25 (Liu &
& Muse, 20
005). Power
r Marker wa
as also used
d to generat
te a distanc
e matrix
based o
on Nei (Nei
, 1972) dist
tance. The
genetic dist
tance matric
ces were th
hen subjecte
ed to the
neighbo
or-joining m
method (Sait
tou and Nei
i, 1987) of t
tree formati
ion. MEGA
A 5.0 (Tamu
ura et al.,
2007) w
was used t
to produce
graphical
trees. Ext
ternal valid
dation (boot
tstrapping)
of tree
wherev
er performe
ed was done
e by the pro
ogram Phyli
ip ver. 3.69
(Felsenstei
in, 2009) w
ith 1000
permuta
ation and co
o-phenetic c
correlation c
coefficient w
was calcula
ated by NTS
SYSpc prog
gram ver.
2.11 (R
Rohlf, 2005
5) for inter
rnal validat
tion of the
phylogene
etic tree. Pr
rincipal co
ordinate
(PCoA)
) and analy
ysis of mo
lecular var
riance (AM
MOVA) wer
e performe
ed by the p
program
GENAL
LEX ver.6.0
0 (Peakall a
and Smouse
e, 2006). W
Where not me
entioned, so
oftware CO
ONVERT
ver. 1.3
1(Glaubitz,
, 2004) wer
re used to co
onvert the d
data in diffe
erent format
t for the ana
alysis of
various
software pr
rogram.
2.6 Cor
rrelation of
Root Length
h and SSR D
Data
Two dis
ssimilarity
distance m
atrixes wer
re created w
with NTSYS
Spc softwar
re ver. 2.11
(Rohlf,
2005). T
The first m
matrix was p
produced fro
om the stan
ndardized ro
oot length d
data using S
SIMINT
procedu
ure based o
on Average
Taxonomic
c Distance
(i.e. DIST
coefficient
t in the pro
ocedure).
Standar
rdization of
f data was d
done by STA
TAND proce
edure of NT
TSYS. The
second ma
atrix was
generat
ed with hel
p of SSR da
ata using N
Nei genetic d
distance (Ne
ei, 1972). T
The allele fr
equency
data fro
om POWER
R MARKER
R was used t
to export th
he data in bi
inary forma
at (allele pre
esence =
“1” and
d allele abse
ence = “0”)
) for analysi
is with NTS
SYS-PC. C
orrelation b
between roo
ot length
and SSR
R data were
e done by t
the mantel
two-way m
matrix corres
spondence t
test (Mante
el, 1967)
with th
he help of M
MXCOMP
procedure
of NTSYS
S-pc. The s
significance
e of the cor
rrelation
between
n the matric
ces was test
ed using the
e normalize
ed Mantel Z
Z-statistics.
3. Resu
ults
3.1 Mor
rphological
l Characteri
ization
3.1.1 Ro
oot Length
Variation
In the h
hydroponic
screening,
variation in
n root lengt
th of the ge
notypes of
each specie
es under
differen
nt concentra
ations (0, 3
and 10 mM
M) is presen
nted in the
Figure 1. It
t was obser
rved that
many g
genotypes fr
from Ae. sp
peltoides, Ae
e. longissim
ma, Ae. sha
aronensis, A
Ae. searsii,
and Ae.
euvaria
abilis exhib
ited good r
root length
h under the
excess B
concentrati
ions. Wher
reas, Ae.
genicul
lata, Ae. biu
uncialis, Ae
e. triunciali
is, T. dicoc
ccoides and
S. cereale
showed ve
ery poor
root len
ngth with 0m
mM and 10
mM B conc
centration. B
But, surpris
singly these
species gav
ve better
root len
ngth under 3
3 mM B. Am
mong the sp
pecies evalu
uated Ae. sh
haronensis,
Ae. longiss
sima and
Ae. kots
schyi reveal
led better to
olerance ag
gainst exces
s B than th
e other spec
cies (Figure
e 1). Ae.
speltoid
des, Ae euv
variablis an
d Ae. sears
sii also exp
perience tole
erance perf
formance ag
gainst B
toxicity
y. Due to t
the inconsi
stency, no
reliable co
onclusion c
can be draw
wn for any
y of the
genotyp
pes from the
e species Ae
e. kotschyi a
and Ae. bico
ornis.
46