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
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Journal of A
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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
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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