Euphytica (2017)213:126
DOI 10.1007/s10681-017-1914-4
Identification and validation of root length QTLs for water
stress resistance in hexaploid wheat (Titicum aestivum L.)
Habtamu Ayalew . Hui Liu . Guijun Yan
Received: 19 December 2016 / Accepted: 16 May 2017
Springer Science+Business Media Dordrecht 2017
Abstract Thorough understanding of the genetic
mechanisms governing drought adaptive traits can
facilitate drought resistance improvement. This study
was conducted to identify chromosome regions har-
bouring QTLs contributing for water stress resistance
in wheat. A RIL mapping population derived from a
cross between W7984 (Synthetic) and Opata 85 was
phenotyped for root length and root dry weight under
water stress and non-stress growing conditions.
ANOVA showed highly significant (p B 0.01) varia-
tion among the RILs for both traits. Root length and
root dry weight showed positive and significant
(p B 0.01) phenotypic correlation. Broad sense heri-
tability was 86% for root length under stress and 65%
for root dry weight under non-stress conditions. A total
H. Ayalew (&)
Breeding Material Development Unit, Institute of Crop
Science, National Agriculture and Food Research
Organization (NARO), 2-1-2 Kannondai,
Tsukuba 305-8518, Japan
e-mail: habtamu01@gmail.com
H. Ayalew
Department of Horticulture, College of Agriculture and
Natural Resources, Debre Markos University,
P.O. Box 269, Debre Markos, Ethiopia
H. Ayalew  H. Liu  G. Yan (&)
School of Plant Biology, Faculty of Science and The
UWA Institute of Agriculture, The University of Western
Australia, 35 Stirling High Way, Crawley, WA 6009,
Australia
e-mail: guijun.yan@uwa.edu.au
of eight root length and five root dry weight QTLs
were identified under both water conditions. Root
length QTLs Qrln.uwa.1BL, Qrln.uwa.2DS, Qrln.u-
wa.5AL and Qrln.uwa.6AL combined explained 43%
of phenotypic variation under non-stress condition.
Opata was the source of favourable alleles for root
length QTLs under non-stress condition except for
Qrln.uwa.6AL. Four stress specific root length QTLs,
Qrls.uwa.1AS, Qrls.uwa.3AL, Qrls.uwa.7BL.1 and
Qrls.uwa.7BL.2 jointly explained 47% of phenotypic
variation. Synthetic wheat contributed favourable
alleles for Qrls.uwa.1AS and Qrls.uwa.3AL. Two
stable root dry weight QTLs on chromosomes 4AL
and 5AL were consistently found in both water
conditions. Three validation populations were devel-
oped by crossing cultivars Lang, Yitpi, and Chara with
Synthetic W7984 to transfer two of the QTLs identi-
fied under stress condition. The F2.3 and F3.4 validation
lines were phenotyped under the same level of water
stress as RILs to examine the effect of these QTLs.
There were 13.5 and 14.5% increases in average root
length due to the inheritance of Qrls.uwa.1AS and
Qrls.uwa.3AL, respectively. The result indicated that
closely linked SSR markers Xbarc148 (Qrls.uwa.1AS)
and Xgwm391 (Qrls.uwa.3AL) can be incorporated
into MAS for water stress improvement in wheat.
Keywords Abiotic stress  Early season drought 
Marker validation  QTL  Synthetic wheat
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126 Page 2 of 11
Background
Water stress is one of the most pressing environmental
problems in dry land agriculture which can manifest in
various forms at various plant developmental stages
(Blum 2011a, b; Passioura 2012; Tardieu 2012;
Vaughn and Nguyen 2013). The multitude drought
scenarios necessitate understanding the target envi-
ronment and developing the right crop ideotype for
successful drought resistance improvement (Blum
2014; Meister et al. 2014). The nature of prevailing
drought dictates the breeding and selection strategy
making breeding for drought resistance to be one of
the most expensive and labour intensive processes.
The global climate change has made rains more erratic
and less predictable with expansion of aridity globally.
Early season drought is becoming more prevalent as
rains are not starting early in the season and if they do
so, interruptions happen for a few weeks after sowing
(Ortiz et al. 2008; Schlenker and Lobell 2010). This
has a drastic effect on crop establishment and
productivity (Blum et al. 1980; Meister et al. 2014;
Maccaferri et al. 2016). If sowing is delayed till the
actual rains start, the crop cycle will be pushed to
terminal drought as rains tend to cease early in the
season. Enhancing the genetic potential of crops in
relation to their capacity to access more water from
deep soils will result in increase in water use and crop
productivity (Manschadi et al. 2006; Blum 2011a;
Wasson et al. 2012; Uga et al. 2013). Selection for
drought resistance gets even more difficult when the
target is to improve root traits. Root phenotyping has
been one of the most arduous tasks in crop improve-
ment mainly because of the difficulty in accessing root
samples (Tardieu 2012; Tuberosa 2012). Several
studies reported novel root phenotyping techniques
(Hoffmann et al. 2012; Cane et al. 2014) and the
identification of QTL for various traits and crops
(Specht et al. 2001; Zheng et al. 2008; Maccaferri et al.
2016). Much of previous research however, was
mainly concentrated on later stage stress resistance
which has overlooked the importance of seedling
resistance especially of root traits. Root system that
suits the prevailing edaphic and hydrologic nature is
essential for crop establishment and effective water
use adaptation to dry environments (Palta et al. 2011;
Comas et al. 2013). As drought resistance is a highly
quantitative trait, genetic improvement through the
sole use of morphological markers and classical
123
Euphytica (2017)213:126
quantitative genetics is less likely to make a major
leap in improving crop productivity. Breeding for
water stress can be made more precise and agile if the
appropriate molecular tools are incorporated into
conventional plant breeding techniques (Song et al.
2006; Collard and Mackill 2008; Fleury et al. 2010;
Budak et al. 2015). Marker assisted selection through
the application of molecular markers and some
statistical genetic tools is reported to be an effective
strategy (Borner et al. 2002; Semagn et al. 2006).
Therefore, this research was undertaken to (1) study
the genetic control of root length and root dry weight
under water stress and non-stress conditions using
QTL mapping (2) confirm effect of identified QTLs on
other genetic backgrounds other than the mapping
population and (3) validate the applicability of the
closest markers for MAS.
Materials and methods
Plant materials
One hundred and four recombinant inbred lines from
the international Triticeae mapping initiative (ITMI)
mapping population derived from a cross between the
Synthetic hexaploid wheat W7984 (Titicum turgidum
cv. Altar 84Aegilops tauschii Coss. line WPI 219)
and the spring wheat cultivar Opata 85 (Song et al.
2005) were used for the identification of QTLs under
non-stress and water stress conditions. Following the
identification and mapping of QTLs, crosses were
made between Synthetic and three other genotypes
(Chara, Yitpi, and Lang) to validate the phenotypic
effect of these QTLs on other genetic backgrounds.
The closely linked SSR markers to the QTLs were
used to genotype segregating lines from the crosses
mentioned above. All of the hybrids were F3.4 except
the hybrids from Synthetic 9 Yitpi cross which were
F2.3.
Phenotypic evaluation
The RILs were evaluated for root length variation
under osmotic stress and non-stress conditions in a
hydroponic system. A total of 104 recombinant inbred
lines in three replicates (repeats of the experiment)
were used in a completely randomized design.
Osmotic stress of -0.5 MPa was induced using PEG
Euphytica (2017)213:126
6000 (Sinopharm Chemical Reagent Co. Ltd, China).
The final stress level at data collection reached
-0.6 0.1 MPa measured using MP4 dewpoint
potentiameter (Decagon Devices Inc. 2003) creating
a progressive stress senario. Plants were let to grow
with their endosperm reserve food for the first seven
days after which half strength Hoaglands solution and
PEG6000 solution for the treatment and Hoaglands
solution alone for the control respectively we added.
The pH of the solution was adjusted between 5.5 and
5.7 and the relative humidity was between 65 and 70%
while the temperature was 25/22 C day/night. Light
intensity of 300 lmol m-2 s-1 was supplied using
cool florescent lamps in 10/14 dark and light timing
using automatic timer. The solution was constantly
aerated by bubbling air into the solution using electric
bubbler. Data were scored on root length (measured
from the base of the crown to the tip of the longest root
in cm) and root dry weight (mg) of individual plants
14 days after planting (7 days stress period). Roots
samples were dried to constant weight by incubating
them at 70 C for 48 h. The same experimental set up
and level of stress as above was used to evaluate the
root length performance of the three validation
populations.
Molecular markers and linkage map
Molecular marker data and linkage map of the
Synthetic 9 Opata 85 RIL mapping population were
accessed from the GrainGenes database (http://wheat.
pw.usda.gov/cgi-bin/graingenes). This map had a total
of 1475 SSR and RFLP markers distributed across the
21 linkage groups. Of the total available markers 1420
were used for this study with an average marker den-
sity of 1 cM after filtering for 40% missing values.
Genotyping populations for marker validation
Genomic DNA was extracted from validation lines
using Nucleon PhytoPure DNA extraction Kit (GE
Healthcare) following the manufacturers instructions.
Young leaves were ground using tissuelyser (Qiagen)
and their total DNA was isolated. DNA was treated
with 1 mg mL-1 RNase (Qiagen) for 3 h at 37 C to
eliminate RNA contamination. The quantity and
quality of the total DNA samples were measured
using a NanoDrop ND-1000 (ThermoFisher Scien-
tific). Polymerase chain reaction (PCR) was
Page 3 of 11 126
performed in 15 lL volume. Polymorphism of the
closest SSR marker was checked among the parental
lines using ordinary primes and agarose gel band
scoring. All of the forward primers of the two markers
were labelled with fluorescent dyes at their 50 end
(Applied Biosystems) for the final capillary elec-
trophoresis. The forward primers of SSR markers
Xbarc148, and Xgwm391, were labelled at their 50 end
by 6FAM, and VIC fluorescent dyes, respectively. The
reaction mixtures contained 50 ng of genomic DNA,
0.1 lM of each forward and reverse primer, 1.5 mM
of MgCl2, 109 PCR buffer (Fisher Biotic), 200 lM
dNTPs (Fisher Biotic) and one 1 lL of Taq DNA
polymerase (Fisher Biotic) using a thermal cycler
(BIO-RAD). PCR thermal cycling was performed as
follows: 95 C denaturing step for 5 min followed by
35 cycles of 95 C for 30 s, annealing for 30 s and
72 C for 45 s, with a final extension at 72 C for
5 min. PCRs for each marker were run separately and
the products were poolplexed for capillary elec-
trophoresis using DNA analyser (Applied Biosystems
3730xl DNA Analyser). The PCR products were
mixed into a 12 lL sample volume in a 96 well AB-C
PCR plate consisting of 0.5 lL of each PCR product,
10 lL of highly deionized formamide (Hi-Di) (Ap-
plied Biosystems), and 1 lL of LIZ600 size standard.
Allele peaks and fragment sizes were analysed using
GeneMarker software version 2.6.7 (SoftGenetics,
LLC State College, PA-16,803, USA). Five hundred
RFU was used as a minimum relative fluorescence for
allele peak detection.
Statistical analysis
Variance components and heritability analysis
All phenotypic data analyses were performed using
GenStat statistical software 17th edition (VSN Inter-
national 2014). Analysis of variance was conducted
based on the following fixed effects model: Yij = -
l ? gj ? eij, where Yij is observed mean, l general/
population mean; gj effect due to the jth genotype; eij is
random error. Heritability was estimated using the
 
formula: h2 d2g = d2g d2e where d2g and d2e are the
estimated genotypic and error variances, respectively
(Nyquist 1991). The estimated genotypic and error
variances were calculated as: d2g MSgMSe
r while d2e
MSe
r where MSg is mean square of the RILs, MSe is the
123
126 Page 4 of 11
residual error and r the number of replicates. The mean
values of each of the trails across the RILs were used
for QTL analysis. The effects of the acquired QTLs
from the female parent (synthetic) were assessed as
percentage of mean differences between homozygous
lines based on the genotype of linked markers to the
respective QTLs.
QTL analysis
QTL analysis was carried out using composite
interval mapping (CIM) based on Kosambis map-
ping function. Windows QTL Cartographer Version
2.5 was used for the QTL analyses (Wang et al.
2012). The standard QTL analysis model (model 6)
with control marker number of 5 and window size
of 10 cM was used. Backward marker selection
method for background marker inclusion in the
regression model was used with a false discovery
rate of 0.01 and default value of marker inclusion
probabilities of 0.01. The whole genome was
scanned in every 1 cM interval. LOD value of 3
and above was used to declare a QTL for both
stress and non-stress growing conditions. The
graphical representation of linkage groups and
QTL positions were constructed using the Map-
Chart 2.30 software (Voorrips 2002).
Results
Phenotypic variation
The induced water stress caused average reductions of
41% in root length and 27% in root dry weight,
respectively. The phenotypic data analysis revealed
highly (p B 0.01) significant differences among the
RILs. Root length ranged from 5 to 25 cm under stress
and from 7 to 30.3 cm under non-stress growing
conditions (data not shown). The phenotypic distribu-
tions of mean root length and root dry weight (Fig. 1a
d) indicated transgressive segregations on both direc-
tions of the parents, suggesting polygenic inheritance
of both traits in wheat. Synthetic (W-7984) was the
longer rooting parent as compared to Opata 85. High
heritability values of root length were recorded under
non-stress (81%) and stress (86%) conditions, respec-
tively (Table 1). Similarly, root dry weight showed
moderate (65% under non-stress) to high (84% under
123
Euphytica (2017)213:126
stress) levels of broad sense heritability (Table 1).
Root length and root dry weight showed positive and
significant (p B 0.01) phenotypic correlation (data not
shown).
QTL identification
Root length and root dry weight QTLs under non-
stress condition
Four root length QTLs explaining 43% of phenotypic
variation were identified on chromosomes 1BL, 2DS,
5AL, and 6AL (Table 2; Fig. 2). Three root dry weight
QTLs were identified on chromosome arms of 4AL,
5AL and 7DL under non-stress condition. The D
genome of Synthetic wheat contributed the favourable
allele for longer roots under non-stress condition.
Favourable alleles for QTLs Qrln.uwa.1BL, Qrln.u-
wa.5AL and Qrln.uwa.6AL were contributed by the A
and B genomes of Opata 85 (Table 2). Three root dry
weight QTLs were identified and mapped to chromo-
some arms of 4AL, 5AL and 7DL under non-stress
condition. Root dry weight QTLs Qrdws.uwa.4AL and
Qrdwn.uwa.4AL under non-stress, and Qrdws.u-
wa.5AL and Qrdwn.uwa.5AL under stress condition
were common between the two water conditions while
Qrdws.uwa.7DL was specific to the non-stress condi-
tion (Fig. 2). The two root dry weight QTLs on
chromosome 5AL (Qrdws.uwa.5AL and Qrdwn.u-
wa.5AL) were mapped 10 cM away from a root length
QTL (Qrln.uwa.5AL), while these three QTLs have
gained favourable alleles from Synthetic (Table 2).
All root dry weight QTLs except the ones on
chromosome 5AL gained favourable alleles from the
male parent, Opata 85.
Root length and root dry weight QTLs under stress
condition
A total of four root length and two root dry weight
QTLs were identified under stress condition. Root
length QTLs were mapped on chromosome 1AS, 3AL
and 7BL with total phenotypic expression of 47%.
Similarly, two root dry weight QTLs explaining 10
and 15% of phenotypic variation were mapped to
chromosomes 4 and 5AL gaining favourable alleles
form Opata and Synthetic in the same order. Favour-
able alleles of root length QTLs on chromosomes 1AS
and 3AL were contributed by Synthetic while Opata
Euphytica (2017)213:126 Page 5 of 11 126

40 Opata 35 (b) Opata Synthetic

(a)
35 30
30 25
Frequency

25
20
Synthetic 20
15
15
10 10
5 5
0 0
5 8 11 14 17 21 24 14 18 22 26 30 34
Root length (cm) Root length (cm)

60 Opata Synthetic 60 (d)

(c) Opata Synthetic
40
Frequency

40

20 20

0 0
8 12 16 20 24 15 18 21 24 27 30
Root dry weight (mg) Root dry weight (mg)

Fig. 1 Distribution of average root length (a, b) and root dry weight (c, d) among 104 RIL mapping lines tested under -0.5 0.1 MPa
water stress (a, c) and non-stress (b, d) conditions

Table 1 Mean squares, genotypic and phenotypic coefficients of variation, and bread sense heritability of maximum root length and
root dry weight tested under -0.5 0.1 MPa osmotic stress and non-stress growing conditions
Osmotic condition MSg MSe d2g d2e d2p H2

RL stress 58.62** 7.92 16.90 2.64 19.54 0.86

RDW stress 39.42** 6.45 10.99 2.15 13.14 0.84
RL normal 52.15** 9.71 14.15 3.24 17.38 0.81
RDW normal 42.55** 14.77 9.26 4.92 14.18 0.65

MSg mean square of genotype, MSe means square of random error, d2g estimated genetic variance, d2p estimated phenotypic variance,
d2e estimated error variance, H2 heritability in broad sense, RL root length, RDW root dry weight
** Indicates significant difference at p \ 0.01

was source of the favourable alleles for the other two QTL effect confirmation and marker validation
QTLs on chromosome 7BL (Table 2). Two equally
significant QTLs with a phenotypic expression of 13% Three validation populations inheriting Qrls.uwa.1AS
each, were located on chromosomes 3AL and 7BL and Qrls.uwa.3AL from Synthetic wheat were evalu-
gaining their favourable alleles from Synthetic and ated under the same level of stress as the RIL
Opata, respectively (Table 2). The two significant population to examine the effect of these QTLs in
QTLs on the long arm of chromosome 7BL were only other genetic backgrounds. Individuals in each vali-
5.8 cM apart. None of the root length QTLs identified dation population were classified into three groups
under non-stress condition collocated with QTLs based on the combination of allele peaks from the
under stress condition suggesting qualitative QTL by parental lines. Parental lines Synthetic and Lang were
environment interaction. not polymorphic for marker Xgwm391 and similarly

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126 Page 6 of 11 Euphytica (2017)213:126

Table 2 Chromosomal position, marker interval, additive effect and phenotypic expression of QTLs identified on the ITMI Syn-
thetic 9 Opata 85 RILs grown under stress and non-stress conditions
Water status Trait Chromosome arm Flanking markers Position LOD Additive r2 (%)

Non-stress RL 1BL Xbarc81/Xbcd1562 72 3.2 -1.5 11

2DS Xgwm210/Xbcd611 19.4 3.8 1.6 12
5AL Xbarc330/Xbcd235 57.5 3.0 -1.5 9
6AL Xgwm169/Xfba221 100.4 3.4 -1.6 11
RDW 4AL Xgwm350/Xfba231 65.7 3.3 -1.3 9
5AL Xmwg624/Xbarc330 53.8 3.5 1.3 10
7DL Xbarc105/Xmwg975 93.2 4.1 -1.8 17
Stress RL 1AS Xbarc148/Xbarc162 36.6 3.1 1.3 9
3AL Xgwm391/Xbcd1431 92 4.0 1.6 13
7BL Xbarc90/Xbarc176 38.9 4.4 -1.5 12
7BL Xfba301/Xbarc278 44.7 4.9 -1.5 13
RDW 4AL Xcdo475/Xbcd130 65.7 3.4 -1.3 10
5AL Xmwg624/Xbarc330 53.8 4.7 1.5 15
RL root length, RDW root dry weight

1A 1B 2D 3A 4A 5A 6A 7B 7D
0.0 W2I^ XksuD14.1 0.0 Xpsr11(Glu-3) 5.7 Xcmwg682 0.0 Xbarc57 0.0 Xbarc206 0.0 Xbarc122 0.0 Xpsr167 0.0 Xgwm537 0.0 Xcnl6
1.0 XksuD14 11.4 Xbcd15 2.0 Xbcd342 0.7 Xbcd1872
4.4 Xwhs179 8.1 Xbarc124 16.1 Xfbb370 2.5 Xpsr889
3.9 Xgwm136 Xgwm550 Xglk683 4.1 Xgwm154 Xbcd1821
7.3 17.2 4.2 4.5 Xgwm400
5.1 Xpsr11(Glu-3) Xgwm33 10.7 Xbarc297 Xbarc294 4.9 Xbarc316 Xgwm459
10.7 18.3 5.0
Xgwm264 12.0 Xfba83 Xbarc321 5.9 Xgwm205 Xbcd21
11.8 18.6 5.4
7.8 XksuD14.2 13.1 Xcdo388 13.6 Xbcd18 19.5 Xbarc310 6.5 Xcdo476
13.9 Xmwg938 14.7 Xbcd1970 26.0 Xtam47 Xbarc180 6.8 Xgwm334 9.6 Xwg834
11.1
Qrln.uwa.2DS

15.4 Xmwg68 Xbcd718 28.0 Xtam61 12.5 Xfbb1 13.9 Xbcd1871 7.2 Xfba65 Xfba42
17.3 11.4
12.3 Xbarc119 16.3 Xabc156 29.9 Xgwm369 12.9 Xfbb332 15.8 Xgwm304 7.8 Xfbb194
18.7 Xgwm210 Xgwm68 13.1 Xbcd1438
16.6 XGli3 31.7 Xcdo482 13.5 Xfba40 16.5 Xbarc186 9.3 Xfbb147 17.2
XksuF43.1 19.7 Xbcd102 Xcdo460 Xfbb209 20.2 Xbarc65 14.6 Xgwm295
16.9 32.3 14.5 Xfba78 17.1 Xbarc303 10.2
Xgwm33 19.4 XksuF43.2 21.1 Xbcd611 34.3 Xfba366 14.4 Xfba307 22.2 Xgwm573
16.3 16.1 Xgwm4 18.8 Xbarc117
17.3 Xbarc263 21.0 XksuE18 24.1 Xfba400 34.5 Xbarc284 17.5 Xgwm192 20.5 Xgwm129 16.1 Xpsr8(Cxp3) 24.2 Xfbb226
18.0 Xmwg710
18.8 Xcdo426 21.2 XksuE19 25.4 Xcdo1379 37.6 Xabc172.1 17.9 Xgwm165 20.7 Xfba352 17.6 Xmwg67 26.2 Xbarc85
23.8 Xbcd340 27.8 Xgwm484 40.1 Xmwg11 18.4 Xbcd1738 21.0 Xgwm415 18.1 XksuG48 26.6 Xbcd310
24.8 Xrz166 28.7 Xbarc168 40.3 Xbarc179 19.2 XksuF8 21.3 Xcdo749 18.8 Xcmwg652 27.6 Xwg180 21.1 Xbarc154
25.6 Xbcd1124 30.7 Xbcd262 40.5 Xfbb237 19.8 Xbcd1652 21.9 Xfba131 19.1 Xfbb166 28.2 Xcnl8
26.4 Nor-B1 34.2 Xcdo1479 41.1 Xmwg12 20.1 Xcdo959 22.7 Xgwm293 28.8 Xfbb150
25.1 Xabc156 27.3 Xabg373 36.0 Xgwm102 42.4 Xfba91 20.5 Xcdo1387 25.5 Xpsr10(Gli-2) 30.3 Xfba32
30.7 Xfbb195 26.0 Xbarc352
28.1 Xgwm18 36.2 Xfbb279 42.7 Xfbb293.3 20.8 Xfbb227 28.9 Xbarc360
27.5 XGli3 28.5 Xcmwg645 38.9 Xwsu1 45.8 XgbxG406 30.9 Xbcd385
20.9 Xfba320 30.5 Xcdo785 28.3 Xfba152 Xfba85
28.9 Xcdo1340 41.5 Xfba341.1 46.1 Xcdo638 21.4 Xrz574 31.5 Xgwm46 28.9 Xrz2
30.2 XksuE18 31.8 Xbarc100
29.4 Xcdo127 42.0 Xcdo405 47.5 XksuA6 21.9 Xfba359 Xfbb176 Xbarc40 31.6 Xbarc72
33.9
33.4 Xbarc148 29.9 Xbcd762 44.7 Xgwm515 49.1 Xcdo1345 22.7 Xbarc106 Xbarc1 32.0 XksuH4 32.2 Xbcd1338
35.2
Qrls.uwa.1AS

Xbcd98.1 30.1 Xbcd1796 45.5 Xbarc228 49.2 Xfbb293.4 23.0 Xgwm601 Xbcd157 33.3 Xbarc267
34.6 36.5
30.3 Xbcd338 45.9 Xbcd260 49.6 Xbcd1823 23.6 Xbarc138 Xpsr128 33.4 Xbarc255
37.3 Xrz244 36.8
30.4 Xcdo92 47.2 Xfba38 49.7 Xabg471 24.3 Xfba147 Xbcd1355 33.5 Xgwm16 Xgwm297
38.0 XksuG9 37.4
Xbcd200 Xcdo618 47.3 Xbarc145 50.3 Xcdo1164 33.7 Xgwm43

Qrls.uwa.7BL
38.3 Xbarc162 30.6 25.6 Xfba211 39.1 Xgwm156 37.7 Xpsr312 37.5 Xbarc125 Xbarc126
Xwg811 48.0 Xbarc292 51.1 Xbcd706 26.9 Xfba43 Xcdo412 34.6 Xfba371 Xfbb193
40.5 Xgwm164 39.8 42.3 Xfbb145
30.8 Xcdo1173 49.8 Xgwm249 51.7 Xmwg22 29.8 Xbcd8 Xbarc165 34.7 Xbcd98
40.6 Xabg373 40.3 44.3 Xcdo1315
31.0 Xcdo98 53.0 Xbcd120 51.9 Xbcd1532 30.4 Xgwm610 Xgwm186 35.0 Xcdo551
40.8 Xabg452 Xwg605 42.3 47.4 Xbarc3
31.2 Xwg605 53.4 Xbcd111 52.1 Xcdo1435 31.2 Xbcd402 Xbcd1949 35.5 Xbcd349 42.6 Xfba377
40.9 Xcdo98 43.2 48.2 Xcdo270
31.5 Xgwm273 Xgwm413 54.2 Xbarc11 53.1 Xabg460 33.5 XksuE2 Xbcd926 36.3 Xfba311 43.9 Xgwm44
41.2 Xbcd1072 43.5 48.4 Xbarc3
32.2 Xmwg837 54.9 Xgwm157 53.9 Xbarc45 36.2 XksuG12 43.9 Xbcd1088 36.8 Xabc455
41.9 Xcdo580 50.2 Xfba148

Qrls.uwa.7BL
32.7 Xbcd1449 57.8 Xfbb99 54.9 Xgwm2 38.4 Xwg622 44.7 Xbcd981 37.4 Xbarc90
42.2 Xbcd98.2 50.8 Xgwm494
32.9 Xcdo278 58.3 Xfba74 56.3 Xfba175 39.9 Xfba65 45.3 Xbarc141 37.8 Xgwm644
43.2 Xbarc120 56.1 Xcdo1428
33.1 Xbcd12 58.9 Xtam8 57.3 Xpsr903 41.3 Xgwm397 46.9 Xcdo57 38.9 Xbcd178 49.2 Xbarc214
44.3 Xmwg67 56.8 Xcdo29
Qrdwn.uwa.5AL
Qrdws.uwa.5AL

33.2 Xmwg837 59.6 Xgwm539 57.7 Xbarc356 45.0 Xgwm637 48.0 Xcdo1090 40.0 Xgwm333
46.6 Xbarc28 57.6 Xfba234
33.5 Xgwm11 61.8 Xgwm608 58.5 Xgwm32 47.6 Xbarc170 48.8 Xmwg522 40.6 Xrz476
47.5 Xcdo473 58.0 Xbarc171
33.6 Xcmwg758 Xfba341.2 Xfba61 58.9 Xgwm5 48.6 Xbarc170 51.7 Xmwg624 42.3 Xcnl7
48.2 Xgwm357 62.3 58.8 Xbarc195
34.3 Xbarc137 Xfbb284 59.4 Xbcd1127 52.8 Xbarc343 Xbarc197 43.9 Xbarc176
50.8 Xbcd1407 52.6 60.9 Xtam36
34.5 Xbarc187 62.8 Xfba62 59.8 Xfbb332 55.4 Xfba4 44.7 Xwg514
52.3 Xcdo312 53.8 Xgwm639 61.9 Xfbb192 Xfbb215 56.7 Xgwm111
Qrln.uwa.5AL

35.0 Xgwm498 63.4 Xfba111 Xfbb122 60.4 Xbarc67 56.0 Xabg390 Xfba354 Xfbb258
55.2 Xgwm135 54.6 Xfbb2 62.2 Xfba397 Xfbb95 46.0
35.5 Xbarc240 64.0 Xfba64 60.6 Xbarc324 56.7 Xpsr115 58.5 Xbcd707
57.8 Glu 56.5 Xbarc330 62.6 Xbcd758 47.6 Xfba301
36.1 Xbcd386 65.6 Xfbb68 60.8 Xbarc19 57.3 Xbcd1670
57.6 Xgwm617 63.0 Xfba367 48.6 Xfbb179
Qrdwn.uwa.4AL
Qrdws.uwa.4AL

37.2 Xgwm582 67.2 Xfbb9 61.1 Xgwm666.1 58.4 Xmwg549

Xgwm131 Xfba116 Xgwm30 60.0 Xbcd1235.1 63.3 Xbcd1860 49.9 Xbarc278
38.0 67.9 61.4 61.0 Xmwg710
Xbarc181 Xcdo1008 Xfba127 63.8 Xfbb283 50.9 Xfbb175
39.0 68.2 61.7 61.9 Xcdo780 64.0 Xbarc151 64.1 Xbarc26
Xbarc174 Xfbb251 Xabg396 64.1 Xcdo772 53.1 Xfba305
65.9 Xcmwg733 39.3 70.8 62.5 62.7 Xcdo475 64.7 Xfbb255
64.9 Xpsr915 53.3 Xfbb352
Qrln.uwa.1BL

66.6 Xbcd265 41.6 Xbarc302 71.7 Xfbb72 62.8 Xbcd366 64.7 Xgwm350 XksuD9 66.4 Xfba166 67.1 Xgwm437
Xglk136 Xbcd410 Xbcd828 65.7 Xcdo204 54.8 Xwg686
67.3 Xbcd1930 42.6 73.2 63.0 66.3 Xbcd130 67.3 Xwg719
68.3 Xbcd183 66.1 Xbarc113 57.2 Xgwm302
67.8 Xbcd808.1 43.1 Xcdo637 73.9 Xbarc219 63.4 Xcdo1174 66.5 Xfba231 69.3 Xgwm666 66.7 Xcdo204 58.7 Xcdo686 69.8 Xfbb112
68.9 Xbcd1889 46.8 Xbcd1150.1 74.8 Xrz444 63.7 Xmwg802 69.7 Xfbb114
Glu-B1 XksuD23 Xcdo118 67.3 Xbarc107 61.3 Xgwm112
70.8 Xbcd808.2 47.7 75.1 64.2 70.3 Xcdo665
Xbcd1150.2 Xglk558 Xgwm382 XATPase 68.4 Xpsr463(Prk) 65.9 Xfba259
49.5 75.4 64.7 71.7 XksuE3
74.0 XksuE3 Xbarc61 Xgwm268 XksuH9 Xabc172.2 73.8 Xfba190 69.2 Xfbb170 70.5 Xbarc258
54.2 75.7 64.8 74.6 Xtam72
Xcmwg706 Xgwm124 XksuH16 XgbxG034 71.0 Xcdo388 71.2 Xbarc315 74.9 Xcdo775
75.4 54.5 78.2 65.8 75.7 Xcdo545
Xcmwg733 Xgwm301 Xbarc25 Xrz395 71.5 Xbcd506
77.1 XksuH9 55.7 78.8 66.2 77.1 Xgwm160 76.7
Xgwm153 Xgwm349 Xfbb277 74.4 Xbarc204
78.0 XksuH14 56.6 79.1 66.8 78.1 Xbarc172
56.9 Xgwm274 79.7 Xfba314 67.8 XksuH2 79.2 Xfba282
80.7 Xcdo89 57.6 Xbcd441 79.8 Xgwm301 68.1 Xcdo281 Xwg177 80.8 Xbarc230
81.2 Xgwm497 60.1 Xmwg69 80.7 Xbarc159 69.1 Xfbb271 81.2 Xcdo1326 82.1 Xbarc121
82.2 Xgwm570
61.3 Xbcd442 84.1 Xfba311 69.9 Xbcd452 83.2 Xbarc78 83.6 Xfba68
63.5 Xbarc188 89.6 Xfba209 70.9 Xbcd115 Xbcd452 84.5 Xfbb67
64.4 Xgwm403 90.6 Xfba209 71.2 Xbcd2044 86.4 Xbarc319 86.0 Xmwg934
86.6 Xgwm121
69.0 Xbarc81 72.2 Xmwg961 88.1 Xfbb209
Xtam33 88.0 Xcsb112(Dhn5)
73.6 Xbcd1562 72.3 88.6 Xbarc105
Qrls.uwa.3AL

Xfbb199
Qrdwn.uwa.7DL
88.7
74.4 Xbcd508 74.7 Xfbb353 90.9 Xbcd1975
XgbxG499 90.6 XksuD2
79.9 Xbcd1514 75.6 91.2 Xbarc327 91.2 Xfba351
92.3 Xgwm666 76.9 Xtam63 92.2 Xbarc111
80.1 Xbcd1261 Xcdo1189
89.5 Xcdo346 77.4 Xbcd1145 94.3 Xfba111 94.0 Xfba69
93.0 Xgwm259 78.6 Xfba167 95.3 Xfba243
82.5 Xgwm666.2 97.5 Xbarc104 96.9 Xmwg975
96.9 Xbarc52 99.0 Xgwm169
Xgwm480 97.6 Xfbb79
Qrln.uwa.6AL

98.9 XksuE11 84.1

85.3 Xfbb293.1 100.4 Xfbb70 98.4 Xfba264
99.0 Xmwg912
XksuE11 86.3 Xbcd372 100.7 Xfbb330 101.8 Xfbb221
99.9
100.6 XksuI27 87.7 Xfbb293.2 101.8 Xbarc315 102.0 Xabg366 101.9 Xfbb82
87.9 Xbcd1773 103.3 XksuD27 102.6 XksuE18 102.8 Xwg420
103.6 Xcdo1160 102.1 Xgwm140 104.7 Xabg391
103.9 Xbarc80 88.3 Xmwg30 105.3 Xcdo457 105.1 Xfbb191
88.5 Xbarc314 105.3 Xbarc184 105.6 Xfba20 Xfbb40 105.5 Xfbb325
106.4 Xbcd1235.2
90.5 Xfba347 106.8 Xcdo1312 105.7 Xfba8
108.0 Xmwg632 91.1 Xbarc197 108.5 Xbcd129 106.3 Xcdo836
108.6 Xfbb249
91.4 Xgwm391 Xgwm179 106.5 Xbcd1510 109.4 Xcnl2 109.4 Xfba204
110.5 Xbarc213 111.3
111.0 Xgwm99 91.6 Xbcd1431.2 111.7 Xgwm126 107.2 Xgwm427
111.9 Xbarc70 110.1 Xgwm617 112.1 Xgwm611
92.0 Xfbb260 112.7 Xcdo20
93.2 Xbcd1431.1 113.1 Xcdo1528 111.2 Xmwg2053 113.8 Xbarc235
Xfbb250 Xgwm247 113.9 Xwg114 113.4 Xmwg573 115.4 Xcdo414
94.3
94.9 Xwg184 116.8 Xbarc153 116.4 Xgwm577
118.3 Xmwg912 96.4 Xfbb322 118.4 Xgwm344
99.9 XgbxG242 119.7 Xfba21 Xfbb189 119.2 Xgwm428
120.6 XksuE11.1 103.1 Xmwg570 120.7 Xbcd588 120.5 Xgwm6 120.9 Xbarc340
121.7 Xcdo393 121.5 Xbarc182
122.5 Xfba253 122.9 Xgwm595
123.4 XksuE11.2 121.8 Xbarc50
124.5 Xwg241 123.4 Xgwm146
125.2 Xbarc53
125.5 Xrz508
127.2 Xbarc287 127.0 Xmwg2112
128.3 Xgwm291
129.3 Xbarc158
130.4 Xfbb189
132.4 Xgwm410
133.2 Xbarc20

138.1 Xbarc76

141.7 Xgwm37

149.2 XksuE3

Fig. 2 Chromosomal location of root length (RL) and dry stress, Qrls RL QTL under stress and Qrln RL QTL under non-
weight (RDW) QTLs under stress and non-stress conditions. stress conditions followed by institution name (uwa University
Qrdws RDW QTL under stress, Qrdwn RDW QTL under non- of Western Australia)

123
Euphytica (2017)213:126
Synthetic and Yitpi were not either for marker
Xbarc148. Mean performance of genotypes based
on the three types of allele combinations (AA, Aa,
and aa) were used to calculate the phenotypic
effect of the identified QTLs as percentage increase
relative to the homozygous recessive (aa) allele.
Synthetic x Chara progenies showed an average
increase of 11% in root length as a result of
Qrls.uwa.1AS and Qrls.uwa.3AL.
Generally, heterozygous (Aa) genotypes showed
longer roots than their homozygous recessive (aa)
counterparts suggesting that long root is dominant
over short root (Table 3). However, the average
phenotypic performance of the homozygous dominant
types (AA) was not much different from the heterozy-
gous (Aa) types. The highest increase in root length
was found among Synthetic 9 Yitpi progenies as a
result of Qrls.uwa.3AL. Homozygous dominant pro-
genies (AA) from Synthetic 9 Yitpi were the longest
rooting genotypes among all validation lines.
Discussion
Identification and mapping of QTLs that contribute to
drought resistance enable a focussed and well
informed breeding and selection for efficient gene
pyramiding, back crossing, and positional cloning
with the help of molecular markers (Collard and
Mackill 2008; Semagn et al. 2010). The present study
was conducted aiming to identify and map QTLs for
root length and root dry weight for improved water
stress resistance. The ITMI mapping population
derived from Synthetic 9 Opata was phenotyped for
the present mapping study based on results from our
previous screening study (Ayalew et al. 2015) and
other study reports on the potential of Synthetic wheat
and its diploid progenitor Aegilops tauschii for
Table 3 Mean root lengths (cm) of genotypes based on allele combinations of the F2.3 and F3.4 hybrids with the corresponding
phenotypic effect of QTLs no root length under 20.5 0.1 MPa water stress
QTLs Genotype Marker
Qrls.uwa.1AS Synthetic 9 Chara Xbarc148
Qrls.uwa.3AL Synthetic 9 Chara Xgwm391
Qrls.uwa.1AS Synthetic 9 Lang Xbarc148
Qrls.uwa.3AL Synthetic 9 Yitpi Xgwm391
Page 7 of 11 126
drought resistance (Reynolds et al. 2007; Sohail
et al. 2011).
Variation in bi-parental populations is essential for
successful identification of QTL contributing for the
genetic control of a trait under study. In the present
study, phenotypic variation for both root length and
root dry weight and the corresponding broad sense
heritability (H2) values were high (6586%) in both
stress and non-stress growing conditions. The high
levels of broad sense heritability indicated the high
proportion of genetic components of variation in the
mapping population. The average shift in a population
mean towards a desired direction is dependent on the
proportion of additive genes in the observed genetic
variation (Falconer and Mackay 1996). This high level
of genetic variability enabled the identification of
eight root length and five root dry weight QTLs under
both growing conditions.
Root length and root dry weight QTLs identified
under non-stress condition
Deep rooting and early vigour (rapid accumulation of
biomass) are among the most desirable traits for water
use efficiency and ground cover to hinder soil
evaporation loss and smother weeds (Kipp et al.
2014; Zhang et al. 2014). The present study identified
four root length and three root dry weight QTLs
combined explaining 43 and 36% of phenotypic
variation, respectively under non-stress condition.
These early vigour QTLs were distributed across all
the three genomes of wheat (Fig. 2). The findings in
the present study were in agreement with Bai et al.
(2013) who reported co-located root length and seed
morphology QTLs on chromosomes 2D, 5A and 6A in
wheat. The two co-located root dry weight and one
root length QTLs on chromosome 5AL in the present
report were mapped about 20 cM away (marker
AA Aa aa Effect (%)
18.2 16.6 16.5 10.3
18.3 18.3 16.4 11.6
21 19.2 18 16.7
21.8 21.3 18.5 17.8
123
126 Page 8 of 11
distance based on grain genes website) from the
cluster of root volume, thousand grain weight and
shoot dry weight QTLs reported by Bai et al. (2013).
Similarly Parent et al. (2015) reported growth related
QTLs on 1B, 2D, and 5A. Deep rooting at early crop
growth stage is positively correlated with early vigour
which enables better crop establishment for improved
photosynthetic capacity and better biomass production
(Landjeva et al. 2008; Wilson et al. 2015). Seed size
plays vital role during the early development of crops.
The co-location (about 2 cM apart) of seed sphericity
QTL (Breseghello and Sorrells 2007) with a root
length QTL (Qrln.uwa.1BL), and the chromosomal
linkage (about 40 cM apart) between a major kernel
weight QTL (Breseghello and Sorrells 2007) with two
root dry weight QTLs (Qrdwn.uwa.4AL and Qrdws.u-
wa.4AL) in the present study gives some indication
that seed size and root growth might have been
simultaneously regulated. Screening for rapid root
growth might be benefited from selecting larger seeds
along with other root parameters. Breeding programs
need to target improving early vigour to better utilize
available water, one of the scare resources.
Water stress resistance QTLs detected
Four root length and two root dry weight QTLs were
identified under stress condition. Two out of four root
length QTLs (Qrls.uwa.1AS and Qrls.uwa.3AL) and
one of the root dry weight QTLs (Qrdws.uwa.5AL)
identified under stress condition gained positive
alleles from Synthetic. Synthetic hexaploid wheats
have previously been reported to have adaptive genes
to drought environments (Reynolds et al. 2007; Sohail
et al. 2011). Opata contributed the positive alleles to
the two root length QTLs (Qrls.uwa.7BL.1 and
Qrls.uwa.7BL.2) on chromosome 7B and other two
root dry weight QTLs (Qrdws.uwa.4AL and Qrdwn.u-
wa.4AL) on chromosome 4A (Table 2). Several pre-
vious studies reported drought resistance related QTLs
in chromosomes arms of 1A, 3A, 4A and 7B which
corroborate with the present study (Rebetzke et al.
2008; Peng et al. 2011; Parent et al. 2015; Maccaferri
et al. 2016). Qrls.uwa.1AS was located 12 cM away
from a major grain weight QTL explaining 14% of
variation (Williams and Sorrells 2014). Previous
research reported that chromosome 3A was one of
the three homologues to have DREB1A (dehydration
responsive element binding) transcription factors
123
Euphytica (2017)213:126
(TFs) genes (Wei et al. 2009; Edae et al. 2013) These
DREB1A transcription factor genes might be the
driving factors behind the scene for the phenotypic
expression of Qrls.uwa.3AL identified in the present
study. The DREB1A TFs are correlated with heading
date, vegetation index and biomass yield (Budak et al.
2015) which are in turn correlated with development
of long roots (Swain et al. 2013; Ayalew et al. 2016a).
Similarly, the two QTLs on chromosome 7B were
mapped with ABA responsive chromosome regions
which slow plant growth (Barakat et al. 2015). This
might also justify the negative additive value by the
parent Synthetic in this study. The phenotypic effect of
the two positive alleles from the A genome of
Synthetic were equally counterbalanced by its nega-
tive alleles from the B genome (Table 2) pointing to
the need of using markers of these QTLs simultane-
ously for MAS to select for long roots and against
short roots, respectively for optimum improvement.
None of the root length QTLs identified under non-
stress growing condition did collocate with QTLs
under stress condition suggesting the presence of
qualitative QTL by environment interaction. In situa-
tions when the nature of QTL by environment
interaction is significantly and qualitatively different,
selection activities for the two environments shall be
dealt separately (Qu et al. 2008; Yan and Holland
2010; Ayalew et al. 2014). Two stable root dry weight
QTLs were identified on chromosomes 4AL and 5AL
(Fig. 2). The closely mapped (10 cM) root length QTL
(Qrln.uwa.5AL) and root dry weight QTLs (Qrdw.u-
wa.5AL and Qrdws.uwa.5AL) on chromosome 5A
could be simultaneously transferred to other genotypes
as the possibility of crossing over with in this interval
is less frequent. The varying patterns of root length
QTLs based on water regimes in this study conformed
to prior studies explaining the significance of QTL by
environment interaction and the complexity of root
length and drought resistance genetics (Kamoshita
et al. 2002; Li et al. 2005; Qu et al. 2008; Sharma et al.
2011; Parent et al. 2015).
QTL effect and marker validation
Confirming the phenotypic effect of identified QTLs
on genetic backgrounds other than the mapping
population itself, and validating the usability of linked
markers for marker assisted selection is a major step
towards the utilization of identified QTL/genes in
Euphytica (2017)213:126
plant breeding (Zhou et al. 2003; Collard et al. 2006;
Palomeque et al. 2010). In the present study three
hybrid populations, which involve Synthetic (source
parent of QTLs) as one of the parents, were developed
to validate the effect of the identified QTLs in their
progenies. SSR markers closely linked to these QTLs,
Xbarc148 and Xgwm391, were used to track down the
acquired QTLs in F2.3 (Synthetic 9 Yitpi) and F3.4
(Synthetic 9 Chara, and Synthetic 9 Lang) proge-
nies. Synthetic 9 Chara progenies showed an average
increase of 10% in root length as a result of
Qrls.uwa.1AS and Qrls.uwa.3AL. The hybrid (Aa)
genotypes were generally higher in average root
length than their homozygous recessive (aa) parents
suggesting that long root is dominant over short root.
This was in agreement with our previous study on the
genetics of gene actions controlling root length under
water stress (Ayalew et al. 2016b). The highest
increase in root length was found among Syn-
thetic 9 Yitpi progenies as a result of Qrls.uwa.3AL.
This discrepancy in the level of gain from the positive
alleles explains the variation in the proportion of
additive dominant genes in the parental lines. Yitpi
seems to have the highest proportion of additive genes
as compared to the rest of the parental lines as its
progenies were the highest in root length.
In conclusion, the SSR markers validated in this
study can be used to identify genotypes with the
desired root phenotype for water stress resistance.
Verifying the functional relationship between Qrls.u-
wa.3AL and DREB1A genes from Synthetic wheat
will enable assaying genotypes using the SSR marker,
Xgwm391 without the need to develop functional
markers for early stage water stress resistance.
Acknowledgements Australian development scholarship
funded the study of the first author. We thank Dr Chunji Liu
of CSIRO for providing some of the wheat genotypes.
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