Mapping quantitative trait loci associated with rootpenetration ability of wheat in contrasting environments

T. L. Botwright Acuna • G. J. Rebetzke •

X. He • E. Maynol • L. J. Wade

Received: 12 September 2013 / Accepted: 3 March 2014 / Published online: 11 March 2014

� Springer Science+Business Media Dordrecht 2014

Abstract The aim of this research was to investigate

the genetic basis for variation in root penetration

ability and associated traits in the mapping population

derived from the Australian bread wheat cultivars

Halberd and Cranbrook in soil columns containing

wax layers grown in controlled conditions and to

compare this with performance in the field. Root and

shoot traits of the doubled haploid line (DHL) from a

cross of Halberd and Cranbrook were evaluated in soil

columns containing wax layers. Contrasting DHLs

that varied in the ability to penetrate a wax layer in soil

columns were then evaluated for maximum root depth

in the field on contrasting soils at Merredin, Western

Australia. Genetic control was complex, and numer-

ous quantitative trait loci (QTL) (53 in total) were

located across most chromosomes that had a small

genetic effect (LOD scores of 3.2–9.1). Of these QTL,

29 were associated with root traits, 37 % of which

were contributed positively by the Halberd with key

traits being located on chromosomes 2D, 4A, 6B, and

7B. Variation in root traits of DHL in soil columns was

linked with field performance. Despite the complexity

of the traits and a large number of small QTL, the

results can be potentially used to explore allelic

diversity in root traits for hardpan penetration.

Keywords QTL �Traffic pan �Roots � Triticum

aestivum L.

Introduction

Soils are frequently inhospitable for root growth,

presenting a range of physical, biological, or chemical

constraints. Australia, in particular, has geologically

old and weathered soils and a variable climate prone to

water deficit that limits crop growth. As a conse-

quence, the average grain yield of rainfed wheat in

Australia is only 2.5 t ha-1 (Angus et al. 2001). The

ability of roots to access water at depth in the soil

profile can be of great benefit, where an additional

1 mm of subsoil water late in the season is estimated to

Electronic supplementary material The online version ofthis article (doi:10.1007/s11032-014-0063-x) contains supple-mentary material, which is available to authorized users.

T. L. Botwright Acuna (&)

Tasmanian Institute of Agriculture, The University of

Tasmania, Private Bag 54, Hobart, TAS 7001, Australia

e-mail: Tina.Acuna@utas.edu.au

G. J. Rebetzke

CSIRO Plant Industry, GPO Box 1600, Canberra,

ACT 2601, Australia

X. He � E. Maynol

School of Plant Biology M084, The University of Western

Australia, 35 Stirling Highway, Crawley, WA 6009,

Australia

L. J. Wade

EH Graham Centre for Agricultural Innovation, Charles

Sturt University, Locked Bag 588, Wagga Wagga,

NSW 2678, Australia

123

Mol Breeding (2014) 34:631–642

DOI 10.1007/s11032-014-0063-x

contribute around 62 kg ha-1 to grain yield (Kirkeg-

aard et al. 2007). Root traits considered to be of benefit

are diverse and range from faster elongation (White

and Kirkegaard 2010), branching (Manschadi et al.

2008; Christopher et al. 2013), and penetration ability

(Ray et al. 1996; Babu et al. 2001), but there is also

evidence that root traits show consistent patterns of

genotypic variation across a range of soil physical

environments (Botwright Acuna and Wade 2012;

Ehdaie et al. 2012; Botwright Acuna and Wade 2013).

Soil physical constraints to root growth are in

particular common. In Western Australia, for example,

soils with a hardpan or traffic pan alone account for up to

40 % of agricultural land (D. Van Gool, personal

communication). As a result, root access to nitrogen and

soil water at depth in the soil profile can be limited

(Ehdaie et al. 2010). Previously, we have shown that

there is genotypic variability in wheat for the ability to

penetrate a wax layer (Botwright Acuna et al. 2007,

2012), which has also been found in a range of other

cereals, including durum wheat (Kubo et al. 2004) and

rice (Clark et al. 2002) to simulate a hardpan. Further-

more, genotypic variation has been related to field

performance in each cereal (Samson et al. 2002;

Botwright Acuna et al. 2007; Kubo et al. 2008;

Botwright Acuna et al. 2012). In wheat, geno-

type 9 environment interaction has shown to account

for a relatively large amount of variation, with geno-

types showing differential adaptation to growth in soils

that have either a sudden (e.g., associated with a

hardpan) or a gradual increase in soil hardness with

depth (Botwright Acuna and Wade 2012). Of the

genotypes assessed in our research to date, detailed

studies on root growth in soil columns with and without

wax layers and in the field have focussed on five

cultivars that we have shown to vary in growth in these

conditions. Of these, the mid-season wheat cultivar

Halberd has consistently been shown to penetrate wax

layers and is capable of root growth into soil that shows a

gradual increase in soil strength with depth. In contrast,

Cranbrook has the opposite response, with few roots

capable of penetrating wax layers and restricted root

growth in soil with a sudden increase in soil strength due

the presence of a hardpan. Fortuitously, a doubled

haploid population with Halberd and Cranbrook as the

parents has been developed (Kammholz et al. 2002),

which has been assessed for quantitative trait loci (QTL)

for a range of parameters including disease, plant height,

yield, and grain quality, but not for root traits.

To date, there have been few publications on QTL

for root architectural traits in wheat (Sharma et al.

2011; Christopher et al. 2013), with others focussing

on adaptation to nitrogen deficiency, or aluminum (Cai

et al. 2008) or boron (Jefferies et al. 2000) toxicity.

There is only one reported publication, to our knowl-

edge, on QTL for root penetration through wax layers

in durum wheat (Kubo et al. 2007), which linked QTLs

for root penetration and root DM on chromosomes 6A

and 1B, respectively. Instead, the majority of research

on root traits in crops has been undertaken in rice.

Much of these data on root genetic architecture in rice

was recently collated by meta-analysis in an effort to

colocate root QTL (Courtois et al. 2009), which

revealed that the majority of meta-QTL for root

penetration number, thickness, and indices were

located on chromosomes 1–3.

The aim of the current study was to investigate the

genetic basis for variation in root penetration ability

and associated root traits in a mapping population

derived from the Australian bread wheat cultivars

Halberd and Cranbrook in soil columns containing

wax layers grown in controlled conditions. Contrast-

ing doubled haploid lines (DHLs), selected from the

tails of the frequency distribution for hardpan pene-

tration ability in soil columns, was then evaluated for

the expression of maximum root depth in the field on

contrasting soils at Merredin, Western Australia.

Materials and methods

Plant material

The DHLs used in this study were derived by a cross

between wheat cultivars Cranbrook and Halberd, and

reported in Kammholz et al. (2002).

Experiment 1: Preliminary evaluation

of penetration of selected DHLs through thin wax

layers in controlled conditions under water deficit

The experiment had two replicates of 41 DHLs in a

randomized complete blocks design (RCBD) and was

augmented with four replicates of the Cranbrook and

Halberd parents. Wax layers (WV, 35:65 paraffin wax to

petroleum jelly, equivalent to a strength of 0.45 MPa),

150 mm in diameter and 3 mm thick, were prepared and

placed at a depth of 0.25 m in split soil columns, 0.15 m

632 Mol Breeding (2014) 34:631–642

123

in diameter and 1.0 m tall, with soil packed in layers at a

bulk density of 1.35 cm3 g-1 (Botwright Acuna and

Wade 2005; Botwright Acuna et al. 2007). The soil was a

commercial mix of loam, coarse river sand ([250 lm),

and sawdust (50:40:10), amended with 490 mg kg-1

Ca(H2PO4)2, 82 mg kg-1 slow-release fertilizer (16 –

8 - 11 ? 2MgO ? TE, 3–4 months), 300 mg kg-1

NH4NO3, and 325 mg kg-1 CaSO4�2H2O. Seeds were

pre-germinated at 4 �C overnight in a Petri dish lined

with moist filter paper and sown at a depth of 20 mm.

Plants were grown in a controlled environment chamber

at a 20/15 �C day/night temperature, with a 10-h day

length and 70 % RH and amended every 2 days with

50 ml nutrient solution containing 480 mg N L-1,

97 mg P L-1, and 160 mg K L-1 until 14 days after

sowing (DAS) when water was withheld. At harvest at 56

DAS, shoots were cut at the soil surface and leaf stage and

tiller number recorded. Columns were split, and roots

were washed from the soil column at depths of

0.0–0.24 m above the wax layer and below the wax

layer. The numbers of seminal and nodal root axes were

counted in each section. Root and shoot dry mass was

measured after drying in an oven at 70 �C for 24 h.

Experiment 2: root depth of selected DHLs grown

in the field in contrasting soil types

The field experiment evaluated root depth during early

reproductive growth of selected DHLs (16) shown to

vary in root penetration ability, the parents (Cranbrook

and Halberd) and check wheat cultivars (Bonnie Rock

and C18) grown on contrasting soil types known to

differ in the expression of hardpan penetration ability

(Botwright Acuna et al. 2007). Experiments were

conducted at Merredin (31�290S: 118�120E; altitude

315 m above sea level) in Western Australia in 2007 at

two sites with contrasting soil properties, described here

as a loamy sand overlying a mottled sandy clay with

ferruginous nodules (‘‘sandy duplex’’) that contain a

hardpan at a depth of about 0.2 m, and a red sandy loam

overlying a clay loam to clay (‘‘red clay’’) that did not

contain a hardpan but soil strength increases with depth.

Soil physical and chemical characteristics at the two

sites were described previously (Botwright Acuna et al.

2007; Botwright Acuna and Wade 2012).

Seeds were sown 20 mm apart in 2 m long rows

with 0.5 m row spacing on June 15, 2007 at the two

sites, with two replicates of the parents and check lines

and one replicate of each DHL in an incomplete blocks

design. Plots were fertilized with 90 kg ha-1 of urea at

seeding and top-dressed with 40 kg ha-1 at 21 and 70

DAS. Plots were kept free of weeds, pests, and

diseases. Root depth was measured by visually

examining soil cores sampled using a 67-mm-diam-

eter dormer auger within the row at 90 DAS. Soil was

sampled at the soil surface and at depths of 0.15–0.25

and 0.35–0.45 m in all plots for the measurement of

gravimetric soil water content.

Experiment 3: Penetration of roots of a DHL

population through thin wax layers in controlled

conditions

The experiment had two replicates in a randomized

complete blocks design (RCBD) and included the full

complement of 161 DHLs (each DHL was one exper-

imental unit) and three copies of the Cranbrook and

Halberd parents. Wax layers and soil columns were

prepared as described in Experiment 1. The soil was

amended with 100 mg kg-1 CaCO3, 300 mg kg-1

slow-release fertilizer (16 – 8 - 11 ? 2MgO ? TE,

3–4 months), 200 mg kg-1 CaSO4�2H2O, and

100 mg kg-1 FeSO4�7H2O. Cultural details were sim-

ilar to Experiment 1, except that plants were grown in a

controlled environment chamber at a 21/16 �C day/

night temperature under well-watered conditions and

were amended weekly with 50 ml nutrient solution. The

use of controlled conditions was to ensure that extrane-

ous variation was minimized so that only penetration

ability was expressed. At harvest at 28 DAS, shoots were

cut at the soil surface and leaf stage and tiller number

recorded. Columns were split, and roots were washed

from the soil column at depths of 0.0–0.24 m above the

wax layer and below the wax layer. The numbers of

seminal and nodal root axes were counted in each

section. Root and shoot dry mass was measured after

drying in an oven at 70 �C for 24 h.

Statistical and genetic analyses

Data were first checked for normality and homogene-

ity of error variance across environments. Residuals

plotted against fitted values revealed a random distri-

bution (data not shown) indicating there was no need

for data transformation. Variance components and

their standard errors were estimated following analysis

by the method of restricted maximum likelihood using

the SAS procedure MIXED (Littell et al. 1996).

Mol Breeding (2014) 34:631–642 633

123

Analyses were then repeated to obtain best linear

unbiased estimators (BLUEs) for subsequent QTL

mapping. Narrow-sense heritability (h2) and genotypic

coefficients of variation (GCV) were calculated from

the variance components.

QTL mapping using the DHL population

The genetic map of the Cranbrook 9 Halberd doubled

haploid population is described by Lehmensiek et al.

(2005) and subsequently updated to contain between

400 and 800 microsatellite, AFLP, DArT, morpho-

logical, and biochemical markers. QTL analysis was

undertaken for the experiment described in ‘‘Experi-

ment 2: root depth of selected DHLs grown in the field

in contrasting soil types’’ section, above, using BLUEs

and mixed linear composite interval mapping in

MultiQTL (Korol et al. 2007). Composite interval

analysis was undertaken using forward–backward

stepwise, multiple linear regression with a probability

into and out of the model of 0.05 and window size set

at 10 cM. Significant thresholds for QTL detection

were calculated for each dataset using 1,000 permu-

tations and a genome-wide error rate (a) of 0.10

(suggestive) and 0.05 (significant). Location of genetic

effects of individual QTL was identified from maps

drawn using MapChart 2.1 (www.multiqtl.com), and

95 % CI for each QTL location was obtained through

jackknifing 1,000 times in MultiQTL.

Results

Preliminary evaluation of shoot and root traits

of a subset of DHLs

A preliminary experiment (1) was undertaken with 41

DHLs, 16 of which contrasted in ability to penetrate a

wax layer in soil columns under conditions of water

deficit. The DHLs in this subset all had similar above-

ground DM of 2.8 g per plant. However, the ‘‘positive’’

group showed around twofold higher root DM and

30 % greater root length below wax layer compared

with the ‘‘negative’’ group (Supplementary Table S1).

Characterization of environments

Total rainfall from March to October at Merredin was

close to the long-term average, although May and June

were particularly dry and September wet (Supple-

mentary Table S2). Soil strength of the red clay at

anthesis increased gradually with depth, reaching a

maximum of 4 MPa at 0.6 m, while soil on the sandy

duplex site contained a distinctive hardpan of 4 MPa

at a depth of 0.15–0.25 m, with a subsequent gradual

decline in soil strength with increasing depth (data not

shown). Soil water availability was on average 13 and

6.3 % on the red clay and sandy duplex, respectively.

Hence, both soil types were sufficient or exceeded

field capacity (9.7 % for the red clay and 6.9 % for the

sandy duplex) required for plant growth.

Root depth in the field

Plants grown on the red clay were on average 10 cm

taller and had twice the above-ground DM than those

on the sandy duplex soil (Supplementary Table S3).

Above-ground DM varied from 2- to 4-fold for the

DHL progeny, but did not exceed that of the parents.

Root depth of the DHLs varied from 30 to 60 cm

(Fig. 1). DHLs identified with improved ability of roots

to penetrate the wax layer (Supplementary Table S1),

the Halberd parent, and Bonnie Rock had deep roots at

both sites (Fig. 1). In contrast, DHLs with less ability to

penetrate the wax layer in soil columns (Supplementary

Table S1), the Cranbrook parent, and C18 produced

Root depth on red clay (cm)

20 30 40 50 60

Roo

t dep

th o

n sa

ndy

dupl

ex (

cm)

20

30

40

50

60

BRC18

CBK

Hal

D103

D108

D114

D119

D13

D22

D23

D29D38

D5

D64

D67

D71D74

D91

D9

Fig. 1 Root depth of selected doubled haploid lines from the

Cranbrook 9 Halberd mapping population, the parents, and

controls (C18 and Bonnie Rock) on the red clay and sandy

duplex soils at Merredin at 90 DAS. DHLs in the upper right

quadrant had superior root penetration ability in the pot

experiment shown in Table 1

634 Mol Breeding (2014) 34:631–642

123

deep roots on the sandy duplex but not the red clay

(Fig. 1). No DHLs had shallow roots in both soil types,

or deep roots in the red clay combined with shallow

roots on the sandy duplex (Fig. 1). Individual DHLs

worth reporting include D108 and D29, which had the

deepest roots on the sandy duplex and red clay,

respectively, while D23 performed well at both sites.

Line D22 tended to have the shallowest roots at both

sites and was equal to or less than the Cranbrook parent.

Phenotypic characterization of shoot and root traits

of the DH population

In Experiment 3, Halberd produced fewer yet wider and

longer leaves to produce greater shoot DM than

Cranbrook (Supplementary Table S4). There was no

difference in the number of tillers or shoot height of the

parents. However, all shoot traits differed significantly

among DH lines (Supplementary Table S4). The mid-

DH mean deviation was significant (P \ 0.05) for shoot

height, leaf length, and shoot DM, suggesting the

potential for additive 9 additive epistasis for these

traits in the DH population. Genetic variation for total

shoot DM and number of tillers was around twice that of

the other variants or traits (Table 1). The non-genetic

variance for all traits was relatively small as few

environments were sampled. Narrow-sense heritabilitys

were subsequently moderate to large for all shoot traits.

For root traits above the wax layer, Halberd

produced longer and greater nodal roots than Cran-

brook, which also had shorter seminal roots. Cranbrook

had a higher proportion of seminal root axes and total

root DM above the wax layer than Halberd, consistent

with poorer penetration ability (Table 2). Few roots of

Cranbrook penetrated the wax layer hence means for all

root traits below the wax layer favoured Halberd. Total

root DM was greater in Halberd than Cranbrook, and

there was no difference in the root/shoot ratio between

the parents (Table 1). All root traits differed signifi-

cantly among DH lines (Table 1). The mid-DH mean

deviation was not significant (P [ 0.05) for all traits

with the exception of root DM above the wax layer,

suggesting little additive-based epistasis for these traits

in the DH population (Table 1). Distributions of

Table 1 Variance components, heritability’s and genotypic coefficients of variances (GCVs, %) for shoot and root traits shown in

Tables 2 and 3

Trait rG2 ± se rG9E

2 ± se rRes2 ± se hLM

2 hSP2 GCV (%)

Shoots

Shoot height (cm) 1.0 ± 0.14 0.24 ± 0.03 0.02 ± 0.01 0.89 0.68 0.83

Leaf 3 length (cm) 6.04 ± 0.91 1.71 ± 0.60 0.68 ± 0.22 0.84 0.72 8.3

Leaf 3 width (cm) 0.009 ± 0.001 0.001 ± 0.0003 0.003 ± 0.0004 0.83 0.70 9.0

Number of tillers 1.94 ± 0.35 0.07 ± 0.02 1.49 ± 0.37 0.71 0.55 17.6

Total shoot DM (g) 0.015 ± 0.001 0.0002 ± 0.0002 0.003 ± 0.0002 0.90 0.82 20.9

Above wax layer

Seminal length (cm) 54.1 ± 9.9 33.5 ± 14.1 13.0 ± 2.9 0.70 0.54 12.2

Nodal axes 9.4 ± 1.4 0.8 ± 0.1 2.7 ± 1.2 0.84 0.73 30.6

Nodal length (cm) 31.8 ± 4.9 11.5 ± 3.4 14.9 ± 1.8 0.71 0.55 19.4

Root DM (g) 0.005 ± 0.0006 0.001 ± 0.0003 0.001 ± 0.0001 0.84 0.72 20.2

% Root DM 0.0041 ± 0.0009 0.0044 ± 0.0017 0.0014 ± 0.0004 0.60 0.43 6.9

Below wax layer

Seminal axes 0.63 ± 0.11 0.24 ± 0.12 0.23 ± 0.04 0.73 0.57 72.1

Seminal length (cm) 87 ± 22 51 ± 23 91 ± 19 0.55 0.38 84.7

Root DM (g) 0.0009 ± 0.0002 0.0008 ± 0.0003 0.0002 ± 0.0001 0.64 0.48 102

% Root DM 0.0041 ± 0.001 0.0044 ± 0.0017 0.0014 ± 0.0004 0.60 0.43 90.3

Total root DM (g) 0.007 ± 0.0009 0.002 ± 0.0003 0.001 ± 0.0003 0.83 0.71 23.9

Root/shoot ratio 0.003 ± 0.0004 0.0006 ± 0.0001 0.0001 ± 0.00002 0.89 0.81 14.2

All variance component and heritability estimates were significantly different from zero at P = 0.05. Data are from Experiment 3

LM line mean, SP single plant

Mol Breeding (2014) 34:631–642 635

123

genotypic means for selected root traits were approx-

imately Gaussian for nodal root number and DM above

the wax layer, but showed some evidence of bimodality

for seminal root number and DM below the wax layer.

Given the higher heritabilities under these controlled

conditions, these distributions suggest that more than

one gene affects the genetic expression of each trait

(Supplementary Figure S5). Further validation is

however required across a range of field environments.

For root traits above the wax layer, genetic variation

was the greatest for the number of nodal axes and fairly

large for nodal length and root DM. Despite this, the

proportion of roots above the wax layer was the smallest

of all root traits above the wax layer (Table 2). In contrast,

genetic variation was very large for all root traits below

the wax layer. However, the narrow-sense heritability

tended to be higher for root traits above the wax layer, due

to smaller non-genetic variances compared with those

below the wax layer. Consequently, genotype 9 envi-

ronment interactions and potential for sampling variation

are more likely for root traits below the wax layer.

Genetic mapping of shoot and root traits

Repeatable genetic variance contributed to the iden-

tification of significant QTLs for root and shoot traits.

For shoot traits, a total of five and eight QTLs were

identified for number of tillers and above-ground DM,

respectively (Table 3). The 2DS and 5AL QTLs for

number of tillers and above-ground DM, respectively,

mapped to genomic locations associated with plant

height and days to anthesis (Fig. 2). Alleles for

improved above-ground DM were transmitted by the

Halberd parent, with gain of function associated with

QTLs for this trait on chromosomes 2DL, 3DS, and

6BS.

For roots, 5–10 QTLs were identified for each of the

seminal root DM above the wax layer, seminal root

DM below the wax layer, and total root DM traits

(Table 3). Three QTLs associated with a gain in

function through the Halberd parent were identified on

group 3 and two QTLs with the group 4 and group 7

chromosomes. In contrast, six QTLs associated with a

loss in function through the Cranbrook parent were

identified on the group 1, two on group 2, and three on

group 3 chromosomes. The 2BL QTL for seminal DM

below the wax layer mapped to the genomic location

for plant height and days to anthesis, while the 7AL

QTL for total root DM similarly mapped to the region

associated with days to anthesis on that chromosome.

Eight and three QTLs for ratios for seminal root

DM below/above and the root/shoot ratio,

Table 2 Minima, maxima, and means for DHL progeny and parent means for root traits (note that seminal no above the wax layer

was invariant)

Parameter Above wax layer Below wax layer Total

root

DM (g)

Root/

shoot

Seminal

length

(cm)

%

Seminal

axes

Nodal

axes

Nodal

length

(cm)

Root

DM

(g)

%

Total

root

DM

Seminal

axes

Seminal

length

(cm)

Root

DM

(g)

%

Total

root

DM

Progeny

Minimum 37 0.21 3 3 0.22 0.70 0.0 0 0.00 0.00 0.22 0.243

Maximum 75 0.63 19 42 0.65 1.00 3.5 34 0.14 0.30 0.65 0.528

l.s.d. 13 0.10 4 8 0.06 0.15 1.4 12 0.06 0.15 0.05 0.058

Mean 60 0.36 10 29 0.35 0.93 1.1 11 0.03 0.07 0.38 0.380

Parents

Cranbrook 56 0.40 9 19 0.38 1.00 0.3 3 0.00 0.00 0.38 0.375

Halberd 75 0.27 14 34 0.39 0.84 2.3 25 0.07 0.16 0.47 0.357

l.s.d. 13 0.08 3 7 ns 0.14 1.2 12 0.05 0.14 0.05 ns

Midparent

versus

progeny

ns ns ns ns * ns ns ns ns ns ns ns

Data are from Experiment 3

636 Mol Breeding (2014) 34:631–642

123

respectively, were identified (Fig. 2). For the ratio

of seminal root DM below/above, three QTLs on

1BS, 4AL, and 7BL were associated with a gain in

function through the Halberd parent. QTL for the

root/shoot ratios was all associated with the Cran-

brook parent.

Discussion

QTL and root traits

Across all traits, genetic control was largely complex

reflected in numerous QTL (53 in total) of small

Table 3 Estimated genetic (additive) effects, nearest linked molecular marker and chromosomal location (with corresponding 95 %

confidence intervals) for shoot and root trait QTL measured on random progeny from the Cranbrook/Halberd wheat population

Population/chromosome LOD Nearest

marker

QTL position (cM)A a-Genetic

effect (�C)

Percent

variance (rA2)

Shoot dry weight

2DL 3.4 wmc18 110 (107–125) 0.053 5

3DS 3.1 gwm456 64 (49–71) 0.047 4

4BS 3.4 Rht-B1 48 (31–63) -0.044 6

4DS 4.5 wmc48b 7 (2–21) -0.060 7

5AL 3.1 psr426 133 (128–143) -0.046 4

5BS 5.4 gwm234 22 (13–31) -0.067 9

7AS 5.1 wmc247 93 (84–104) -0.071 9

Above-wax seminal dry weight

1BS 4.6 Glu-B3 4 (0–14) -0.038 7

1DS 4.5 scuM02 54 (48–71) -0.042 8

3AL 3.8 RGA61.2 96 (82–108) 0.036 5

3BS 3.7 cdo395 44 (38–47) -0.020 4

3DL 5.4 gwm3 121 (112–125) 0.049 12

5AL 3.2 abg397 48 (24–53) -0.029 6

7DS 4.8 psr1606 4 (0–7) 0.047 9

Below-wax seminal dry weight

1BS 3.8 gwm666 16 (11–29) -0.019 5

1DS 4.5 wPt-0077 92 (73–99) -0.026 8

2BS 4.6 psr596 84 (72–97) -0.027 9

3BS 3.3 cdo395 54 (34–67) -0.016 5

4AL 5.3 wmc262 136 (133–141) 0.032 12

Total root weight

1BS 4.8 Glu-B3 0 (0–14) -0.026 6

1DS 9.1 wPt-0077 87 (77–93) -0.061 12

2AS 4.4 wPt-0921 54 (49–65) 0.035 5

2BS 8.8 gwm388 101 (94–105) -0.056 11

3BS 7.8 wPt-8238 49 (45–54) -0.050 9

3DL 6.1 gwm3 152 (136–157) 0.053 9

4AL 3.7 wmc262 136 (121–144) 0.025 4

6DL 3.4 stm537acag2 96 (72–111) -0.033 5

7AS 4.8 wmc83 87 (78–94) -0.042 6

7DS 4.5 cdo1400 32 (21–40) 0.041 6

Positive additive effects indicate that the Cranbrook allele with ‘‘a’’ the additive effect estimated as one-half the difference in

homozygotes carrying either parental allele. Data are from Experiment 3A Distance from the tip of the short arm of the chromosome

Mol Breeding (2014) 34:631–642 637

123

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638 Mol Breeding (2014) 34:631–642

123

genetic effect (LOD scores of 5.2–9.1) located across

most chromosomes. Of these QTLs, 29 were associ-

ated with root traits, around 37 % of which were from

the gain of function parent Halberd with key traits for

root DM below the wax layer located on chromosomes

2D, 4A, 6B, and 7B. Of these, QTL for seminal root

DM below the wax layer was colocated with shoot DM

on both the 2D and 6B chromosomes. Despite a large

number of small QTL, the results can be potentially

used to explore allelic diversity in root traits for

hardpan penetration.

In wheat, a range of root traits have been evaluated for

their impact on crop growth and development, e.g., as

reviewed by de Dorlodot et al. (2007). Until now, there

was only one report of QTL for the ability of roots to

penetrate a wax layer (Kubo et al. 2007), where QTL was

located for root DM on 1B, and penetrating root number

and RP index on 6A. However, only total root DM from

Cranbrook on 1B was collocated with the QTL reported

by Kubo et al. (2007). This location is significant: Sharma

et al. (2011) have reported QTL for root biomass and

length on the T1BL.1RS chromosome in bread wheat,

which is collocated with QTL for total root DM, seminal

root DM above and below the wax layer and seminal root

ratio. The 1RS translocation has been associated with

increased root weight under well-watered conditions

(Ehdaie et al. 2012), although the observed response

varies with the intensity of water deficit.

Other reports for QTL for other root traits of wheat

have generally focussed on more easily accessed

traits from seedling screens such as root number and

angle (Sanguineti et al. 2007; Hamada et al. 2012;

Christopher et al. 2013; Liu et al. 2013). While our

QTL analysis has focussed on the integrative trait of

root DM, at least one of the root number QTL on

chromosome 4A reported by Christopher et al. (2013)

was collocated with QTL for total root DM and

seminal root DM below the wax layer. Similarly, a

root number QTL reported by Ren et al. (2012) on

chromosome 3B was collocated with a QTL for

seminal root DM above the wax layer.

Reanalysis of Kubo et al. (2007) shows some

consistency with their reports of QTL for number of

penetrating roots in durum wheat on chromosome 6A.

While we mapped the number of penetrating roots

below the wax layer, there were little QTL and none

on chromosome 6A. It is perhaps unlikely this level

of similarity would be identified, given difference not

only in DH population but also species. In contrast,

root traits in rice have been extensively studied, with

some 28 traits collated from a range of publications in

a meta-analysis (Courtois et al. 2009). Of these, key

root penetration traits and their location on the rice

genome include penetrating root number on chromo-

some 2 (Ray et al. 1996; Ali et al. 2000; Price et al.

2000; Zheng et al. 2000), root DM on chromosome 5

(numerous citations), and a few reports for penetrat-

ing root DM on chromosomes 9 and 12 (Zhang et al.

2001; Nguyen et al. 2004). Comparative genomics

between wheat and rice reveals a reasonable degree

of synteny between the rice chromosomes 2 and 5

(plus 10) with wheat chromosomes 6 and 1, respec-

tively (Sorrells et al. 2003). Of these, total root DM

from the Cranbrook parent was located on chromo-

somes 1B and D, which may be comparative to the

root DM QTLs for rice on chromosome 5. For

example, there have been numerous reports of QTLs

for penetrating root number of rice on chromosome 2,

which has a high degree of synteny with wheat

chromosome 6. While this discussion highlights the

opportunities for trait and gene identification when

crossing among donors and species, additional evi-

dence is required.

QTL collocated with other major genes

The Cranbrook 9 Halberd mapping population was

developed by Kammholz et al. (2002) and is polymor-

phic for traits including plant height, tolerance to boron

and aluminum, and rust reaction. Halberd, for example,

is mostly tolerant to boron while Cranbrook is sensitive.

Jefferies et al. (2000) have reported QTL for whole

shoot boron concentration at the top of chromosome 7B

and another on chromosome 7D for leaf score symp-

tom. In our study, small QTL for seminal root DM

below the wax layer and seminal root ratio was reported

for the positive (Halberd) allele but at the distal end of

chromosome 7B. Two QTLs for total root DM and

seminal root DM above the wax layer were collocated

with the leaf score symptom QTL on chromosome 7D,

which is thought to be involved in the translocation of

boron in leaf tissue (Jefferies et al. 2000). The results

presented here point to the potential involvement of the

root system in this response, but finer mapping and

more detailed study on root physiology in relation to

boron toxicity would be required.

Mol Breeding (2014) 34:631–642 639

123

Trait heritability

For shoot traits, the numbers of leaves and tillers for

the Cranbrook and Halberd parents were similar to our

previous observations under well-watered conditions

(Botwright Acuna et al. 2007, 2012). An exception

was above-ground DM, which was larger in the

Halberd parent and was significantly different from

the midparent–progeny mean, indicating potential for

epistasis. In contrast, the lack of difference in leaf

width between the midparent and progeny is consis-

tent with Rebetzke et al. (2001). There was high

heritability for most seedling shoot components,

except for number of tillers and plant height. However,

tiller number and above-ground DM both had low

repeatability.

For root traits, phenotypic relationships and heri-

tability were complex. In the preliminary experiment

in soil columns, DHLs were identified as either

positive or negative for root traits associated with

DM distribution below the wax layer and root length;

yet, there was no difference in these traits between the

parents. This contrasts with our previous observations

where typically few roots of Cranbrook penetrate the

wax layer (Botwright Acuna et al. 2007, 2012). The

experiment was undertaken in water-deficit conditions

for a longer period of time, and the wax layer may have

been damaged. In the experiment with the full set of

DHLs under well-watered conditions, root traits

generally favoured the Halberd parent while few to

no Cranbrook roots penetrated the wax layer, which is

consistent with our previous observations (Botwright

Acuna et al. 2007, 2012). As a result, seminal root

number below the wax layer showed a bimodal

distribution (Fig. 1). Midparent–progeny means were

the same for all traits with the exception of root DM

above the wax layer, which possibly indicated epi-

static inheritance for this trait. Variance components

showed a relatively high contribution of geno-

type 9 environment interaction, albeit in the con-

trolled environments used in these experiments,

compared with genotypic variance for all root traits.

This is not surprising given the plasticity of root

systems (Ehdaie et al. 2012) and is consistent with our

own and others field experiments. For example, we

have reported significant genotype 9 environment

interaction for root depth of wheat cultivars across

environments with contrasting soil characteristics

including a hardpan (Botwright Acuna and Wade

2012, 2013). In the field data presented herein, there

was a very promising relationship between DHLs

varying for root penetration ability through wax layers

under water deficit in pots with field response. Both the

DHLs selected for penetration ability in pots and the

Halberd parent all had deeper roots in the clay soil,

while Cranbrook and DHLs with relatively poor

penetration ability in soil columns with few roots or

less root DM under the wax layer tended to have

deeper roots in the sandy duplex. The next stage in this

research would be to evaluate DHLs identified with

transgressive segregation from the larger DHL popu-

lation in a range of field environments such as those

identified in (Botwright Acuna and Wade 2012, 2013),

to establish whether this relationship is maintained.

Heritabilities were greater for above-ground traits

than below-ground ones, especially when expressed

on a single plant basis. There were generally high

GCVs that indicated large genetic variation for most

root traits. The majority of root traits showed trans-

gressive segregation, as seen in the frequency distri-

bution for these traits and the QTL, where both parents

contributed positive and negative alleles. Evidence for

transgressive segregation suggests potential for com-

bining positive alleles from either parent together in

developing improved progeny for different root traits.

Conclusions

This paper represents a first attempt to dissect traits for

hardpan penetration by wheat roots. While the larger

QTL accounted for only about 10 % of the variation,

this was used to explore allelic diversity. The traits

were genetically complex and many QTLs were

identified for related traits indicating different mech-

anisms or contributions for the trait of penetration. An

improved understanding of the response of root traits

to environmental constraints may underpin the iden-

tification of QTL with greater accountability. In

addition to validation in a range of field environments,

this may require better germplasm or a more refined

screen that is better targeted to the particular types of

constraints or root traits, or both. Further, trait

dissection is required for this complex trait, whose

expression is modified through time and the progres-

sion of water deficit.

640 Mol Breeding (2014) 34:631–642

123

Acknowledgments We thank N. Song Ai and L.W. Bell for

their assistance; M. Zhou for his helpful comments on the paper;

the University of Western Australia for access to controlled

environment rooms; and the Department of Agriculture and

Food in Western Australia for field sites. This project was

supported by the Australian Grains Research and Development

Corporation (UWA00090).

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