Published April 13, 2017
Research
Pea Phenology: Crop Potential
in a Warming Environment
Shaoming Huang, Krishna K. Gali, Bunyamin Tar’an,
Thomas D. Warkentin,* and Rosalind A. Bueckert
Abstract
One hundred and seven recombinant inbred
lines (RILs) were developed from the cross of
field pea (Pisum sativum L.) cultivars CDC Centennial ´ CDC Sage with the objectives of evaluating phenology and yield components, and to
map the quantitative trait loci (QTLs) responsible
for these traits. Experiments were conducted in
2013 using normal seeding date at Saskatoon
and Rosthern in Saskatchewan, and in 2014
using both normal and late seeding. The late
seeding date was used to expose the plots to a
more heat stressful environment, analogous to
that experienced in warmer regions of the North
American prairies. Days to flowering termination (DTFT) was positively correlated with final
seed yield under both normal and late seeding
conditions. Among the yield components, pod
number (PN) was most positively associated
with seed yield, followed by thousand seed
weight (TSW) and seed number per pod (SNPP).
A genetic linkage map consisting of 1024 loci
with a total coverage of 1702 cM was developed
using SNP markers. Ten QTLs were found consistent over more than one environment, five
for flowering traits and five for yield component
traits. A stable QTL at Linkage Group 6b for
days to flowering was detected over four environments. The QTLs for flowering duration, TSW
and reproductive node number were different
between normal and late seeding, which implies
different mechanisms were involved under the
contrasting environments.
Department of Plant Sciences, University of Saskatchewan, Saskatoon,
SK, Canada. Received 3 Dec. 2016. Accepted 2 Feb. 2017. *Corresponding author ([email protected]). Assigned to Associate Editor Heathcliffe Riday.
Abbreviations: cM, centi-Morgan; DOF, duration of flowering; DTF,
days to flowering; DTFT, days to flowering termination; GBS, genotyping-by-sequencing; H 2, broad-sense heritability; LG, linkage group;
LOD, logarithm of odds ratio; MAS, marker assisted selection; PN, pod
number; QTL, quantitative trait loci; RCBD, randomized complete
block design; RIL, recombinant inbred line; RNN, reproductive node
number; SNP, single nucleotide polymorphism; SNPP, seed number
per pod; TSW, thousand seed weight.
F
ield pea is an annual herbaceous plant, and is a cool season
crop widely grown in the temperate zones of the world. The
annual growing area and production of field pea in the world
ranges from 6 to 7 million ha and 10 to 13 Tg, respectively, which
makes it the fourth most important pulse crop after dry bean
(Phaseolus vulgaris L.), chickpea (Cicer arietinum L.), and cowpea
[Vigna unguiculata (L.) Walp.] in terms of area and third in terms
of production after dry bean and chickpea, globally (FAOSTAT,
2014). Historically, pea has been grown in cool, temperate regions,
but its production has been expanding in the past two decades in
the drier and warmer regions of the Canadian and U.S. prairies.
Heat stress is one of the major abiotic stresses limiting the
world’s agricultural production. Lobell and Field (2007) reported
an 8.3% yield reduction in maize, in response to every 1°C rise
in temperature for the period 1961 to 2002. Similarly in wheat,
every 1°C above the optimal temperature could shorten the flowering and grain-filling duration by 5%, respectively, thus reducing grain yield accordingly (Lawlor and Mitchell, 2000). For
every 1°C increase in the growing season temperature, 53 kg
Published in Crop Sci. 57:1–12 (2017).
doi: 10.2135/cropsci2016.12.0974
© Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA
This is an open access article distributed under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
crop science, vol. 57, may– june 2017 www.crops.org1
ha–1 yield loss occurred in chickpea in India (Chanders
et al., 2008). Field pea production in the Mediterranean
region decreased by 600 kg ha–1 as a response to every 1°C
increase of average temperature during flowering (Ridge
and Pye, 1985). As pea has poorer heat tolerance than
chickpea and lentil (Siddique, 1999), lowered grain yield
was reported in Australia when the maximum temperature exceeded 25°C during flowering (Sadras et al., 2012).
The world’s land annual temperature has increased 1.6°C
since the 19th century (IPCC, 2014), and is expected to
rise because of the continued emission of greenhouse
gases. Therefore, there is an urgent need to breed pea cultivars with better heat tolerance.
Depending on the intensity and duration, high temperature affects pea’s reproductive organs in different
ways. A mild heat stress (25–30°C) did not lead to an
immediate abscission of reproductive organs in pea, but
did result in abortion of organs carried by the upper nodes
(Guilioni et al., 1997). Mild heat stress resulted in reduced
seed number due to decreased growth rate from flowering
to seed-filling (Guilioni et al., 2003). A short period of
extreme high temperature at anthesis can cause detrimental and direct damage to yield. Jiang et al. (2015) found
that when pea plants at anthesis were exposed to 36/18°C
day/night for 7 d in a growth chamber, the percentage
pollen germination, pollen tube length, pod length, seed
number per pod, and the seed/ovule ratio dropped dramatically compared to pea exposed to normal conditions of 24/18°C. However, the above research was not
conducted under field conditions where temperature and
environmental conditions are natural and realistic. One
well-accepted and economical approach that was used in
this study was to grow crops at different seeding dates in
anticipation of receiving heat stress at different stages in
the plant’s cycle, and this approach has been used in several crop breeding programs including pea (French, 1990),
chickpea (Krishnamurthy et al., 2011), and summer Brassica (Morrison and Stewart, 2002).
Visual selection for physiological traits linked to plant
response to high temperature, empirical selection for yield
and more recently marker-assisted selection (MAS) are
the principal selection methods used to improve heat tolerance through breeding (Howarth, 2005). Advances in
QTL mapping of heat tolerance have been made in major
crops including wheat (Triticum aestivum L.) (Kumar et al.,
2010; Mason et al., 2010; Pinto et al., 2010), rice (Oryza
sativa L.) ( Jagadish et al., 2010; Ye et al., 2012) and maize
(Zea mays L.) (Ottaviano and Gorla, 1991; Frova and SariGorla, 1994). Among legume crops, similar research has
only been reported in cowpea (Lucas et al., 2013). Any
QTLs related to heat tolerance have not been reported
in pea to date. However, advances have been made in
mapping QTLs for resistance to abiotic stresses in pea,
including for drought (Iglesias-Garcia et al., 2015), frost
2
(Lejeune-Hénaut et al., 2008; Klein et al., 2014), and
salinity (Leonforte et al., 2013). With the use of next generation sequencing technology and high-density genetic
maps of pea (e.g.,, Sindhu et al., 2014; Leonforte et al.,
2013; Duarte et al., 2014), identification of QTLs linked
to heat tolerance traits will become possible.
The overall objectives of this research were to evaluate flowering, yield components, and grain yield in a field
pea recombinant inbred population expected to segregate
for tolerance to heat at flowering, identify potential heat
tolerance traits, and identify the genomic regions associated with these traits through QTL analysis.
Material and Methods
Plant Materials
A population of 107 recombinant inbred lines (RILs), which
was named as PR-11, was derived from a cross between CDC
Centennial ´ CDC Sage made in 2008 at the Crop Development Centre, University of Saskatchewan, Canada. These
two cultivars demonstrated variation in flowering time, and
CDC Centennial produced greater yield in warm seasons as
observed in preliminary experiments. CDC Centennial which
was developed by the Crop Development Centre, University of
Saskatchewan, has white flowers, seeds with yellow cotyledons,
a relatively large seed weight (260 g 1000 seed–1), and high yield
(Warkentin et al., 2007). CDC Sage was also developed by the
Crop Development Centre has white flowers, seeds with green
cotyledons, medium seed weight (197 g 1000 seed–1), and high
yield (Warkentin et al., 2006). The F2 plants were advanced to
F7 by single seed descent in the Agriculture Greenhouse, University of Saskatchewan to develop the RILs.
Field Experiments
All 107 RILs were planted in 2 yr (2013 and 2014) for field
evaluation. In 2013, the field trials were conducted at Saskatoon (52°12 N, 106°63 W; Dark Brown Chernozem) and
Rosthern (52°66 N, 106°33 W; Orthic Black Chernozem)
in Saskatchewan, Canada, using normal seeding dates for
field pea in Saskatchewan, that is, 13 May for Saskatoon, and
15 May for Rosthern. In 2014, the field trial was conducted
only at Saskatoon but with two seeding dates, that is, normal
and late. The normal seeding date was 14 May, and the late
seeding date was 4 June. The late seeding date was expected
to expose the RILs to greater heat stress during the flowering stage, which occurred in mid-July and early August when
maximum daytime temperature was likely to be the greatest
(highest maximum temperature could rise up to 35°C). The
RILs were planted in a randomized complete block design,
with two blocks per seeding date and location. Each RIL was
planted in a 1 by 1 m microplot with three rows. Weeds were
controlled using recommended best management practices for
field pea production in Saskatchewan.
Phenotyping
Once flowering started, every second day plots were evaluated for flowering related traits: days to flowering (DTF), days
to flowering termination (DTFT), and duration of flowering
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crop science, vol. 57, may– june 2017
(DOF). For DTF 50% of plants within the microplot had an
open flower. For DTFT, 50% of plants within the microplot
reached flower termination. Then DOF was calculated as
DTFT minus DTF (Maurer et al., 1966).
When RILs reached maturity, two representative plants for
each micro-plot were hand harvested for the evaluation of yield
component traits. These component traits were reproductive
node number (RNN), pod number (PN), seed number per pod
(SNPP), and thousand seed weight (TSW) from the main stem
and the mean from these two subsampled plants was used. In
addition, the grain yield of each micro-plot was measured after
the plot was combined at harvest maturity.
Genotyping and Single Nucleotide
Polymorphism Allele Calling
The mapping was done using an Illumina 1536 GoldenGate
array (Sindhu et al., 2014) and genotyping-by-sequencing
(GBS; van Oeveren et al., 2011), separately, in which 106 RILs
were genotyped through GoldenGate and 90 RILs were genotyped through GBS. The final linkage map of PR-11 was based
on the mapping information of the common 90 RILs.
For SNP array genotyping, the standard GoldenGate Protocol was followed, (http://www.illumina.com/technology/
goldengate_genotyping_assay.ilmn) where arrays were scanned
using an Illumina Hiscan, and Genomestudio software was
used for calling SNP alleles.
For GBS genotyping, DNA of each RIL was quantified
using picogreen and DNA concentrations were normalized to
20 ng L –1. DNA of each individual was digested with restriction enzymes PstI and MspI, then ligated with unique four to
eight base pair barcode adapters. Ligated samples were pooled
and PCR-amplified in a single tube, producing a single library
from 96 samples. Paired-end sequencing of individual libraries was carried out on one lane each of an Illumina Hiseq
sequencer using V4 sequencing chemistry at Sickkids Hospital,
Toronto. The raw Illumina reads were assigned to individual
RILs based on the four to eight base pair adapters ligated to
individual DNA. Following deconvolution, barcode sequences
were removed from the sequence and reads were trimmed for
quality with Trimmomatic-0.33. Filtered reads were mapped
to the draft genome assembly provided through the pea genome
sequencing consortium (Madoui et al. 2016) using Bowtie22.2.5. Samtools-1.1 and BCFtools-1.1 were utilized to declare
variants and output them in variant call format.
Statistical Analysis
The Shapiro–Wilk test of residuals’ normality was conducted
for each collected trait (P  0.05). ANOVA was conducted via
SAS Mixed Procedure (SAS Institute, 2015), in which genotype, environment, and their interaction were considered as
fixed factors, and block was considered as random. The broadsense heritability (H 2) was estimated for four environments
with all factors considered random (genotype, environment,
and the genotype ´ environment interaction) via SAS Proc
Mixed procedure, using the formula:
H 2 = s2g / s2p = s2g / êés2g + s2g´e / m + s2residual / (m´b)úù
ë
û
crop science, vol. 57, may– june 2017 (Falconer and Mackay, 1996) where s2g , s2g´e , and s2residual
are the variance of genotype, genotype ´ environment interaction, and the residual, respectively; m is the total number of
environments number, which was four in this study, and b is
the block number within each environment, which was two
in this study.
Pearson’s correlation among the eight measured traits at
normal and late seeding was determined through the SAS Proc
Corr procedure. In order to discover the relative contribution
of four yield component traits (RNN, PN, SNPP, and TSW) to
the main-stem seed yield at normal and late seeding, a path coefficient analysis (multiplying the ordinary regression coefficient
by the standard deviation of the corresponding variable) was
conducted using the SAS Proc Calis procedure. Similar to the
correlation analyses above, the average of each RIL evaluated at
2013 and 2014 normal seeding dates was used to represent the
final RIL value under the normal seeding dates. Likewise, the
mean of each RIL over two blocks at the 2014 late seeding was
used to represent the final RIL value at late seeding.
Linkage Map Construction
and Quantitative Trait Loci Analysis
For linkage mapping, all the polymorphic SNPs identified from
both genotyping assays were filtered for missing values (<15%),
allele frequency based on Chi-square values at P = 0.05, and
used for linkage analysis using MstMap program using a LOD
of 9.0 and Kosambi mapping function (Wu et al., 2008). The
detection of QTLs related to flowering and yield components
at each environment was conducted separately. For each QTL
analysis, a preliminary detection of putative QTLs with a LOD
value >3 was conducted by an interval mapping method (a
single-QTL model) using MapQTL 6 (Kyazma B.V., Wageningen, the Netherlands) (Van Ooijen, 2009). Secondly, 1000
permutations were tested in order to determine the threshold
of LOD at which QTLs were significantly correlated with the
trait. Finally, for any given linkage group (LG) with more than
one LOD peak above the threshold value, markers having the
highest LOD score on that LG would be selected as cofactors,
and a multiple-QTLs model mapping (which is equivalent to
composite interval mapping) was rerun to identify the true
QTLs for the traits of interest.
Results
Phenotypic Analysis of the Measured Traits
The mean daily temperature during the vegetative stage
ranged from 14.7°C (2014 Saskatoon normal seeding) to
16.0°C (2013 Rosthern normal seeding), and the mean
temperature during flowering varied from 16.8 to 19.3°C
(Fig 1.) During the flowering stage, RILs in 2014 Saskatoon late-seeding experiment received the most heat stress
because they experienced both the highest average daily
maximum temperature (25.5°C) and highest number
of days with maximum temperature exceeding 27°C (6
days), followed by RILs in 2013 Saskatoon normal seeding
(24.5°C, 3 d), 2014 Saskatoon normal seeding (23.9°C, 2
d), and 2013 Rosthern normal seeding (22.7°C, 2 d).
www.crops.org3
Fig. 1. Daily temperature and precipitation during the growing season in each experimental trial. The red line on the x axis in each panel
is the flowering period for the recombinant inbred lines (RILs), on average. The daily precipitation data of 2013 Rosthern is not shown
due to a lack of official source, the monthly precipitation at this location from May to August was 9, 154, 4, and 1 mm, respectively (data
retrieved from 2013 rainfall summary, Ministry of Agriculture, Government of Saskatchewan). The daylength was above 16 h at each
experimental site when plants started to flower.
The average value of days to flowering (DTF), days to
flowering termination (DTFT) and duration of flowering
(DOF) among RILs in the 2014 late-seeding experiment
were lower than the average of RILs in the normal seeding experiments (Table 1). Among the yield components
and final plot yield, only the seed number per pod (SNPP)
was significantly different between the 2014 late seeding
experiment compared to the three normal seeding experiments. No difference in PN was observed across the four
experimental trials. For RNN, TSW, and plot grain yield,
the variation over the four trails were more attributed to
the two growing seasons (2013, 2014) rather than the
locations (Saskatoon, Rosthern) or seeding dates (normal,
late). Although when only comparing the average difference between 2014 normal and late seeding experiments
via t test, SNNP (t value = –5.98, P < 0.0001), TSW (t
value = 5.29, P < 0.0001), and plot yield (t value = 5.65, P
< 0.0001) demonstrated significant differences.
Under normal seeding date environments, CDC Centennial began to flower 2 d later than CDC Sage with a
longer duration of flowering in two of three environments.
CDC Centennial also had greater PN on the main-stem
4
and grain yield in two of three environments, as well as
higher TSW than CDC Sage, whereas CDC Centennial had a lower SNPP than CDC Sage. No difference in
RNN was observed in the parental lines. At late seeding,
CDC Centennial had greater TSW but fewer SNPP than
CDC Sage, whereas no substantial difference was observed
in DTF, DOF, PN, or grain yield between them (Table 1).
Broad-Sense Heritability
For each trait, except RNN and SNPP, the genetic variance
was much larger than the variance of genotype ´ environment (Table 2). A significant genetic variation among RILs
existed for all measured traits (ANOVA results not shown).
The broad-sense heritability (H2) ranged from 0.38 to 0.90
(Table 2). Among the flowering traits, DTF had the highest heritability (H2 = 0.90), followed by DTFT (H2 = 0.67)
and DOF (H2 = 0.48). Among the yield components, TSW
was most heritable (H2 = 0.84), RNN had the lowest heritability (H2 = 0.38), other components were moderately
heritable with their heritability around 0.6.
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crop science, vol. 57, may– june 2017
¶ Proc Mixed procedure was used to compare CDC Centennial and CDC Sage across the four experimental trials. For traits of DTFT, TSW and SNPP, different letters were given to CDC Centennial and CDC Sage as they demonstrated
significant differences.
§ Proc Mixed procedure was used to compare the means across the four experimental trials. For any given trait, the RIL mean values sharing any common letter, indicate that they do not differ.
‡ Cent, CDC Centennial; Sage, CDC Sage.
† DTF, days to flowering; DTFT, days to flowering termination; DOF, duration of flowering; RNN, reproductive node number on the main stem per plant; PN, pod number on the main stem per plant; SNPP, seed number per pod; TSW,
thousand seed weight (g per 1000 seed); Yield, plot yield (g m –2).
51.1
68.4b
17.4
5.7
6.8
5.5a
221.6b
496.6
52.4
71.2a
18.8
5.5
7.4
4.4b
280.0a
569
46.4
62.0
15.6
6.6
6.9
5.7
197.6
446.2
47.3
62.5
15.3
5.9
6.9
4.6
240.1
457.4
47.3 ± 1.6d
62.5 ± 1.5c
15.3 ± 1.91c
6.4 ± 1.0a
7.2 ± 1.4a
5.14 ± 0.6a
223.7 ± 32.7b
425.7 ± 85.3c
55.9
72.7
16.8
6.2
7.7
5.4
217.2
435.9
56.0
75.0
19.0
7.0
9.0
4.0
264.9
487.6
55.6 ± 1.7a
74.0 ± 2.9a
18.4 ± 2.8b
6.3 ± 1.0a
7.4 ± 0.7a
4.8 ± 0.7b
238.1 ± 22.5b
473.8 ± 90.7c
55.3
80.5
25.3
4.1
6.9
4.6
312.3
770.5
DTF
DTFT
DOF
RNN
PN
SNPP
TSW
Yield
50.3 ± 2.0c§
67.4 ± 3.7b
17.1 ± 3.6bc
5.4 ± 1.4b
7.0 ± 1.7a
4.7 ± 0.8b
271.4 ± 29.3a
554.0 ± 122.0b
51.0
67.0
16.0
4.8
7.1
4.2
302.9
560.5
49.5
67.5
18.0
5.1
5.7
5.6
244.6
560.5
52.5 ± 2.1b
73.5 ± 6.0a
21.0 ± 5.50a
4.5 ± 1.2c
6.7 ± 1.6a
4.9 ± 0.7ab
277.8 ± 28.7a
694.8 ± 183.1a
52.5
71.5
19.0
4.8
6.9
5.3
227.0
594.0
Sage
Cent
Cent‡
Trait†
RIL
Cent‡
Sage‡
RIL
Sage‡
RIL
Cent‡
Sage‡
RIL
Cent‡
Sage‡
Means over
4 site-years¶
2014 Saskatoon
late seeding
2014 Saskatoon
normal seeding
2013 Rosthern
normal seeding
2013 Saskatoon
normal seeding
Table 1. Data for the measured traits of parental lines and recombinant inbred lines (RILs) at each experiment in 2013 and 2014.
crop science, vol. 57, may– june 2017 Correlation and Path Coefficient Analysis
Because no significant genotype ´ environment effect
was observed in any measured trait, correlations among
these traits under normal seeding environment were based
on the averaged data of the three normal seeding environments in 2013 and 2014. The 2014 Saskatoon late seeding
data were used to calculate correlations among all eight
traits at late seeding. Some common relationships among
flowering and yield traits were found under both normal
and late seeding environments (Tables 3 and 4). The DTF
was positively correlated with DTFT (R 2 = 0.58, 0.29).
The DTFT was positively correlated with DOF (R 2 =
0.82, 0.42), RNN (R 2 = 0.24, 0.20) and yield (R 2 = 0.47,
0.29). Positive correlations were also observed between
DOF and RNN (R 2 = 0.31, 0.23), DOF and PN (R 2 =
0.18, 0.20), RNN and PN (R 2 = 0.58, 0.74), whereas negative correlations were observed between DOF and SNPP
(R 2 = –0.27, –0.21), PN and SNPP (R 2 = –0.35, –0.28).
Path analysis, also known as structural equation modelling, was conducted in order to discover the cause-andeffect relationships between yield components and seed
yield. Results revealed that PN, SNPP, and TSW had
direct positive effects on main-stem seed yield, RNN had
a direct negative effect on the main-stem seed yield at both
sowing dates (Fig. 2 and 3). These results demonstrated that
the variation of seed yield on the main stem was principally
derived from PN (r = 0.77, 0.73), followed by TSW (r =
0.54, 0.34) and SNPP (r = 0.48, 0.12), though the proportion of yield variation explained by each component was
not exactly the same between the two seeding dates. However, the main-stem yield at both seeding dates was not
significantly associated with its final plot yield, meaning
that side branches had a significant influence on plot yield,
given that all the RILs had similar germination rate and
stand establishment. However, indirect effects were not
accounted for in the model, so the residual (unaccounted
for) error would be inflated, which would diminish the
relative effect of unaccounted for branches on yield.
Genetic Mapping and Quantitative
Trait Loci Detection
Ninety of the 107 RILs and two replications of the parents CDC Centennial and CDC Sage were genotyped
using both Golden-Gate and GBS methods. A total 3920
polymorphic SNPs, of which 366 were from GoldenGate
assays, were selected for linkage analysis. The total of 13
linkage groups (LGs), which included four segments for
LGI, two segments for LGV, VI, and VII, and one segment
for LGII, III, and IV, were built and aligned with the seven
LGs according to the field pea consensus map reported by
Sindhu et al, (2014). The total distance covered by this map
was 1702 cM within which 1024 loci were included, and
the average distance between loci was 1.4 cM (Table 5).
The length of individual linkage groups ranged from 89.7
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Table 2. Broad-sense heritability and the mean squares of partial variance components for flowering and yield traits of PR-11
among four environments.
Variance
components
2g‡
2ge
2residual
 2p
H2§
DTF†
DTFT†
DOF†
RNN†
PN†
SNPP†
TSW†
Yield†
1.9 ± 0.3
0.1 ± 0.1
1.5 ± 0.1
2.11
0.90
3.2 ± 0.7
0.7 ± 0.6
11.1 ± 0.8
4.76
0.67
1.5 ± 0.5
0.9 ± 0.6
11.1 ± 0.8
3.11
0.48
0.1 ± 0.0
0.1 ± 0.1
1.1 ± 0.1
0.26
0.38
0.3 ± 0.1
0.0 ± 0.0
1.9 ± 0.1
0.54
0.56
0.1 ± 0.0
0.1 ± 0.0
0.4 ± 0.0
0.18
0.57
317.9 ± 52.6
12.2 ± 26.1
474.1 ± 33.0
380.21
0.84
3319.4 ± 679.0
764.0 ± 667.1
11907.0 ± 815.5
4998.38
0.66
† DTF, days to flowering; DTFT, days to flowering termination; DOF, duration of flowering; RNN, reproductive node number on the main stem per plant; PN, pod number on
the main stem per plant; SNPP, seed number per pod; TSW, thousand seed weight (g per 1000 seed); Yield, plot yield (g m –2).
‡ The genotypic variance of all the traits was significant at the 0.01 probability level, block variance was not significant and is not shown.
§ H 2 = s2g / s2p = s2g / és2g + s2g´e / m + s2residual / (m´b)ù
ëê
ûú
Table 3. Correlations between days to flowering (DTF), days to flowering termination (DTFT), duration of flowering (DOF), reproductive node number on the main stem per plant (RNN), pod number on the main stem per plant (PN), seed number per pod
(SNPP), thousand seed weight (TSW) (g per 1000 seed) and plot yield (g m –2) in PR-11 under normal seeding date environments
in 2013 and 2014.
Trait
DTF
DTFT
DOF
RNN
PN
SNPP
TSW
Yield
DTF
DTFT
DOF
RNN
*
0.58***
*
0.01
0.82***
*
–0.03
0.24*
0.31**
*
PN
–0.27**
–0.01
0.18*
0.58***
*
SNPP
TSW
Yield
0.32**
–0.04
–0.27**
–0.29**
–0.35***
*
0.12
0.13
0.08
0.02
–0.21*
–0.15
*
0.36***
0.47***
0.33***
0.13
0.01
0.04
–0.01
*
* Significant at the 0.05 probability level.
** Significant at the 0.01 probability level.
*** Significant at the 0.001 probability level.
Table 4. Correlations between days to flowering (DTF), days to flowering termination (DTFT), duration of flowering (DOF), reproductive node number on the main stem per plant (RNN), pod number on the main stem per plant (PN), seed number per pod
(SNPP), thousand seed weight (TSW) (g per 1000 seed) and plot yield (g m –2) in PR-11 in 2014 late seeding date experiment.
Trait
DTF
DTFT
DOF
RNN
PN
SNPP
TSW
Yield
DTF
DTFT
*
0.29**
*
DOF
–0.70***
0.42***
*
RNN
PN
SNPP
TSW
Yield
–0.01
0.20*
0.23*
*
–0.05
0.10
0.20*
0.74***
*
0.11
–0.13
–0.21*
0.01
0.28**
*
0.04
0.01
–0.07
0.02
–0.14
0.23*
*
0.14
0.29**
0.08
0.08
0.21*
–0.19*
–0.04
*
* Significant at the 0.05 probability level.
** Significant at the 0.01 probability level.
*** Significant at the 0.001 probability level.
(LGV) to 344.6 cM (LGIII). The highest number of loci
(232) were on LGVI, while LGV had the lowest (55).
For each measured trait, a separate QTL analysis was
conducted for each experimental trial. A total of 25 QTLs
were identified, among which 11 QTLs (six related to
flowering, five related to yield components) were identified in more than one trial (Table 6, Fig 4). Six out of these
11 QTLs were located on LGIII, three on LGIV and one
each on LGII and VIb. Four QTLs on LGIII were clustered
6
in the region 100 to 160 cM, three QTLs on LGIV were
clustered at 220 to 260 cM. Some QTLs such as DTF-2,
DOF-3, and PN-1 were specific to year, DOF-2 was specific to location (only identified at Saskatoon over 2013
and 2014), whereas other QTLs such as SNPP-2 (found
in 2013 Saskatoon and 2014 late seeding), TSW-2 (found
in 2013 Rosthern and 2014 Saskatoon normal seeding)
were not specific to either year or location. No consistent pattern was found as to whether those QTLs were
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crop science, vol. 57, may– june 2017
Fig. 2. Path coefficient analysis of four yield component traits (reproductive node number [RNN], pod number [PN], seed number per pod
[SNPP], and thousand seed weight [TSW]) on main-stem seed yield under normal seeding environment. The partial coefficients, r, are
the numbers above the arrows, and the residual error unaccounted for error is e.
Fig. 3. Path coefficient analysis of four yield component traits (reproductive node number [RNN], pod number [PN], seed number per pod
[SNPP], and thousand seed weight [TSW]) on main-stem seed yield under late seeding environment. The partial coefficients, r, are the
numbers above the arrows, and the residual error unaccounted for error is e.
Table 5. General features of the genetic linkage map using
single nucleotide polymorphism (SNP) markers based on 90
recombinant inbred lines (RILs) of PR-11 population.
Linkage
group
LGIa
LGIb
LGIc
LGId
LGII
LGIII
LGIV
LGVa
LGVb
LGVIa
LGVIb
LGVIIa
LGVIIb
Total
Size
cM
85.9
16.4
10.5
37
234.4
344.6
307.8
28.2
61.5
45.5
237.4
262.0
30.8
1702
Loci
number
57
10
9
37
147
226
216
16
39
27
205
194
21
1204
Average loci
distance
cM
1.5
1.6
1.2
1.0
1.6
1.5
1.4
1.8
1.6
1.7
1.2
1.4
1.5
1.4
more specific to year, location, or different seeding treatment. A QTL associated with DTF (DTF-3) was consistently detected over all four trails on LGVIb, explaining
18.0 to 29.5% of the phenotypic variation depending on
the site-year. Another stable QTL over all three normal
seeding trials for DTFT (DTFT-1) was detected on LGIII
(R 2 = 16.2–22.4%). Additionally, a major QTL for TSW
(TSW-2) was located on LGIV (R 2 = 30.9–40.9%). Some
QTLs specific to late seeding were also identified, such as
DOF-4 at LGVIIa (R 2 = 14.0%, LOD = 3.3), RNN-3 at
LGVIIb (R 2 = 15.2%, LOD = 3.2) and Yd-5 at LGIII (R 2
= 22.1%, LOD = 3.8).
crop science, vol. 57, may– june 2017 Discussion
Heat Stress in Canadian Field Pea
Production Region
Both the average temperature of the growing season and
during pea’s vegetative stage ranged from 15 to 16°C among
the normal and late seeding date experiments, which did
not exceed the mean seasonal temperature threshold (17.5
°C) for pea yield reduction in Canada (Bueckert et al.,
2015), and were in the optimal temperature range for pea
growth (Mahoney, 1991). Besides, regardless of seeding
date, the average daily temperature during flowering stage
sat within the ideal range. However, correlation analysis
showed that the average yield of RILs was negatively associated with the daily maximum temperature at antheis (r
= –0.85) and the number of days with temperature over
27°C (r = –0.80), implying that yield reduction related
to heat stress was mainly due to the increased number of
hot days at the flowering stage. A similar yield response to
the frequency of hot days in the growing season was also
reported (Bueckert et al., 2015).
The approach of growing crops at different seeding
dates in anticipation of receiving heat stress at different
stages in the plant’s cycle has been used in several crops
when breeding for heat tolerance, including pea (French,
1990), chickpea (Krishnamurthy et al., 2011), and summer
Brassica (Morrison and Stewart, 2002). The mean daily
maximum temperature during flowering reached 25.5°C
compared to 23.7°C across the other three normal seeding
date trials (Fig. 1), and it surpassed the broadly accepted
upper optimal temperature threshold of 25°C (Pumphrey
and Raming, 1990). The number of days with the maximum temperature over 27°C was also higher than the
www.crops.org7
Table 6. Quantitative trait loci (QTLs) for phenotypic traits of recombinant inbred lines (RILs) in four environments over 2013
and 2014.
QTL symbols†
Trait
Linkage group
Environment‡
Position§
LOD¶
PVE#
Add††
DOF-1
DOF-2
DOF-2
DOF-3
DOF-3
DOF-4
DTF-1
DTF-1
DTF-2
DTF-2
DTF-3
DTF-3
DTF-3
DTF-3
DTFT-1
DTFT-1
DOF
DOF
DOF
DOF
DOF
DOF
DTF
DTF
DTF
DTF
DTF
DTF
DTF
DTF
DTFT
DTFT
3
3
3
4
4
7a
2
2
3
3
6b
6b
6b
6b
3
3
2013 ROS
2013 SAS
2014 NOR
2013 SAS
2013 ROS
2014 LAT
2013 SAS
2014 LAT
2013 SAS
2013 ROS
2013 SAS
2013 ROS
2014 NOR
2014 LAT
2013 SAS
2013 ROS
cM
61.9
135.1
100.0
214.0
241.7
224.0
109.6
109.6
132.7
143.7
72.3
94.6
65.5
94.6
66.9
61.9
3.9
4.5
4.6
3.3
3.6
3.3
2.8
3.1
3.6
2.9
8.4
4.2
5.6
4.3
3.5
5
18.2
18.5
20.8
13
16.9
14
8.5
10.8
11
10.4
29.5
19.7
25
18
16.2
16.6
1.7
1.1
1
–0.9
–1.7
–0.6
0.6
0.9
–0.6
–0.5
–1
–0.8
–0.8
–0.6
1.1
2.2
DTFT-1
DTFT-2
DTFT-3
PN-1
PN-1
RNN-1
RNN-2
RNN-3
SNPP-1
SNPP-2
SNPP-2
SNPP-3
SNPP-3
SNPP-3
TSW-1
TSW-1
TSW-2
TSW-2
TSW-3
Yd-1
Yd-2
Yd-3
Yd-4
Yd-5
DTFT
DTFT
DTFT
PN
PN
RNN
RNN
RNN
SNPP
SNPP
SNPP
SNPP
SNPP
SNPP
TSW
TSW
TSW
TSW
TSW
Yd
Yd
Yd
Yd
Yd
3
4
6b
3
3
3
6b
7b
2
3
3
4
4
4
3
3
4
4
6b
3
3
4
6b
3
2014 NOR
2013 ROS
2013 SAS
2014-NOR
2014 LAT
2014 NOR
2014 NOR
2014 LAT
2014 NOR
2013 SAS
2014 LAT
2013 SAS
2013 ROS
2014 LAT
2013 ROS
2014 NOR
2013 ROS
2014 NOR
2014 NOR
2013 SAS
2013 ROS
2013 ROS
2013 SAS
2014 LAT
68.1
241.7
79.8
116.2
132.0
114.7
45.7
23.0
2.0
148.2
133.0
224.7
259.0
262.0
160.0
165.6
249.0
214.0
190.7
43.0
82.2
104.2
220.3
148.8
5
3.1
3
5.6
4.3
7.2
3
3.2
4
6.7
4.2
3.8
2.9
4.2
3.7
4.3
12.3
9.3
3.2
3.7
3.4
3
3.1
3.8
22.4
11.4
14.4
24.8
20.1
31
9.8
15.2
18.3
26.2
14.8
13.8
14
14.6
9.8
12.5
40.9
30.9
7.7
15.8
16.1
11.9
13.2
22.1
1.1
–1.6
–1
0.5
0.5
0.4
–0.2
–0.3
0.3
–0.3
–0.2
0.2
0.2
0.2
–7.9
–6.9
16.2
10.7
5.4
39.5
57.1
–51.9
–36
28.3
† DTF, days to flowering; DTFT, days to flowering termination; DOF, duration of flowering; RNN, reproductive node number on the main stem per plant; PN, pod number on
the main stem per plant; SNPP, seed number per pod; TSW, thousand seed weight (g per 1000 seed); Yield, plot yield (g m –2).
‡ 2013 SAS, 2013 Saskatoon site; 2013 ROS, 2013 Rosthern site; 2014 NOR, 2014 Saskatoon normal seeding site; 2014 LAT, 2014 Saskatoon late seeding site.
§ Genetic distance of maximum logarithm of odds (LOD) value for specific trait.
¶ Maximum LOD value. The threshold value of a significant QTL ranged from 2.8 to 3.2 for different traits.
# PVE, phenotypic variance explained by the QTL detected for the trait.
†† The additive value associated with CDC Centennial Allele. A positive value means CDC Centennial increase the value of trait.
three normal seeding date experiments. As a result, the
warmer environment appeared to accelerate flowering
and maturity, as previously reported (Guilioni et al., 1997,
2003). However, multiple years’ experiments would be
needed for further verification.
8
Phenotypic Trait Assessment
Among the flowering and yield-related traits in this
study, the broad-sense heritability (H 2) of days to flowering (DTF) was highest (H 2 = 0.90), the heritability of
other traits was also moderately high, ranging from 0.38
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crop science, vol. 57, may– june 2017
Fig. 4. Confidence interval of quantitative trait loci (QTLs) for measured traits at normal and late seeding date environments in pea recombinant inbred line population-11. Red bars are the QTLs detected in more than one environment. Detailed information of each QTL is
summarized in Table 6. DTF, days to flowering; DTFT, days to flowering termination; DOF, duration of flowering; RNN, reproductive node
number on the main stem per plant; PN, pod number on the main stem per plant; SNPP, seed number per pod; TSW, thousand seed
weight (g); Yield, plot yield (g m –2).
to 0.84 (Table 2). This finding was in agreement with
previous publications (Timmerman-Vaughan et al., 2005;
Fikreselassie, 2012). Correlations among the four yield
components were relatively weak, which was similarly
reported in Krajewski et al. (2012). Only PN showed a
significant positive association with RNN, and negative
correlations with SNPP, and TSW under both normal and
late seeding date environments (Tables 3 and 4). Likely
the increase in PN was offset by the decrease in SNPP,
and a negative relationship between PN and SNPP was
displayed in our study and also in previous research (Moot
and McNeil, 1995; Krajewski et al., 2012). This finding
may be attributed to competition for assimilates during
reproductive growth, or specifically when environmental
stresses occur in the reproductive stage.
Further, the path coefficient analysis was carried out
to discover the relative contribution of yield components
to the grain yield on the main stem under normal and late
seeding date environments. Both results revealed that the
variance of the main-stem seed yield primarily derived
from the variance of pod number (Fig. 2 and 3). Consistent
results have been reported by French (1990) and Ayaz et
al. (2004), which stated that seed yield per plant was most
positively correlated with the number of pods per plant. As
crop science, vol. 57, may– june 2017 well, Sarawat et al. (1994) concluded that grain yield heterosis was mainly due to more pods per plant in hybrids. The
variation in seed yield on the main stem appeared not to
be the cause of the major variation in final grain yield on a
plot scale (no significant correlation was observed between
these two). The reasons may be either the difference of
emergence rate in each plot or yield gains from branches.
Given that the emergence rate among RILs was similar,
this finding indicated that the final grain yield was derived
also from seed yield on the branches. The importance of
the contribution of the basal branches on yield has already
been emphasized by Singh et al. (2011). However, studies regarding the initiation of branches are less well documented than for the main stem, and previous authors concluded that the ability to produce basal branches mainly
depended on pea genotype and plant density (Spies et al.,
2010). Besides yield components, phenological traits like
DTF and DOF also affected grain yield in pea. Also, DTF
was positively associated with grain yield in this study, as
well as in others (Timmerman-Vaughan et al., 2005; Singh
et al., 2011; Bueckert and Clarke, 2013). The reason might
be that delayed onset of flowering may allow for greater
assimilate production through vegetative development
which could be used for the reproductive development.
www.crops.org9
Linkage Map Quality and Quantitative Trait
Loci for Flowering and Yield Traits
The total coverage of the linkage map was 1702 cM (Table
5), which was in the range of the previous published SNPderived maps for pea, ranging from 358.02 to 1916 cM
(Deulvot et al., 2010; Leonforte et al., 2013; Duarte et al.,
2014; Sindhu et al., 2014). The average inter-locus interval
in this map was only 1.4 cM, much less than the published
SNP-derived maps, indicating the high resolution of this
map. This high resolution allowed the confidence interval
of many detected QTLs in an individual experiment site
to narrow to 1 cM, facilitating the opportunity to identify
the candidate gene in the interval.
Six QTLs related to flowering traits (three for DTF,
one for DTFT, two for DOF) were identified in more
than one environment (Table 6, Fig 4). Of these QTLs
located at different regions on LG II, III, IV and VIb, only
DOF-2 and DTF-2 shared part of overlapping region on
LGIII. To our knowledge, this is the second report related
to QTLs associated with DOF and DTFT in pea, following Klein et al. (2014). However, we failed to find any
common QTL as these authors; the daylength difference
in the growing season (latitude was 54° N in this study,
45° N for theirs) might be part of reason. In addition, the
lack of repetition over multiple site-years in their study
made their result less reliable. Identification of QTLs for
DTF on LGII, III, VI was also previously reported by
Fondevilla et al. (2008); however, the positions of these
QTLs were quite different. Prioul et al. (2004) based an
analysis on F2–derived RILs from the cross of JI296 (a
white-flowered, early flowering cultivar) ´ DP (a purple-flowered, late flowering cultivar), found three QTLs
associated with days to flowering, and one of these was
situated at a similar position on LGVI as DTF-3 reported
here. DTF-3 was consistently detected across all four
experimental sites, and explained an average of 20% of the
phenotypic variation. DTF-1 (LGII) overlapped with the
locus related to nodes of first flower identified by Timmerman-Vaughan et al. (2005). Several QTLs for DTF on
LGIII were also mapped, however their relative positions
on the LGIII varies in the published pea linkage maps.
Prioul et al. (2004) reported the position at 18 to 26 cM
(full length of their LGIII was 194 cM), Jha et al. (2016)
reported its position at around 60 cM (full length of LGIII
was 101 cM), Klein et al. (2014) found its position at the
tail of LGIII, whereas in this study its position (DTF-2)
was at 125 to 135 cM in the 340 cM LGIII. No QTL with
a major effect was identified (highest R 2 was 0.26 for DTF)
in this research indicating that the control of flowering
time is multi-genic. Only five stable QTLs were identified associated with PN, SNPP, and TSW, three of them
clustered on LGIII at the interval from 110 to 170 cM, the
other two QTLs overlapped on LGIV at the interval of
210 to 260 cM. The number of QTLs related with yield
10
and yield components in this study were low compared
to that reported by Timmerman-Vaughan et al. (2005),
similar as Krajewski et al. (2012) and Klein et al. (2014).
TSW-2 and SNPP-3 (both on LGIV) in this study shared
similar positions with the loci for seed weight and seed
number detected by Timmerman-Vaughan et al. (2005),
as well as Klein et al. (2014). The localization of a QTL
for grain yield on LGVII was quite similar between Timmerman-Vaughan et al. (2005) and Tar’an et al. (2003).
Timmerman-Vaughan et al. (2005) identified six QTLs
for TSW on LGI (2), IIIa (1), IV (2), and VII (1); four of
them overlapped with the loci for seed number. Krajewski
et al. (2012) detected a common QTL for SNNP over two
different populations.
Conclusion
A stable QTL on LGVIb over all four experimental environments was associated with days to flowering. Under
normal seeding environment, a major QTL for thousand
seed weight on LGIV was identified, which co-localized
with loci for days to flowering termination, duration of
flowering and seed number per pod.
Correlation and path coefficient analysis revealed that
late flowering termination and high pod number per plant
were promising and helpful indices for high yield potential under both normal and heat stress environments. In
Canada, a short period of extreme high temperature is the
most pervasive type of heat stress. It can cause the abscission of reproductive organs in pea, and cultivars with late
termination of flowering could take advantage of indeterminate growth habit and flower again.
Conflict of Interest Disclosure
The authors declare there to be no conflict of interest.
Acknowledgement
This research was funded by Agriculture Development Fund
of Saskatchewan, Western Grains Research Foundation, Saskatchewan Pulse Growers Association, and the NSERC-CRD
program. We would like to thank Brent Barlow, Scott Ife,
Alison Sackville, Donna Lindsay, and other staff of the Crop
Development Centre pulse crop breeding for seed preparation
and field maintenance, and Rob Stonehouse at the pulse crop
molecular genetics lab for technical assistance.
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