Download Interval mapping of QTLs controlling yield

J. Appl. Genet. 45(3), 2004, pp. 297–306
Interval mapping of QTLs controlling yield-related traits
and seed protein content in Pisum sativum
Lidia Irzykowska1, Bogdan Wolko2
1
2
Department of Phytopathology, August Cieszkowski Agricultural University of Poznañ, Poland
Institute of Plant Genetics, Polish Academy of Sciences, Poznañ, Poland
Abstract. A linkage map of garden pea was constructed on the basis of 114 plants (F2 generation) derived from
a cross combination Wt10245 × Wt11238. The map, consisting of 204 morphological, isozyme, AFLP, ISSR,
STS, CAPS and RAPD markers, was used for interval mapping of quantitative trait loci (QTLs) controlling seed
number, pod number, 1000-seed weight, 1000-yield, and seed protein content. Characterization of each QTL
included identification of QTL position with reference to the flanking markers, estimation of the part of variance
explained by this QTL, and determination of its gene action. The yield-related traits were measured in F2 plants
and in F4 recombinant inbred lines (RILs). The interval mapping revealed two to six QTLs per trait,
demonstrating linkage to seven pea chromosomes. A total of 37 detected QTLs accounted for 9.1-55.9% of
the trait’s phenotypic variation and showed different types of gene action. As many as eight and ten QTLs
influencing the analysed traits were mapped in linkage groups III and V, respectively, indicating an important role
of these regions of the pea genome in the control of yield and seed protein content.
Key words: interval mapping, Pisum sativum, QTL, seed protein content, yield.
Introduction
The garden pea (Pisum sativum L.) is one of
the most important legume crops in Europe.
The majority of agriculturally significant traits
in pea, as in other plant species, are inherited quantitatively. The complexity of these phenotypic
traits exhibiting continuous variation arises from
the segregation of alleles at many interacting loci
(quantitative trait loci = QTLs), whose effects depend on the environment. Recent advances in molecular genetics and statistic methods make it
possible to create genetic maps covering the whole
genome and identify the chromosomal regions
where these QTLs are located. The interval mapping method postulates a single QTL at the position on the chromosome corresponding to
the maximum ratio of the likelihood of the QTL at
that point to that of no QTL at that point (Hackett
1997). Using statistic analysis, the variation
of a quantitative trait can be divided into the effect
of individual QTLs linked to markers on a genetic
map.
The genetic map of pea has been developed
gradually, starting with the first version given by
Lamprecht in 1948, up to the latest version finished in 1998 by the Pisum Mapping Committee
(Weeden et al. 1998). Nevertheless, the studied
range of quantitative traits in pea is still limited.
A few reports in this field concerned seed weight
(Timmerman-Vaughan et al. 1996), seed cotyledon color (McCallum et al. 1997), stem length
and internode number (Irzykowska et al. 2002),
and Aphanomyces root rot resistance (Pilet-Nayel
et al. 2001).
The objective of the presented study was to
identify and localize QTLs affecting yield-related
traits and seed protein content, with the use of
an earlier constructed pea genetic map
(Irzykowska et al. 2001).
Received: December 8, 2003. Accepted: May 13, 2004.
Correspondence: L Irzykowska, Department of Phytopathology, August Cieszkowski Agricultural University of Poznañ,
D¹browskiego 159, 60-594 Poznañ, Poland; e-mail: [email protected]
298
L Irzykowska, B Wolko
Material and methods
Plant material
A total of 114 F2 plants derived from the Wt 10245
× Wt 11238 cross combination and F4 recombinant inbred lines (RILs) obtained by selfing of F2
and F3 individuals were used in this analysis.
The parental lines were selected on the basis of
contrasting monogenic morphological characters
as well as significant differences in quantitative
trait expression. The maternal line, Wt 10245, is
a large-seeded cultivar and the paternal line,
Wt11238 (=WL1238), is a small-seeded line, often exploited in our genetic studies, with the morphological and isozyme markers distributed in all
chromosome linkage groups.
Plants were evaluated in field trials. The F2 pea
seeds were sown in two rows with a distance
of 40 cm between rows and 20 cm between plants.
All plants were provided with supports to prevent
lodging, in order to minimize differences in growing conditions of plants and to provide convenient
access to plants during observations and measurements. The F3 and F4 RIL populations were derived from 10 seeds collected from plants of
the previous plant generation and were grown on
field plots in similar conditions as described
above.
Marker analysis and genetic map construction
In total, 240 markers were used for the linkage
analysis. The marker analysis and genetic map
construction was performed as previously described (Irzykowska et al. 2001). Goodness-of-fit
to the co-dominant 1 : 2 : 1 or dominant 3 : 1 ratio
was tested by c2 analysis with the computer program Linkage-1 (Suiter et al. 1987). The most
probable order and map distances among markers
were determined by multiple linkage analysis using MAPMAKER/Exp. 3.0 software (Lander et al.
1987). The threshold value of the logarithm of
odds (LOD) for assigning markers to linkage
groups were set at LOD ³ 3.0, with a maximum
distance of 35 cM.
QTL mapping
The studied quantitative traits were selected on
the basis of their significance for pea seed yield.
In the F2 (114 plants) and/or F4 (104 RILs) plant
populations, the following measurements were
made:
– pod number and seed number in two growing
seasons in F2 and F4 plant populations;
– 1000-seed weight (± 0.1 g), calculated for normally developed seeds from F2 and F4 plants
– seed yield, estimated by weighing all seeds from
F2 and F4 plants;
– total seed protein content, determined in bulked
seeds collected from F4 plants, using the Kjeldhal
method (the Kjel-Foss nitrogen analyser).
Each plant was measured in the F2 generation
and the mean value from five plant measurements
was calculated for each trait estimated in the F4
generation. Trait means and analysis of variance
were determined with Genstat software (Genstat 5
Committee 1993). QTLs were detected by interval
mapping using MAPMAKER/QTL 1.1 (Lincoln
et al. 1993). The significance level required to assess marker-QTL associations was set at
LOD ³ 2.1.
The QTLs mapped were characterized by
five statistic parameters (Tanksley 1993):
LOD = logarithm of odds, i.e. maximal value of
the LOD curve for the particular QTL;
V E = percentage of total trait variance explained by the QTL; a = additive effect;
d = dominance effect; and d/a = coefficient describing the type of gene action. The d/a value
of 1.0 is considered to reflect complete dominance (d/a = D) and a heterozygote is identical
to the parental homozygote that shows a greater
value of the particular trait. On the other hand,
a value of –1.0 is considered to reflect a recessive
gene action (d/a = R) and in this case the heterozygote would be identical to the lesser homozygote. If the d/a value is close to zero, then
the gene action is additive (d/a = A) and the heterozygote is intermediate between the two parental
homozygotes.
Results
Phenotypic analysis
Analyses of all examined traits showed continuous
variation with approximately normal frequency
distributions. The results obtained from analysis
of variance of 104 inbred lines (F4) derived from
the cross combination Wt 10245 × Wt 11238 for
yield-related traits and seed protein content, were
presented in Table 1. The measurements of all
quantitative traits were analysed statistically to localize the genetic components of the trait.
Interval mapping
All the 54 QTLs determining five quantitative
traits were localized on a previously constructed
Interval mapping of QTLs in Pisum sativum
Table 1. Mean values and F-statistics from analysis
of variance for traits observed in the F4 generation
of Pisum sativum
Quantitative
trait
Minimum
Mean
Maximum
F
statistics
Seed number
13.2
59.1
163.7
2.5*
Pod number
2.8
16.2
37.8
3.1*
94.4
219.5
443.1
17.2*
Seed yield (g)
2.1
12.6
29.6
3.4*
Seed protein
content (%)
23.7
28.3
35.2
559.3*
1000-seed
weight (g)
Table 3. Locations of putative QTLs affecting
yield-related traits in the F4 generation of Pisum
sativum
Trait
Yield-related traits
The interval mapping revealed 11 QTLs controlling yield-related traits in the F2 population
and 21 QTLs in the F4 RIL population (Tables 2
and 3). From among 32 QTLs identified, eight
were mapped in LG III, seven in LG V, four per LG
I and LG II, and three per LG IV, LG VI and LG
VII (Figure 1). The number of QTLs estimated in
the F2 progeny varied from two for seed weight to
four for seed number (Table 4). The number of
QTLs scored in the RIL population for
yield-related traits was higher: five for all analysed
traits except seed yield, for which six loci were
mapped.
Seed number was determined by four QTLs
in the F2 generation (LG III, IV, V and VII)
and five loci mapped in the F4 RIL population
Table 2. Locations of putative QTLs affecting
yield-related traits in the F2 generation of Pisum sativum
Trait
QTL Linkage
symbol group
Map interval*
Distance **
cM
Seed
number
1sn-1
1sn-2
1sn-3
1sn-4
III
IV
V
VII
afp13i - afp14a
afp9e - C1516b
Acp1 - Pgdc
Est1 - Est2
8.0
9.0
0.0
1.0
Pod
number
1pn-1
1pn-2
IV
V
B873c - P628
Pgdc - afp12e
4.0
6.0
Seed
yield
1yd-1
1yd-2
1yd-3
III
IV
VII
afp8g - afp4i
afp9b - Cs1516b
Est1 - Est2
8.0
10.0
1.0
Seed
weight
1tgw-1
1tgw-2
I
V
afp12a - afp11e
afp11a - afp6b
0.0
4.0
* The nearest marker was marked in bold type
** The distance was measured from the nearest marker to the maximum LOD peak of a QTL.
QTL Linkage
symbol group
Map
interval*
Distance**
(cM)
Seed
number
2sn-1
2sn-2
2sn-3
2sn-4
2sn-5
I
II
V
VI
VII
afp1g - afp6d
afp9i - afp9g
OPG9b - B807b
Pl - afp8e
Est1 - Est2
4.0
0.0
0.1
1.0
1.0
Pod
number
2pn-1
2pn-2
2pn-3
2pn-4
2pn-5
I
II
III
V
VI
afp11c - afp16g
afp9i - afp9g
Lap1 - afp10h
OPG9b - B807b
Pl - afp8e
0.0
0.0
3.0
0.1
1.0
Seed
yield
2yd-1
2yd-2
2yd-3
2yd-4
2yd-5
2yd-6
I
II
III
III
III
VI
afp11c - afp16g
afp9i - afp9g
afp2a - Cs1516a
afp4j - afp11h
afp4e - afp13i
Pl - afp8e
0.0
0.0
6.0
0.0
15.0
1.0
1000seed
weight
2tgw-1
2tgw-2
2tgw-3
2tgw-4
2tgw-5
II
III
III
V
V
wb - afp7e
OPC16d - afp2a
b - afp10e
tl - r
te - afp1b
6.0
4.0
4.0
0.0
4.0
* significant at a = 0.01
genetic map (Irzykowska et al. 2001). The regions
responsible for inheritance of analysed traits were
identified in seven of nine constructed linkage
groups (LGs) (Figure 1).
299
* The nearest marker was marked in bold type
** The distance was measured from the nearest marker to the maximum LOD peak of a QTL.
(LG I, II, V, VI and VII). The LOD values of all
QTLs mapped were low (Table 4) except one:
2sn-4 (LOD = 4.0). The portion of total trait variance explained (VE) was the highest for two
QTLs: 2sn-3 (VE = 44.3%) and 2sn-4
(VE = 55.9%). For most of QTLs mapped, the additive effect of the allele from the paternal line
was strongly negative, but for three of them
(1sn-3, 2sn-1, 2sn3) the additive effect was positive. Only in one case, the loci mapped using
the F2 (1sn-4) and F4 population (2sn-5) were located in the same position in LG VII (Tables 4
and 5). They showed the same additive effect
and inheritance type (recessive) and similar values of LOD (2.4 and 2.2) and VE (11.1%
and 10.4%). The loci 1sn-3 and 2sn-3 were
mapped close to each other, in the same fragment
of LG V, and their confidence intervals partly
overlap, but large differences in VE and d parameters implicate different types of gene action manner.
Two QTLs determining pod number in the F2
generation were mapped in LG IV (1pn-1)
and LG V (1pn-2). Both of them showed low
LOD and VE values. For the 1pn-1 locus, the additive effect of the allele from the paternal line was
negative, but for the 1pn-2 locus, the Wt11238 allele increased the trait value and showed recessiveness (Table 4).
afp16g
afp11c
afp1r
afp1c
afp10f
Cs1508b
ENOD40
O PG 9a
Cs1526
afp1k
Cs1513
afp1f
afp2f
afp7d
afp6d
afp1g
*
*
*
*
*
afp3k
afp7e
s
wb
afp9i
afp9g
afp2j
afp16a
k
afp14i
Cs1508a
afp5d
afp11b
afp3j
afp6e
afp2c
afp5e
afp6c
afp9f
B835b
afp14d
afp4d
afp15h
a
afp5c
afp13l
Aat-p
afp1a
afp1n
O PC09
LG II
*
*
*
*
L109
Cs1506a
afp14a
afp13d
afp13i
afp4e
afp10h
Lap-1
afp4i
afp6a
Cs1532
afp15a
afp13b
afp4j
afp11h
afp1p
afp4c
afp8i
b
afp10
afp8g
afp7a
B808
afp1
afp14k
Cs1516a
O PJ14b
Lap-2
afp2a
*
*
*
B827b
afp9b
B868a
afp1e
afp1m
O PJ14a
afp8b
Cs1506b
afp2i
afp16f
afp10b
afp16d
O PC16
*
*
*
*
**
**
*
*
B807b
afp2b
afp11a
afp6b
afp8f
gp
afp16h
cp
te
afp1b
afp11d
afp13j
afp5b
afp14b
afp14l
Acp-1
Pgd-c
afp12e
U
afp4g
O PG 9b
afp10i
afp7k
B873b
afp12b
tl
r
afp9h
P108
Cs1504
afp6f
afp14c
afp7m
Acp-2
LG V
seed number F 2
seed number F 4
pod number F 2
pod number F 4
seed protein content F 4
P393
P628
C1516b
B873c
C1507a
afp13f
afp9e
Est-S
O PG 12
afp7l
afp11g
afp16e
afp16b
afp3b
LG IV
afp7g
LG III
seed
seed
seed
seed
*
*
weight F 2
weight F 4
yield F 2
yield F 4
afp8e
Pl
afp1h
B873a
afp9c
afp14e
Cs1507b
afp15c
afp15i
afp7c
afp2e
afp14f
afp10a
afp14h
afp10g
afp11f
afp8a
Zd10
LG VI
Est-1
Est-2
afp4b
afp13a
afp13e
afp4f
afp1o
O PF09a
O PF03
B807a
afp15e
Cs1503b
O PG 11
afp2d
afp12d
B835a
afp8d
afp8h
afp14g
afp8c
Unattached
group VIII
**
*
afp13h
afp10c
afp14j
afp2h
afp5h
afp1d
Pgd-p
afp7j
LG VII
50
0
cM
Figure 1. Linkage map constructed for a mapping population derived from the Wt 10245 × Wt 11238 cross combination (Irzykowska et al. 2001), with localized QTLs controlling
yield-related traits and the seed protein content. The linkage groups and reference markers (* on the left side of linkage groups) were designed according to a previous pea genome linkage
map (Weeden et al. 1998).
*
**
afp12a
afp11e
afp1l
afp4a
afp4k
afp2g
afp5a afp9a
afp9j
O PF09
afp4h
afp15g
d
Idh
afp10d
afp1i
afp13g
afp12f
afp3e
afp3g
afp3i
LG I
Interval mapping of QTLs in Pisum sativum
Table 4. Characteristics of QTLs affecting
yield-related traits in the F2 generation of Pisum
sativum
QTL
symbol
a
d
d/a
LOD
VE
(%)
1sn-1
1sn-2
1sn-3
1sn-4
2.3
2.5
2.2
2.4
14.0
20.4
9.4
11.1
–27.3
–22.9
16.4
–18.1
–7.5
–30.6
22.0
25.6
0.27
1.34
1.34
–1.41
1pn-1
1pn-2
2.2
2.5
12.9
12.2
–5.2
6.8
–6.5
0.8
1.25
0.11
1yd-1
1yd-2
1yd-3
2.4
2.2
2.5
15.7
12.9
11.7
0.9
–5.2
–3.7
8.4
–6.5
5.9
9.33
1.25
–1.59
1tgw-1
1tgw-2
2.4
2.6
10.9
21.0
–21.2
–29.3
23.1
37.1
–1.09
–1.27
LOD = logarithm of odds; VE = variance explained; a = additive
effect; d = dominance effect
Table 5. Characteristics of QTLs affecting
yield-related traits in the F4 generation of Pisum
sativum
QTL
symbol
a
d
d/a
LOD
VE
(%)
2sn-1
2sn-2
2sn-3
2sn-4
2sn-5
2.1
2.5
2.1
4.0
2.2
10.7
10.8
44.3
55.9
10.4
12.3
–11.2
21.0
–19.8
–11.3
6.1
7.4
–22.1
–29.2
9.4
0.49
–0.66
–1.05
1.47
–0.83
2pn-1
2pn-2
2pn-3
2pn-4
2pn-5
2.6
2.8
3.6
2.8
3.9
38.2
12.2
51.0
51.5
55.4
5.9
–3.4
6.1
6.0
–5.5
–3.6
0.9
–6.2
–6.2
–7.2
–0.61
–0.26
–1.02
–1.03
1.31
2yd-1
2yd-2
2yd-3
2yd-4
2yd-5
2yd-6
2.0
2.7
2.7
2.1
2.5
2.4
9.1
11.6
36.7
10.7
37.4
47.8
2.0
–2.6
–4.3
1.4
3.3
–4.1
2.2
1.5
–4.7
3.2
–5.7
–6.0
1.10
–0.58
1.09
2.29
–1.73
1.46
2tgw-1
2tgw-2
2tgw-3
2tgw-4
2tgw-5
2.1
3.6
2.3
2.6
3.2
11.2
24.2
12.1
10.9
15.4
–18.7
–29.7
7.2
–21.3
–15.3
16.7
–20.1
31.4
16.9
30.3
-0.89
0.68
4.36
0.79
–1.98
LOD = logarithm of odds; VE = variance explained; a = additive
effect; d = dominance effect
Five loci influencing pod number were localized in the F4 population, each of them in a different linkage group (Table 3). The VE value ranged
from 12.2% to 55.4% and LOD from 2.6 to 3.9
(Table 5). The estimation of the additive effect of
the Wt11238 allele showed that it increased pod
number in the 2pn-1, 2pn-3 and 2pn-4 loci
and negatively influenced trait values in the remaining QTLs. The d/a coefficient values indicate
dominance for the 2pn-5 locus and recessiveness
for the 2pn-3 and 2pn-4 loci. It was impossible to
301
determine unambiguously the type of gene action
in loci 2pn-1 and 1pn2 (additive or recessive).
Two loci, 1pn-2 and 2pn-4, were mapped in LG V.
Their LOD peaks were not localized in the same
map interval but the confidence interval estimated
for the 1pn-2 locus includes the confidence
interval determined for the 2pn-4 locus. Moreover,
the estimated additive effects of the Wt11238
allele are similar, but the inheritance mode in each
locus is probably different.
Three QTLs determining pea seed yield were
identified in the F2 population. They had low
values of LOD (2.2–2.5) and explained only about
10% of total phenotypic variability of the trait
(Table 4). The additive effect of the Wt11238
allele in locus 1yd-1 was positive and the d/a
parameter value was very high (9.33), indicating
the possibility of overdominance. The allele
Wt11238 decreased seed yield in the 1yd-2
and 1yd-3 loci and suggested dominance in
the 1yd-2 locus and recessiveness in the 1yd-3
locus.
The QTL mapping of seed yield, based on
the F4 generation, showed the activity of six loci
with relatively low LOD values (2.0-2.7). Three of
them: 2yd-1, 2yd-2 and 2yd-4, explained about
10% of total trait variability. The VE values for
2yd-3 and 2yd-5 were 36.7% and 37.4%,
respectively, and for the 2yd-6 locus over 47%
(Table 5). The estimation of the additive effect
of the Wt11238 allele showed that it influenced
positively the seed yield in the 2yd-1, 2yd-4
and 2yd5 loci, but it decreased the seed yield in
the other loci discovered. The values of the parameter d/a suggested a dominance model of gene
action for the 2yd-1 and 2yd-3 loci,
and overdominance for the 2yd-4 and 2yd-6 loci.
The remaining two loci, 1yd-2 and 2yd-5,
displayed recessive inheritance.
None of the QTLs mapped in the F4 population
had the same location as loci mapped in the F2
population. However, the location of 1yd-1, 2yd-3,
2yd4 and 2yd-5 loci in LG III can suggest that
genes from that group can have an essential impact
on pea seed yield.
Two loci determining 1000-seed weight were
mapped in the F2 generation in LG I and V (Table 2), and five QTLs were localized in F4 RILs
in LG II, III (2) and V (2) (Table 3). None of the F2
QTL locations were confirmed in the F4 population. The LOD values of only two loci (2tgw-2 and
2tgw-5) exceeded 3 and they controlled 24.5%
and 15.4% of the total variability of the trait, respectively (Table 5). All QTLs localized showed
302
L Irzykowska, B Wolko
a high negative additive effect of the allele from
the WT11238 line, except 2tgw-3, which showed
a moderate positive additive effect. The d/a values
for the Wt11238 allele of genes mapped in the F2
population showed recessiveness. In the case of
QTLs localized in the F4 population the d/a values
indicated overdominance of the Wt11238 allele in
the 2tgw-3 locus, dominance in 2tgw-2
and 2tgw-4, and recessiveness in 2tgw-1
and 2tgw-5.
probable was the additive type of gene action.
It was impossible to assess unambiguously
the gene action for the prot4 locus (recessive or
additive). Our results indicate that loci mapped
in LG V influenced the trait most significantly,
due to their high LOD and VE values.
Discussion
Construction of the genetic map
Seed protein content
Five QTLs were found for protein content (Table 6). Three of them were located in LG V (prot2,
prot3 and prot4). They had high LOD values
(4.4-5.3) and controlled a large portion of the total
trait variability (18.3-25.5%). Each of them revealed a positive additive effect of the allele from
the Wt11238 line and it could increase the protein
content even by 1.5% (Table 7). Two other QTLs
mapped – prot1 in LG II and prot5 in LG VII –
showed lower LOD (2.2 and 2.4) and VE values
(13.1% and 15.7%). Both of them revealed a negative additive effect of the allele from the Wt11238
line and they decreased the trait value (1.1%
and 0.1% respectively).
The high value of the parameter d/a suggested
overdominance in the prot5 locus. For genes
mapped in the prot1, prot2 and prot3 loci, the most
Table 6. Locations of putative QTLs determining the
total protein content in pea seeds observed in the F4
generation of Pisum sativum
Trait
QTL
symbol
Linkag
e group
Map interval*
Distance**
(cM)
Seed
protein
content
prot1
prot2
prot3
prot4
prot5
II
V
V
V
VII
afp4d - C1508a
r-t
lafp6f - afp14c
afp8f - gp
afp13a - afp13e
4.5
0.0
3.8
6.0
4.0
* The nearest marker was marked in bold type.
** The distance was measured from the nearest marker to the maximum LOD peak of a QTL
Table 7. Characteristics of QTLs determining the total
protein content of pea seeds observed in the F4
generation of Pisum sativum
QTL
symbol
prot1
prot2
prot3
prot4
prot5
LOD
VE
(%)
a
d
d/a
2.2
4.4
5.3
4.9
2.4
13.1
18.3
25.5
24.3
15.7
–1.1
1.4
1.5
1.3
–0.1
–0.1
–0.1
0.2
–1.0
–1.7
0.09
–0.07
0.13
–0.77
17.00
LOD = logarithm of odds; VE = variance explained; a = additive
effect; d = dominance effect
A genetic linkage map consisting of 204 markers
(140 AFLPs, 24 RAPDs, 10 ISSRs, 5 CAPSs,
1 STS, 11 isozymes and 13 morphological markers) has been developed by mapping a hybrid population Wt 10245 × Wt 11238 (Irzykowska et al.
2001). The created map spans 2416 cM, with
an average distance between adjacent markers
of 12 cM (Figure 1). However, the length of almost 50% map intervals was shorter than 10 cM
and only 1.5% intervals were longer than 30cM.
Out of nine linkage groups (LGs) discovered,
eight have been related to the latest model map of
the Pisum genome (Weeden et al. 1998) by means
of 30 reference markers (morphological markers,
allozymes, STS and CAPS markers). The distribution of markers on the map was generally uniform,
except for LG VI, which consisted of two subsets
of markers. One LG found did not have any reference marker and therefore could not be assigned to
an appropriate chromosome.
The QTL localization was carried out by the interval mapping method, with LOD ³ 2. This was
justified considering the number of individuals in
the mapping population. Higher LOD values undoubtedly increase the reliability of QTL mapping
(Paretson 1995; Shibaike 1998) but on the other
hand it decreases the statistic chance to detect minor genes (with a low VE value), especially in
the experiments where the number of individuals
in the tested population is low (Tanksley 1993).
Other authors working with populations of a similar size established the same threshold value of
LOD. Timmerman- Vaughan et al. (1996) used
LOD ³ 2 in QTL mapping studies with a population of 102 pea F2 plants and 51 RILs. Maughan
et al. (1996), working with an F2 population
of 150 plants, used the same LOD value in comparative QTL mapping among three legume species. Higher LOD values were applied
(LOD ³ 2.5) for QTL mapping studies when population size reached 200-250 F2 plants
(Schafer-Pregl et al. 1999; Lan, Paterson 2000;
Interval mapping of QTLs in Pisum sativum
303
Lippman, Tanksley 2001). In our study, as in all studies mentioned above, quantitative traits were analysed
by MAPMAKER/ QTL software.
tified in LG V in both generations but in different
map intervals (1sn-3 and 2sn-3).
Number of QTLs identified
In F2 and F4 populations, two and five QTLs for pod
number were mapped, respectively. The approximate
localization in LG V was confirmed only for the 1pn2
and 2pn-4 loci. It is also interesting that in the 2pn-2
locus region, the gene fn determining flower number
has been mapped (Weeden et al. 1996). Its precise localization in LG II has not been described, so it is impossible to verify the identity of these genes.
The seed yield obtained from an individual
plant was tested as an element characterizing
the structure of pea seed yield. Three QTLs were
identified on the basis of F2 plant observations.
The exact localization of none of them was confirmed in the RIL population. Out of nine QTLs
identified during our 2-year study, four were
localized in LG III.
It is worth emphasizing that the 2pn-2 locus determining pod number, the 2sn-2 locus determining seed number, and the 2yd-2 locus determining
seed yield were localized exactly in the same map
interval in LG II. Similarly, the 2sn-4, 2pn-5
and 2yd-6 loci were mapped within a distance of
1 cM from the afp8e marker in LG VI
and the 1sn-4, 2sn-5 and 1yd-3 loci occupy
the same map interval in LG VII. Furthermore,
the 2pn-1 and 2-yd-1 loci were mapped at the same
interval of LG I. Such a convergence in QTL localization determining different quantitative traits indicates a clear relationship between these elements
of the pea yield structure and possibly gene
pleiotropy.
Yield-related traits
Four elements determining pea seed yield, i.e.
seed and pod number per plant, 1000-seed weight
and seed yield, were analysed in the F2 and F4 RIL
generation of the mapping population Wt10245
× Wt11238. For most traits neither the number,
nor the QTL localization was confirmed between
the two generations examined. Moreover, more
QTLs were discovered in the F4 RIL population
than in the F2 plant generation for each trait examined. Other authors reported similar results of
QTL mapping in pea (Timmerman et al. 1996),
corn (Veldboom, Lee 1996), tomato (Paterson et
al. 1991) and sugar beet (Schafer-Pregl et al.
1999). The different numbers of QTLs scored in
the F2 and RIL populations could be a consequence of environmental factors fluctuating over
the years (Paterson et al. 1991). The detected
QTLs do not explain all of the phenotypic variance
of the traits since some of the variance is due either
to environmental effects or to QTLs too small to
be detected with the number of F2 and F4 plants
analysed. Soller and Beckman (1990) suggest that
the difference between the assessment of trait expression in an F2 individual plant and a mean value
for several plants in the RIL population could be
the cause of those discrepancies. They proved that
for discovering a QTL with a minor phenotypic effect, the necessary number of F2 plants is higher
than that of recombinant lines. It seems probable
that all factors mentioned above could exert an impact on QTL identification in the presented study.
The quantitative traits were observed in different
generations and the number of F2 plants was comparable with the number of recombinant lines analysed. Furthermore, the observations of
quantitative trait expression were conducted in
field conditions in different growing seasons.
Seed number per plant
Four QTLs were localized in the F2 population,
whereas five in the RIL population. Completely
compatible are positions of the 1sn4 and 2sn5 loci
in a map interval of 3.1 cM in length in LG VII.
Considering the localization identity, similar VE
value, consistent additive effect and the same gene
action, those loci probably denote the same gene.
Another QTL determining seed number was iden-
Pod number and seed yield per plant
1000-seed weight
In the light of the relationship between seed number, pod number and seed yield, it is interesting
that position of none of tgw loci agrees with other
QTLs determining the remaining elements of
the pea yield structure. Ramsay et al. (1995) reported such a convergence in QTL mapping studies of broad bean. The seed weight loci have been
mapped for several legume species, such as broad
bean (Ramsay et al. 1995; Vaz Patto et al. 1999),
soybean (Maughan et al. 1996), cowpea (Menendez et al. 1997) and pea (Timmerman-Vaughan et
al. 1996). At least two of the five QTLs mapped on
the basis of the F4 generation measurements,
namely 2tgw-2 and 2tgw-3 (LG III), were localized in similar positions as reported by
Timmerman-Vaughan et al. (1996). The 2tgw-4
was mapped in the r-tl interval (LG V), which can
indicate an influence of the r gene (wrinkled seed)
on seed weight in pea.
304
L Irzykowska, B Wolko
Seed protein content
One of the major tasks in pea breeding is the production of cultivars with a high protein content of
seeds. Considering the great variability of storage
proteins identified in seeds, a polygenic character
of the total protein content trait was expected.
Variability of this trait was observed in seeds
yielded by the F4 plant population. Five QTL determining the total protein content of pea seeds
were mapped in three linkage groups. The prot1
locus was localized in LG II. Matta and Gatehouse
(1982) mapped the convicilin gene (cvc)
in the same group but its position on the map is
very distal from the prot1 locus. Interesting is
the relationship among three QTLs mapped
in LG V and the localization of genes encoding
the storage protein fractions. It is probable that
the prot2 locus mapped in a similar position as
the r gene (0 cM) and the vicilin gene Vc-1 localized at the same locus as the r gene by Smirnova
and Eggi (1994), are the same gene. Mahmoud
and Gatehouse (1984) reported an interesting relationship between the r gene (determining the wrinkled seed) and the Vc-1 locus. They postulated
the pleiotropic effect of the r gene not only on
the complex starch grain formation but also on
vicilin synthesis. Furthermore, the second protein
locus – prot3 in LG V – was localized close to
the Lg-1 gene, encoding the acidic subunit of
legumine, the main fraction of seed storage protein
in pea (Matta, Gatehouse 1982; Weeden et al.
1998). The next protein QTL – prot4 in LG V –
was mapped at a distance of 12 cM from the gp
marker. The vicilin gene Vc-2 has been mapped in
the same region (Ellis et al. 1993; Weeden et al.
1998), which may suggest a connection between
these loci. The alleles originating from
the large-seeded cultivar Wt10245 on each of
the loci mapped in LG V increased the protein
content of seeds by about 1.5%. The last of protein
QTLs – prot5 – was mapped in LG VII (map interval Est1-Est2). There is no information about localization of any genes encoding seed proteins in
this linkage group.
It is impossible to decide about the identity
of protein genes with QTLs discovered at this
stage of research. However, the convergence in
the earlier localization of protein genes and mapping results of genetic components of the total protein content trait suggests a significant role of
LG V in pea storage protein inheritance.
Number of QTLs localized in the same map
interval
We distinguished 10 map intervals, where two to
three QTLs were localized. Some other authors
reported similar results in QTL mapping studies
(Paterson et al. 1991; Veldboom and Lee 1996;
Shibaike 1998). There are many possible
explanations of this phenomenon. If on the basis of
2-year observations two QTLs were mapped in
the same map interval determining the same trait
and having a similar gene action type and additive
effect, this may be one gene, whose expression
weakly depends on the environment (e.g. 1sn-4
and 2sn-5).
However, in our study most of overlapping
QTLs determined different quantitative traits.
For example, on the basis of the observations of F4
plants, three QTLs determining different components of seed yield (2sn-2, 2pn-2 and 2yd-2) were
mapped in the same interval (afp9i-afp9g).
The peaks of LOD curves overlap the position of
the afp9i marker. The allele of Wt11238 decreased
the trait values and the gene action was determined
tentatively (A or R) for all QTLs. Similar observations concern the loci 2sn-4, 2pn-5 and 2yd-6
mapped in the interval Pl-afp8e of LG VI. This can
suggest a pleiotropic activity of one gene (Paterson et al. 1991). Pleiotropy is an important feature
of the genetic architecture of any quantitative trait
because most loci participate in multiple biochemical pathways. However, the activity of gene clusters controlling separate traits cannot be precluded
(Veldboom and Lee 1996). The genetic resolution
afforded by this experiment does not permit us to
distinguish between linkage and pleiotropy with
QTL mapping. Probably, a more numerous mapping population and more closely spaced markers
in the map are needed to determine whether
the QTLs correspond to one gene with pleiotropic
effects or separate but closely linked genes, each
controlling a single character (Shibaike 1998).
Assessment of gene action
One of the most common parameters describing
the type of gene action with reference to quantitative traits is the d/a ratio (Mather and Jinks 1982).
Many authors have used this approach (Edwards
et al. 1987; Paterson et al. 1991; Tanksley 1993;
Timmerman-Vaughan et al. 1996; McCallum et al.
1997; Lan and Paterson 2000; Lippman and
Tanksley 2001). It was also applied for characterization of QTLs mapped in the presented study.
In contrast to monogenic traits the alleles of
loci determining quantitative traits rarely show
complete dominance or recessiveness. The gene
actions described in respect to specific alleles vary
from complete dominance or even over-dominance to complete recessiveness, covering all
Interval mapping of QTLs in Pisum sativum
intermediate values, because of continuous
variability of quantitative traits.
However, results reported by many authors
(Edwards et al. 1987; Paterson et al. 1991;
Timmerman-Vaughan et al. 1996) as well as those
presented in our study show that in some cases it is
impossible to determine gene action unambiguously. It is difficult to determine the type of gene
action when the d/a value is intermediate
(e.g. d/a = 0.7 or d/a = –0.6). In fact, the estimation
of gene action based on results obtained in the F4
generation can be misleading because heterozygosity is reduced by half with each generation
of plant selfing.
The estimated d/a values for 6 QTLs mapped
indicate overdominance. Those loci represent
more than 18% of all QTLs mapped. Veldboom
and Lee (1996) in their QTL studies of corn
reported overdominance effects of 40% loci
mapped. As overdominance events used to be very
rare, they postulated to consider the pseudo-overdominance effect. It occurs when two tightly
linked dominant genes in the repulsion phase are
active, from which only one was identified by
interval mapping (Edwards et al. 1987; Veldboom
and Lee 1996; McCallum et al. 1997).
Utility value of results of this study
The indication of a closely linked marker was one
of the elements of QTL characterization. For 18
QTLs the linkage to the closest marker did not exceeded 1 cM. The others were mapped in a distance of 1-15 cM. Marker loci discovered could be
used in the future for mass selection, especially
those linked closer than 1 cM (Mohan et al. 1997).
However, their application in breeding practice
should be preceded by a few years’ testing of
the coefficient of QTL heritability in different environments and plant populations (Lande
and Thompson 1990; Velboom and Lee 1996).
Moreover, it is essential how many of the parameters determining the quantitative trait expression
have been identified. The number of discovered
QTLs depends to a large extent on the mapping
population size (Young 1999). The value of QTLs
identified for practical applications will be verified in the following years.
Conclusions
Our research provides some insight into the genetic complexity of traits and identifies regions of
the pea genome interesting for further investiga-
305
tion. It is noteworthy that QTLs localized in these
regions were distinguished by a high VE value.
The majority of such QTLs were discovered in
the F4 generation, e.g. 2sn-3 and 2sn-4 loci for
seed number or 2pn-3, 2pn-4, 2pn-5 for pod number (Table 5). This knowledge could be useful for
the identification, cloning and sequencing of
genes representing the particular QTL. However,
it must be emphasized that even close linkage between an agronomic trait and a marker on the genetic map does not guarantee a close physical
distance between them. Therefore, the integration
of the genetic map with the physical map seems to
be necessary (Jones et al. 1997).
Acknowledgement. The financial support of
this study as a part of the State Committee for
Scientific Research project No. 5 P06A 023 18
is gratefully acknowledged. The authors would also
like to thank Wojciech K. Œwiêcicki for plant
material supply and help in the maintenance of
the field experiment.
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