4.3 Materials and methods
5.4.2 Association mapping
Association analysis was performed with 692 molecular markers and phenotypic means of 6 environments using two models: (1) GLM and (2) K model which incorporated kinship matrix (K) as a random effect. The average relative kinship between any two genotypes was 0.057, with 76%
of the pairwise kinship estimates ranged between 0 to 0.05, indicating a low level of relatedness (Figure 5.1). Numbers of significant markers detected in both models at FDR of 20% are shown in Table 5.5. With GLM model, between 3 and 154 significant markers were found for 12 of the 14 traits. No significant marker was found associated with oleic acid content and seed weight. By taking kinship coefficient between individuals into account, the K model reduced the number of significant markers to between 1 and 11 for 6 of the 14 traits. The significant markers identified in K model were all detected in GLM. Under the assumption that the observed phenotype is not associated with the tested markers, the quantile-quantile plot should depict a uniform [0,1]
distribution from thePvalues of the association tests. As shown in Figure 5.2, the distribution was strongly skewed towards significance for GLM as compared with K model, indicating that K model would be more suitable for association analysis.
Figure 5.1: The distribution of pairwise relative kinship values between 81 canola quality winter rapeseed varieties and breeding lines.
Results of association analyses from K model are shown in detail in Table 5.6 and summarized in Table 5.7. The number of significant markers ranged from 1 (brassicasterol and total phytosterol) to 11 (24-methyl:24-ethyl sterol and oil content). The significant markers are distributed on 13 linkage
Table 5.5: Number of significant markers at FDR = 0.20
Protein of defatted meal 5
-Seed weight -
-groups: A01, A02, A03, A04, A05, A06, A07, C01, C03, C04, C05, C07 and C09. In cases where more than one marker was found on the same linkage group in a trait, significant LD between marker pairs (P≤1.4×10−7) were used as an indication that the marker pairs represent the same QTL.
For instance, 9 markers associated with campesterol that were located between 5.2 to 26.4 cM on linkage group C09 were considered to represent the same QTL because the LD was significant between the marker pairs (Table 5.6). Among the 9 associated markers on C09, marker E36M50-184 which has the lowestP-value was selected to represent QTL AM-Camp.1.
In total, between one and seven QTL were revealed by the significant markers for the 6 respective traits (Table 5.7). More QTL were detected for 24-methyl:24-ethyl sterol ratio (seven QTL) and oil content (six QTL) as compared to one QTL detected for brassicasterol, campesterol, total phytosterol, and 24-methyl sterol contents. By using one representative marker per QTL in multiple linear regression analysis, the minimum phenotypic variance explained by the QTL ranged from 14% for total phytosterol content to 47% for 24-methyl:24-ethyl sterol (Table 5.7).
From a total of 27 associated markers identified, 18 showed significant associations with one trait while 9 were found associated with more than one trait (Table 5.8). Markers that were associated with more than one trait were all located on C09 and the phenotypic effects of the shared markers were all positive for campesterol, 24-methyl sterol, total phytosterol, and oil content. Most notably, marker E45M53-229 was found associated with all the four traits.
5.4 Results 115
Figure 5.2: Quantile-quantile plots of both GLM and K model for all traits (continued on next page)
Figure 5.2: (continued from previous page) Quantile-quantile plots of both GLM and K model for all traits
5.4Results117 Table 5.6: Results of association analysis with K model
LGa Marker Marker Pos.c PEd P-value R2 Multi QTL Linkage disequilibrium (r2)f
no.b (cM) regr.e Marker. no
1 2 3 4 5 6 7 8
Brassicasterol (mg 100 g−1seed)
A04 1 E40M51-198 21.0 −9.30 2.41e−5 0.20 x AM-Bra.1
Campesterol (mg 100 gseed−1 )
C09 1 E33M47-288 5.2 35.56 2.49e−4 0.18 AM-Camp.1
-C09 2 E36M51-141 9.2 35.41 1.68e−4 0.20 AM-Camp.1 0.90
-C09 3 E45M53-229 10.6 37.39 1.24e−4 0.20 AM-Camp.1 0.81 0.90
-C09 4 E36M50-184 10.6 35.83 1.05e−4 0.21 x AM-Camp.1 0.72 0.80 0.90
-C09 5 E33M59-100 14.2 34.22 1.33e−4 0.20 AM-Camp.1 0.82 0.90 0.81 0.90
-C09 6 E32M49-403 19.5 26.88 2.26e−3 0.12 AM-Camp.1 0.53 0.60 0.68 0.60 0.53
-C09 7 E38M49-127 22.4 28.95 1.63e−3 0.13 AM-Camp.1 0.45 0.51 0.57 0.66 0.59 0.75
-C09 8 E32M60-396 25.2 35.88 9.92e−4 0.15 AM-Camp.1 0.34 0.39 0.44 0.53 0.48 0.52 0.74
-C09 9 E34M53-139 26.4 37.74 1.14e−3 0.14 AM-Camp.1 0.40 0.44 0.50 0.44 0.40 0.60 0.66 0.89
Total sterol (mg 100 gseed−1 )
C09 1 E45M53-229 10.6 44.44 1.29e−3 0.14 x AM-Total.1
24-methyl sterol (mg 100 g−1seed)
C09 1 E33M47-288 5.2 28.89 1.26e−4 0.20 AM-Methyl.1
-C09 2 E36M51-141 9.2 28.81 7.85e−5 0.22 x AM-Methyl.1 0.90
-C09 3 E45M53-229 10.6 30.53 1.22e−4 0.20 AM-Methyl.1 0.81 0.90
-C09 4 E36M50-184 10.6 26.86 3.57e−4 0.17 AM-Methyl.1 0.72 0.80 0.90
-C09 5 E33M59-100 14.2 25.63 1.98e−4 0.19 AM-Methyl.1 0.82 0.90 0.81 0.90
-C09 6 E32M49-403 19.5 22.71 1.47e−3 0.14 AM-Methyl.1 0.53 0.60 0.68 0.60 0.53
24-methyl:24-ethyl sterol
A02 1 E32M51-122 161.7 7.63 1.07e−3 0.14 x AM-MEratio.1
A05 1 E42M55-165 90.1 −8.98 1.96e−3 0.13 x AM-MEratio.2
Continued on next page
118
LGa Marker Marker Pos.c PEd P-value R2 Multi QTL Linkage disequilibrium (r2)f
no.b (cM) regr.e Marker. no
1 2 3 4 5 6 7 8
A06 1 E37M62-236 56.4 10.72 1.60e−3 0.13 AM-MEratio.3
-A06 2 E32M59-334 56.9 11.52 1.52e−4 0.20 x AM-MEratio.3 0.71
-A06 3 E32M49-205 56.9 −11.28 1.34e−3 0.14 AM-MEratio.3 0.68 0.45
A06 4 E39M49-368 56.9 −11.27 1.98e−3 0.13 AM-MEratio.3 0.75 0.51 0.90
-A06 5 E33M51-174 63.1 10.16 3.92e−3 0.11 AM-MEratio.3 0.55 0.46 0.53 0.46
A07 1 E40M50-127 93.0 −8.22 2.98e−3 0.12 x AM-MEratio.4
C03 2 E36M56-172 15.0 −5.92 1.84e−3 0.13 x AM-MEratio.5
C05 1 E36M57-089 50.8 −7.38 2.29e−3 0.12 x AM-MEratio.6
C07 2 E39M62-176 50.6 7.19 3.72e−3 0.11 x AM-MEratio.7
Oil (%)
A01 1 E39M62-071 74.0 1.72 1.86e−3 0.13 x AM-Oil.1
A03 1 E38M59-213 61.9 1.72 2.87e−3 0.12 x AM-Oil.2
-A03 2 E38M51-252 100.4 −1.02 1.13e−3 0.14 x AM-Oil.3 0.05
C01 1 E36M51-355 106.7 0.92 2.53e−3 0.12 x AM-Oil.4
C04 1 E32M47-182 35.7 −1.08 8.40e−4 0.15 x AM-Oil.5
C09 1 E45M53-229 10.6 1.86 1.12e−3 0.14 AM-Oil.6
-C09 2 E36M50-184 10.6 2.05 1.31e−4 0.20 x AM-Oil.6 0.90
-C09 3 E38M49-127 22.4 1.70 5.93e−4 0.16 AM-Oil.6 0.57 0.66
-C09 4 E32M60-396 25.2 2.10 2.41e−4 0.18 AM-Oil.6 0.44 0.53 0.74
-C09 5 E34M53-139 26.4 1.91 2.20e−3 0.13 AM-Oil.6 0.50 0.44 0.66 0.89
-C09 6 E39M55-408 31.3 1.97 6.66e−4 0.16 AM-Oil.6 0.44 0.53 0.74 0.79 0.68
aLinkage group;
bMarker number on each linkage group
cPosition of the marker on the respective linkage group;
dPhenotypic effect (See Table 5.3 for units of measurements); positive sign: visible marker allele increases trait; negative sign: visible marker allele decreases trait;
eMarkers selected (marked with ‘x’)to represent the QTL in muliple regression analysis;
fLinkage disequilibrium between marker pairs on the same linkage group. Significantr2values are indicated in bold
5.4 Results 119
Table 5.7: Summary of association analysis
Trait K model Multiple regression
No. of No. of Phenotypic effectc No. of Adj.
markersa LGb Min Max QTLd R2
Phytosterols (mg 100 g−1seed)
Brassicasterol 1 1 9.30 9.30 1 0.19
Campesterol 9 1 26.88 37.74 1 0.21
Sitosterol - - - - -
-Avenasterol - - - - -
-Total sterol 1 1 44.44 44.44 1 0.14
24-methyl sterol 6 1 22.71 30.53 1 0.22
24-ethyl sterol - - - - -
-Campesterol:sitosterole - - - - -
-24-methyl:24-ethyl sterole 11 7 5.92 11.52 7 0.47
Other traits
-aNumber of significantly associated markers
bNumber of linkage groups with significant markers
cAbsolute value of minimal and maximal phenotypic effect of significant markers
dNumber of representative markers used to determine the minimal phenotypic variance explained by the QTL
eoriginal value (ratio)×100
120
LGa Markers Pos.c Traits
Brassica- Campe- Total 24-methyl 24-methyl:24- Oil
sterol sterol sterol sterol ethyl sterol
PEc QTL PE QTL PE QTL PE QTL PE QTL PE QTL
A01 E39M62-071 74.00 1.72 AM-Oil.1
A02 E32M51-122 161.70 7.63 AM-MEratio.1
A03 E38M59-213 61.90 1.72 AM-Oil.2
E38M51-252 100.40 −1.02 AM-Oil.3
A04 E40M51-198 21.05 −9.30 AM-Bra.1
A05 E42M55-165 90.10 −8.98 AM-MEratio.2
A06 E37M62-236 56.40 10.72 AM-MEratio.3
E32M49-205 56.90 −11.28 AM-MEratio.3
E32M59-334 56.90 11.52 AM-MEratio.3
E39M49-368 56.90 −11.27 AM-MEratio.3
E33M51-174 63.10 10.16 AM-MEratio.3
A07 E40M50-127 93.00 −8.22 AM-MEratio.4
C01 E36M51-355 106.7 0.92 AM-Oil.4
C03 E36M56-172 15.00 −5.92 AM-MEratio.5
C04 E32M47-182 35.7 −1.08 AM-Oil.5
C05 E36M57-089 50.80 −7.38 AM-MEratio.6
C07 E39M62-176 50.60 7.19 AM-MEratio.7
C09 E33M47-288 5.20 35.56 AM-Camp.1 28.89 AM-Methyl.1
E36M51-141 9.20 35.41 AM-Camp.1 28.81 AM-Methyl.1
E36M50-184 10.60 35.83 AM-Camp.1 26.86 AM-Methyl.1 2.05 AM-Oil.6
E45M53-229 10.60 37.39 AM-Camp.1 44.44 AM-Total.1 30.53 AM-Methyl.1 1.86 AM-Oil.6
E33M59-100 14.20 34.22 AM-Camp.1 25.63 AM-Methyl.1
E32M49-403 19.50 26.88 AM-Camp.1 22.71 AM-Methyl.1
E38M49-127 22.40 28.95 AM-Camp.1 1.70 AM-Oil.6
Continued on next page
5.4Results121
Table 5.8–Continued from previous page
LGa Markers Pos.b Traits
Brassica- Campe- Total 24-methyl 24-methyl:24- Oil
sterol sterol sterol sterol ethyl sterol
PEb QTL PE QTL PE QTL PE QTL PE QTL PE QTL
E32M60-396 25.20 35.88 AM-Camp.1 2.10 AM-Oil.6
E34M53-139 26.40 37.74 AM-Camp.1 1.91 AM-Oil.6
E39M55-408 31.30 1.97 AM-Oil.6
aLG: linkage group
bMarker position on the respective linkage group
cPE Phenotypic effect. Positive sign: visible marker allele increases trait; negative sign: visible marker allele decreases trait