Phenotypic evaluation and correlation
In both groups, the absolute values of kurtosis and skewness were less than 1 (Table 1), and the five traits conformed to a normal distribution (Figures S1 and S2). The HB2 of the five traits, averaged across 2 years, ranged from 62.19 % to 93.48 %. The coefficient of genetic variation of the five traits ranged from 13.21 % to 43.16 % (Table 1). In general, four of the vigor traits (excluding HL) under the improved lines had higher values than those under soybean landraces.
Table 1
Analysis on the variation of vigor traits of 257 improved lines and 104 landraces soybean accessions
Traits
|
Years
|
257 improved lines accessions
|
104 landrace accessions
|
Mean ± SD
|
K
|
S
|
CV (%)
|
HB2 (%)
|
Mean ± SD
|
K
|
S
|
CV (%)
|
HB2 (%)
|
FW (g)
|
2018
|
2.06 ± 0.37
|
0.12
|
0.12
|
18.08
|
82.04
|
1.82 ± 0.33
|
2.05
|
0.41
|
18.19
|
93.48
|
2019
|
2.04 ± 0.37
|
0.10
|
0.17
|
18.05
|
1.80 ± 0.32
|
2.25
|
0.62
|
17.92
|
SL (cm)
|
2018
|
8.88 ± 1.76
|
0.01
|
-0.15
|
19.76
|
90.81
|
8.61 ± 1.63
|
0.18
|
-0.27
|
18.95
|
87.35
|
2019
|
8.54 ± 1.56
|
0.47
|
-0.26
|
18.32
|
8.41 ± 1.49
|
0.57
|
-0.27
|
17.67
|
RL (cm)
|
2018
|
19.20 ± 4.06
|
-0.30
|
0.24
|
21.17
|
90.71
|
17.89 ± 3.82
|
0.53
|
0.53
|
21.37
|
93.05
|
2019
|
18.01 ± 4.07
|
-0.07
|
0.24
|
22.61
|
17.50 ± 3.78
|
1.33
|
0.58
|
21.60
|
HL (cm)
|
2018
|
9.13 ± 1.21
|
-0.31
|
-0.11
|
13.21
|
84.17
|
9.22 ± 1.70
|
-0.55
|
-0.18
|
18.43
|
87.88
|
2019
|
9.23 ± 1.36
|
0.16
|
0.02
|
14.71
|
9.42 ± 1.82
|
-0.65
|
-0.10
|
19.32
|
GP (%)
|
2018
|
61.24 ± 21.76
|
-0.57
|
-0.39
|
35.54
|
74.40
|
59.71 ± 21.18
|
-0.22
|
-0.54
|
35.47
|
62.19
|
2019
|
58.38 ± 16.83
|
-0.38
|
-0.27
|
28.83
|
42.95 ± 18.53
|
-0.44
|
0.23
|
43.16
|
Abbreviations: FW, seedling fresh weight; SL, seedling length; RL, main root length; HL, hypocotyl length; GP, germination percentage; SD, standard deviation; K, Kurtosis; S, Skewness; CV, coefficient of variation; HB2, heritability in the broad sense |
The Pearson correlation of the five traits showed there was a significant positive and negative correlation between the five traits of improved lines and soybean landraces (Table 2). There was no significant correlation between HL and FW, SL, whereas HL and RL were significantly negatively correlated (Table 2).
Table 2
Pearson correlation of five traits of improved lines and landraces soybean accessions
Populations
|
Traits
|
FW
|
SL
|
RL
|
HL
|
Improved lines
|
SL
|
0.378**
|
|
|
|
RL
|
0.373**
|
0.373**
|
|
|
HL
|
−0.101
|
−0.023
|
−0.155*
|
|
GP
|
0.436**
|
0.258**
|
0.446**
|
−0.131*
|
Landraces
|
SL
|
0.336**
|
|
|
|
RL
|
0.385**
|
0.220*
|
|
|
HL
|
−0.057
|
0.109
|
−0.218*
|
|
GP
|
0.340**
|
0.462**
|
0.306**
|
0.079
|
Note: * and ** denote significant differences at the 0.05 and 0.01 probability levels, respectively |
Molecular Markers And Genetic Structure
Of the 175 SSR markers that were polymorphic, a total of 844 alleles were detected. The number of alleles ranged from 2 to 12, with a mean of 4.82 alleles per marker. Genetic diversity ranged from 0.01 to 0.85, with a mean of 0.50. Polymorphic information content (PIC) ranged from 0.01 to 0.85, with a mean of 0.45 (Table S3). In comparison, higher values were observed for the numbers of alleles, genetic diversity, and PIC in soybean landraces than those in improved lines (Table S4).
STRUCTURE software analysis showed that the 175 markers were relatively independent. The log-likelihood function [Ln P(D)] has no obvious inflection point (Fig. 1A). Therefore, the suitable value of K was determined using Delta K; here, three subpopulations were detected (Fig. 1B). For improved lines, the PopⅠ, PopⅡ, and PopⅢ subpopulations contained 62, 81, and 114 accessions (Fig. 2A), respectively. For soybean landraces, the PopⅠ, PopⅡ, and PopⅢ subpopulations contained 41, 38, and 25 accessions (Fig. 2B), respectively.
Linkage Disequilibrium
LD of allelic variation between SSR loci is the basis of association analysis. Considering that group mixing leads to the enhancement of LD, an LD analysis of both the improved lines and the soybean landraces was carried out. For the improved lines, a total of 14,992 pairs were detected, which included both inter-and intra-chromosomal combinations (Fig. 3A). The SSR pairs of loci with LD supported by P < 0.01 occupied 19.66 % of the total population (Fig. 3A). The D' of linked marker pairs, supported by the P < 0.01 mean, was 0.38 (Table 3). For the soybean landraces, a total of 14,834 pairs were detected, which included both inter-and intra-chromosomal combinations (Fig. 3B). The SSR pairs of loci with LD, supported by P < 0.01, occupied 6.38 % of the total population (Fig. 3A). The D' of linked marker pairs, supported by the P < 0.01 mean, was 0.44 (Table 3). The study revealed that the improved lines had more LD locus pairs than soybean landraces, whereas soybean landraces had higher D' values than improved lines. This suggests that the history of soybean landraces has undergone more reorganization, retaining higher LD.
Table 3
Comparison of D’ of LD for pairwise SSR loci between improved lines and landraces soybean accessions
Populations
|
No. of LDa locus pairs
|
Frequency distribution of D'b (P < 0.01)
|
Mean of D'b
|
0-0.2
|
0.2–0.4
|
0.4–0.6
|
0.6–0.8
|
0.8-1
|
Improved lines
|
208(29.71 %)
|
15
|
127
|
38
|
21
|
7
|
0.38
|
Landraces soybean
|
83(11.93 %)
|
1
|
34
|
36
|
9
|
3
|
0.44
|
Note: a LD means linkage disequilibrium; b D' means standardized disequilibrium coefficients |
Regression analysis of the D' value and the genetic distance shows that the attenuation of the D' value follows the Y=b ln (x) ་ c derivative. The D' between SSR loci decreased with increasing genetic distance for improved lines and soybean landraces (Figs. 4A and 4B). The minimum (0.5) distance of LD decay for improved lines and landraces was 2.53 cM and 1.46 cM, respectively. It can thus be seen that the LD decay rate of soybean landraces was slow, whereas that of improved lines was fast.
Association Mapping
Using the GLM program of marker-trait association, and based on the threshold (p < 0.01), 19 and 10 SSR loci were revealed in the two populations in 2018 and 2019, respectively (Table S5). Two years were co-detected. The SSR markers for improved lines were seven for FW, two for SL, two for RL, one for HL, and two for GP, and the SSR markers for soybean landraces were three for FW, two for SL, two for RL, one for HL and two GP (Table 3).
Association mapping identified seven markers for FW in the improved lines. The range of phenotypic variation explained (PVE) was from 5.95 % to 12.08 %, of which Sat_256_Chr7 accounted for maximum phenotypic variations: 12.08 % in 2018 and 11.32 % in 2019 (Table 4). There were two markers associated with mapping for SL; the range of PVE was from 6.35 % to 13.35 %, of which Satt441_Chr7 accounted for the maximum phenotypic variations, viz. 13.35 % in 2018 and 2019 (Table 4). There were two markers of association mapping for RL. The range of PVE was from 7.09 % to 12.11 %, of which Satt329_Chr8 accounted for maximum phenotypic variations: 12.11 % in 2018 and 11.32 % in 2019 (Table 4). There was one marker of association mapping with HL, located on nine chromosomes, which accounted for phenotypic variations of 8.58 % in 2018 and 10.22 % in 2019 (Table 4). There were two markers of association mapping for GP. The range of PVE was from 6.98 % to 10.79 %, of which Satt303_Chr18 accounted for maximum phenotypic variations: 10.79 % in 2018 and 6.98 % in 2019 (Table 4). Three markers were co-associated with two or three vigor traits: Aw277661, Satt441, and Satt606 (Table 4).
Table 4
Simple sequence repeat (SSR) marker significantly associated with five traits (P < 0.01)
Traits
|
SSR
marker
|
Chr.
|
Position
(cM)
|
Start position
(bp)
|
End position
(bp)
|
2018
|
2019
|
P value
|
PVE(%)
|
P value
|
PVE(%)
|
Improved lines
|
FW
|
Sat_266
|
3
|
47.27
|
36046708
|
36046763
|
0.0085
|
5.95
|
0.0031
|
6.57
|
Aw277661
|
3
|
74.49
|
32803245
|
32803313
|
0.0004
|
6.25
|
0.0005
|
6.37
|
Sat_256
|
7
|
74.52
|
17606889
|
17606932
|
0.0001
|
12.08
|
0.0008
|
11.32
|
Satt441
|
9
|
46.2
|
11271676
|
11271771
|
0.0001
|
8.59
|
0.0001
|
8.17
|
Satt577
|
14
|
6.05
|
668802
|
668837
|
0.0001
|
9.07
|
0.0001
|
9.41
|
Satt606
|
15
|
39.77
|
14918728
|
14918821
|
0.0001
|
8.34
|
0.0001
|
9.23
|
Sat_228
|
16
|
23.91
|
3049971
|
3050050
|
0.0019
|
7.96
|
0.0011
|
8.32
|
SL
|
Aw277661
|
4
|
74.49
|
32803245
|
32803313
|
0.0003
|
7.44
|
0.0016
|
6.35
|
Satt441
|
9
|
46.2
|
11271676
|
11271771
|
0.0001
|
13.35
|
0.0001
|
13.35
|
RL
|
Satt329
|
8
|
110.94
|
21161155
|
21161226
|
0.0001
|
12.11
|
0.0001
|
11.32
|
Satt606
|
15
|
39.77
|
14918728
|
14918821
|
0.0005
|
7.09
|
0.0001
|
11.74
|
HL
|
Satt588
|
9
|
117.01
|
45420963
|
45421065
|
0.0059
|
8.58
|
0.0011
|
10.22
|
GP
|
Satt606
|
15
|
39.77
|
14918728
|
14918821
|
0.0001
|
10.79
|
0.0005
|
6.98
|
Satt413
|
17
|
113.61
|
40043052
|
40043061
|
0.0029
|
8.34
|
0.0031
|
7.84
|
Landraces soybean
|
FW
|
Sat_378
|
8
|
116.62
|
23605177
|
23605222
|
0.0001
|
27.21
|
0.0003
|
23.11
|
Satt262
|
10
|
57.02
|
35510863
|
35510922
|
0.0004
|
18.38
|
0.0005
|
18.44
|
Satt509
|
11
|
32.5
|
6206957
|
6207049
|
0.0072
|
14.18
|
0.0040
|
14.96
|
SL
|
Satt291
|
6
|
45.75
|
7320677
|
7320718
|
0.0061
|
12.74
|
0.0037
|
13.31
|
Sat_378
|
8
|
116.62
|
23605177
|
23605222
|
0.0059
|
16.19
|
0.0093
|
16.47
|
RL
|
Sat_337
|
4
|
32.09
|
4172809
|
4172860
|
0.0070
|
16.27
|
0.0031
|
18.2
|
Satt571
|
20
|
18.5
|
1291809
|
1291850
|
0.0013
|
12.37
|
0.0011
|
12.74
|
HL
|
Satt304
|
14
|
65.55
|
13284499
|
13284588
|
0.0013
|
15.36
|
0.0049
|
12.6
|
GP
|
Sat_378
|
8
|
116.62
|
23605177
|
23605222
|
0.0009
|
21.08
|
0.0001
|
28.13
|
Satt509
|
11
|
32.5
|
6206957
|
6207049
|
0.0022
|
15.07
|
0.0021
|
13.8
|
Abbreviations: FW, seedling fresh weight; SL, seedling length; RL, main root length; HL, hypocotyl length; GP, germination percentage; Chr., chromosome; PVE, phenotypic variations explained |
In the soybean landraces, there were three markers associated with mapping for FW; the range of PVE was from 14.18 % to 27.21 %, of which Sat_378_Chr8 accounted for maximum phenotypic variations of 27.21 % in 2018 and 23.11 % in 2019 (Table 4). There were two markers of association mapping for SL. The range of PVE was from 12.74 % to 16.47 %, and Sat_378_Chr8 accounted for maximum phenotypic variations: 16.19 % in 2018 and 16.47 % in 2019 (Table 4). There were two markers of association mapping for RL. The range of PVE was from 12.37 % to 18.2 %, and Sat_337_Chr4 accounted maximum phenotypic variations of 16.27 % in 2018 and 18.2 % in 2019 (Table 4). There was one marker of association mapping for HL, located on 14 chromosomes, which accounted for phenotypic variations: 15.36 % in 2018 and 12.6 % in 2019 (Table 4). There were two markers of association mapping for GP. The range of PVE was from 13.8 % to 28.13 %, and Sat_378_Chr8 accounted for maximum phenotypic variations: 21.08 % in 2018 and 28.13 % in 2019 (Table 4). Two markers were co-associated with two or three vigor traits, namely Sat_378 and Satt509 (Table 4).
Mining Of Elite Alleles And Carrier Materials
The alleles and carrier materials for the loci associated with the five vigor traits were identified (Table S6). In the improved lines, Sat_256-236bp had the greatest positive effect (+ 0.32 g) for FW. The carrier material was Hefeng 48 (Table S6). Satt441-281bp had the greatest positive effect (+ 1.21 cm) for SL, and the carrier material was Heilong 66 (Table S6). Satt329-262bp had the greatest positive effect (+ 3.58 cm) for RL, and the carrier material was Heinong 44 (Table S6). Satt588-128bp had the greatest positive effect (+ 0.99 cm) for HL, and the carrier material was Jiunong 66 (Table S6). Satt413-196bp had the greatest positive effect (+ 12.94 %) for GP, and the carrier material was Heinong 44 (Table S6). Some of the phenotypic effect values of locus-allelic viz., Aw277661-266bp, Satt588-185bp, and Satt441-294bp had positive effects, and the elite allele frequencies were 78.21 %, 74.30 %, and 73.05 %, respectively. This may be because of strong artificial selection.
In the soybean landraces, Sat_378-168bp had the greatest positive effect (+ 0.18 g) for FW, and the carrier material was Keshandajinhuang (Table S6). Sat_378-156bp had the greatest positive effect (+ 0.62 cm) for SL, and the carrier material was (Table S6). Sat_337-281bp had the greatest positive effect (+ 3.48 cm) for RL, and the carrier material was Longyoutai (Table S6). Satt304-169bp had the greatest positive effect (+ 0.41 cm) for HL, and the carrier material was Keshandajinhuang (Table S6). Sat_378-156bp had the greatest positive effect (+ 7.96 %) for GP, and the carrier material was Fangzhengbailudou (Table S6). Some of the phenotypic effect values of locus-allelic viz., Satt571-135bp, Satt509-189bp, Satt262-256bp, and Satt291-223bp had positive effects, and the elite allele frequencies were 94.23 %, 81.55 %, 76.92 %, and 70.19 %. This may be because of the long-term natural selection.
Design For The Positive Elite Crosses Combinations
The two best cross combinations for improving seed vigor traits were identified (Table 5), based on the elite alleles detected. The positive effect of elite alleles on five seed vigor traits, namely Suinong 26, Hefeng 48, Heilong 44, Dalihuang and Keshandajinhuang emerged repeatedly (Table S6), suggesting that these materials carry superior fragments and are more likely to improve the corresponding characteristics in practical breeding if multiple vigor traits are improved simultaneously, and all superior genes are polymerized in one variety to the greatest extent.
Table 5
The positive elite crosses predicted by association mapping with five traits
Traits
|
Parental combinations
|
No. of elite alleles predicted
|
Fresh weight
|
Suinong 26 × Dalijuang
|
10
|
Suinong 26 × Keshandajinhuang
|
8
|
Seedling length
|
Heimong 66 × Tiejiasilihuang
|
3
|
Heimong 66 × Ninganxiaoheidou
|
3
|
Root length
|
Heinong 44 × Helongyoutai
|
4
|
Heinong 37 × Helongyoutai
|
3
|
Hypocotyl length
|
Jiunong 1 × Keshandajinhuang
|
2
|
Dengke 1 × Keshandajinhuang
|
2
|
Germination percentage
|
Heinong 44 × Fangzhengbailudou
|
2
|
Hefeng 24 × Xiaoheiqi
|
2
|
Association mapping of seed vigor in spring soybean (Glycine max (L.) Merr.) in northeast China |