Phenotypic analyses
The two parent materials showed significant differences in HSW, SA, SP, SL, SW, SC-R, SC-G, SC-B, FC, TC, StC, and LC (Table 1). The statistical results of the phenotypic variations in the seed-related traits among the parents, F2 populations, and F2:3 individuals (Table S1) suggested that HSW, SA, SP, SL, SW, SLWR, PC, StC, FC, LC, and TC showed continuous variation. The absolute values of skewness and kurtosis were almost less than 1, approximately conforming to the normal distribution, meeting the requirements of QTL analysis (Fig. 1; Table 1).
Table 1
Details of average of F2, two environments of F2:3 individuals and their parents
Trait | Parents | Population |
WY7 | TCX7 | Min | Max | Mean | SD | Variance | CV% | Skewness | Kurtosis | |
HSW | 133.05** | 245.54** | 113.62 | 230.34 | 170.20 | 27.72 | 762.07 | 16.29 | 0.23 | -0.69 | |
SA | 290.20 ** | 526.83 ** | 245.00 | 484.25 | 360.25 | 59.28 | 3485.39 | 16.46 | 0.24 | -0.85 | |
SP | 65.46** | 89.15 ** | 55.94 | 87.59 | 73.38 | 6.53 | 42.26 | 8.90 | 0.08 | -0.59 | |
SL | 22.56 ** | 30.61 ** | 19.18 | 29.60 | 25.22 | 2.13 | 4.52 | 8.46 | 0.00 | -0.46 | |
SW | 16.00** | 21.62** | 13.56 | 20.88 | 17.63 | 1.55 | 2.37 | 8.77 | 0.09 | -0.65 | |
SLWR | 1.43 | 1.42 | 1.30 | 1.53 | 1.44 | 0.05 | 0.00 | 3.46 | -0.33 | -0.35 | |
ST | 9.03 | 9.97 | 6.67 | 10.92 | 8.93 | 0.71 | 0.49 | 7.91 | -0.03 | 0.28 | |
SC-R | 64.22** | 136.62 ** | 53.34 | 158.93 | 94.16 | 31.80 | 1002.93 | 33.77 | 0.64 | -1.12 | |
SC-G | 56.89* | 119.36 * | 46.84 | 137.25 | 80.35 | 25.73 | 656.80 | 32.03 | 0.70 | -0.98 | |
SC-B | 61.94 * | 91.37 * | 54.26 | 104.06 | 74.90 | 11.92 | 141.00 | 15.92 | 0.49 | -0.70 | |
FC | 5.67 * | 10.95* | 3.02 | 12.83 | 7.98 | 2.06 | 4.19 | 25.78 | -0.15 | -0.35 | |
TC | 0.53 ** | 0.65 ** | 0.43 | 0.72 | 0.57 | 0.06 | 0.00 | 9.84 | 0.16 | -0.27 | |
StC | 32.66 * | 35.95 * | 29.56 | 37.58 | 33.95 | 1.53 | 2.32 | 4.50 | -0.23 | 0.42 | |
PC | 30.90 | 31.40 | 28.19 | 34.05 | 31.12 | 1.22 | 1.48 | 3.93 | -0.19 | -0.29 | |
LC | 1.30 * | 1.01 * | 0.83 | 1.48 | 1.12 | 0.13 | 0.02 | 11.53 | 0.07 | -0.23 | |
SD standard deviation, CV coefficient of variation, HSW 100-seed weight (g), SA seed surface area (mm2), SP seed perimeter (mm), SL seed length (mm), SW seed width (mm), SLWR seed length and width ratio, ST seed thickness (mm), SC-R seed coat color R value, SC-G seed coat color G value, SC-B seed coat color B value, FC fiber content (%), TC tannin content (%), StC starch content (%), PC protein content (%), LC lipid content (%). Significant differences between two parental lines WY7 and TCX7 are marked by * and **, which were determined by the Student’s t test at P < 0.05 and P < 0.01, respectively. |
Correlation analyses among different traits
Significant Pearson’s correlations (p < 0.01) for the same trait showed a significant positive relationship between the F2 and F2:3 populations in Xueyao and Jiuhua (Table S2). Phenotypic correlations (p < 0.01) among the different traits are shown in Fig. 2. Seed shape traits, including HSW, SA, SP, SL, and SW, were positively correlated with PC, and negatively correlated with StC. Seed coat color traits, including SC-R, SC-G, and SC-B, were positively correlated with FC and SC and negatively correlated with LC. There was no significant correlation between the seed shape traits and seed coat color traits in this study.
Genetic map construction
A total of 121 F2 plants and their parents were genotyped using 130,514 SNPs in the Faba_bean_ 130 K SNP TNGS genotyping platform, showing excellent results, quality, and matching scores (Tables S3 and S4). There were 12,023 SNP-tagged gene microarrays with polymorphism between parents (Table S5), and they were successfully genotyped into “A,” “B,” and “H” types in the population. All co-isolated markers were defined as one bin, and 1106 bin markers were used to construct a genetic map containing 6 LGs. The overall length of the genetic map was 1182.65 cM with an average marker spacing of 0.098 cM. Each LG range was from 157.08–296.82 cM, and the average distance between markers was from 0.079–0.114 cM. LG1 had the largest number of markers with 3325 SNPs. The smallest gap identified in the map was 0.826 cM, the total number of gaps > 5 cM was 9, and the largest gap was 11.78 cM LG6. Additionally, the ratio of marker intervals < 5 cM for all LGs was > 97% (Fig. 3; Table 2).
Table 2
Summary of the consensus reference genetic map of faba bean in this study
Linkage groups | SNP count | Bin count | Length (cM) | Average interval (cM) | Largest gap size (cM) | Numbers of gaps > 5 cM |
LG1 | 3325 | 285 | 296.818 | 0.089 | 5.811 | 1 |
LG2 | 2207 | 173 | 173.630 | 0.079 | 6.651 | 1 |
LG3 | 2121 | 184 | 182.697 | 0.086 | 4.975 | 0 |
LG4 | 1758 | 157 | 157.084 | 0.089 | 4.975 | 0 |
LG5 | 1597 | 156 | 181.963 | 0.114 | 6.650 | 3 |
LG6 | 1015 | 151 | 190.461 | 0.188 | 11.784 | 4 |
Total | 12023 | 1106 | 1182.653 | 0.098 | 11.784 | 9 |
QTL analysis
QTL mapping was performed using QTL lciMapping and QTL-Cart CIM, and 65 (Table S6) and 50 (Table S7) QTLs were identified for all 15 seed-related traits detected in the F2 and F2:3 populations, respectively. Together, these two mapping strategies identified 28 overlapping QTLs (Table S8). Of these, the QTL intervals observed using the CIM method were usually wider, whereas the intervals from the ICIM method were narrower. Consequently, the results obtained using the lCIM method were used in this study. The genetic effect (the explanation rate of phenotyte variance or VG/VP) of the QTLs detected using ICIM for 15 seed-related traits ranged from 4.90–73.99%, with peak LOD values ranging from 4.48–35.25 (Table S6). Among the 65 loci, there were 11 QTLs that were detected for more than two traits (Table S9). There were 39 QTLs identified that individually accounted for > 10% of the phenotypic variation (Table 3) and 1 QTL explained < 5% of the phenotypic variation (Table S6). A total of 41 and 21 QTLs were found to have positive and negative additive effects, respectively.
Table 3
QTLs distribution of 15 seed-related traits with responsible for more than 10% of the explained phenotypic variation
Trait | LG | QTL | environment | Left maker | Right maker | VG/VP (%) | Peak LOD | Add |
name | pos | name | pos |
HSW | 5 | qHSW5.1 | F2(2020) | yDN135233_c2_g1_2027 | 19.5 | yDN120969_c0_g1_320 | 22.5 | 35.84 | 17.81 | 345.255 |
| | | F2:3-JH(2021) | yDN135233_c2_g1_2027 | 19.5 | yDN120969_c0_g1_320 | 22.5 | 38.88 | 15.79 | 214.079 |
| | qHSW5.2 | F2:3-XY(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 25.71 | 10.42 | 228.937 |
SA | 5 | qSA5.1 | F2(2020) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 51.51 | 18.41 | 81.633 |
| | | F2:3-XY(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 40.33 | 15.13 | 54.410 |
| | qSA5.2 | F2:3-JH(2021) | yDN135233_c2_g1_2027 | 18.5 | yDN120969_c0_g1_320 | 22.5 | 39.64 | 24.81 | 49.392 |
| | qSA5.3 | F2:3-JH(2021) | hDN150254_c0_g5_96 | 26.5 | dDN52935_c3_g4_215 | 28.5 | 15.19 | 11.34 | 5.348 |
SP | 5 | qSP5.1 | F2(2020) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 46.06 | 15.55 | 8.962 |
| | | F2:3-JH(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 55.47 | 23.55 | 5.636 |
| | | F2:3-XY(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 40.13 | 14.82 | 5.828 |
SL | 5 | qSL5.1 | F2(2020) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 42.65 | 14.06 | 2.724 |
| | | F2:3-JH(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 48.32 | 28.30 | 1.756 |
| | | F2:3-XY(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 35.96 | 13.79 | 1.904 |
SW | 5 | qSW5.1 | F2(2020) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 39.37 | 14.92 | 1.857 |
| | | F2:3-JH(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 42.93 | 22.55 | 1.165 |
| | | F2:3-XY(2021) | yDN151173_c1_g2_816 | 18.5 | yDN135233_c2_g1_2027 | 19.5 | 36.78 | 12.90 | 1.356 |
| 6 | qSW 6.1 | F2(2020) | yDN128644_c0_g1_417 | 71.5 | hDN154119_c0_g2_776 | 75.5 | 10.69 | 5.07 | 0.866 |
SLWR | 3 | qSLWR 3.1 | F2:3-JH(2021) | yDN154982_c0_g1_462 | 95.5 | yDN150491_c1_g1_2729 | 96.5 | 18.23 | 6.37 | 0.004 |
| | qSLWR 3.2 | F2:3-XY(2021) | hDN149791_c1_g1_342 | 129.5 | yDN133005_c0_g1_512 | 130.5 | 17.24 | 11.11 | -0.036 |
| | qSLWR 3.3 | F2:3-XY(2021) | yDN155504_c1_g2_308 | 147.5 | yDN155504_c1_g2_253 | 149.5 | 16.78 | 9.38 | -0.036 |
| 6 | qSLWR 6.1 | F2(2020) | yDN145987_c0_g1_369 | 67.5 | yDN138086_c0_g1_82 | 71.5 | 25.02 | 7.34 | -0.046 |
| | | F2:3-XY(2021) | yDN145987_c0_g1_369 | 67.5 | yDN138086_c0_g1_82 | 70.5 | 10.40 | 6.21 | 0.032 |
ST | 4 | qST4.1 | F2:3-XY(2021) | hDN131761_c0_g1_1016 | 45.5 | hDN122802_c0_g1_447 | 46.5 | 16.90 | 12.25 | 0.127 |
| 5 | qST5.1 | F2(2020) | yDN131562_c0_g2_1383 | 23.5 | dDN54339_c2_g2_203 | 24.5 | 24.78 | 7.29 | 0.646 |
| | | F2:3-JH(2021) | yDN131562_c0_g2_1383 | 23.5 | dDN54339_c2_g2_203 | 24.5 | 19.19 | 9.38 | 0.452 |
| | qST5.2 | F2:3-JH(2021) | hDN154491_c1_g8_222 | 98.5 | hDN152331_c2_g3_311 | 99.5 | 10.07 | 4.78 | 0.123 |
| | qST5.3 | F2:3-XY(2021) | hDN148575_c1_g1_723 | 7.5 | yDN151173_c1_g2_935 | 9.5 | 10.66 | 8.58 | 0.475 |
SC-R | 1 | qSC-R1.1 | F2(2020) | hDN132853_c1_g2_224 | 71.5 | dDN45140_c0_g1_2482 | 72.5 | 68.60 | 35.13 | 24.007 |
| | qSC-R1.2 | F2(2020) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 12.90 | 11.02 | 11.943 |
| | | F2:3-JH(2021) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 73.99 | 34.76 | 35.411 |
| | | F2:3-XY(2021) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 65.35 | 29.45 | 34.151 |
SC-G | 1 | qSC-G1.1 | F2(2020) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 51.48 | 35.25 | 17.308 |
| | | F2:3-JH(2021) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 66.97 | 28.88 | 25.116 |
| | | F2:3-XY(2021) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 68.34 | 33.01 | 36.541 |
| | qSC-G1.2 | F2(2020) | yDN157063_c3_g3_806 | 295.5 | yDN157063_c3_g3_836 | 296 | 12.36 | 12.19 | -11.891 |
SC-B | 1 | qSC-B1.1 | F2(2020) | yDN147029_c0_g1_601 | 56.5 | yDN142452_c3_g4_331 | 57.5 | 24.62 | 15.73 | 9.483 |
| | qSC-B1.2 | F2(2020) | hDN135643_c3_g1_514 | 63.5 | yDN151467_c2_g1_440 | 64.5 | 22.55 | 16.11 | 7.153 |
| | qSC-B1.3 | F2(2020) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 10.63 | 8.93 | -6.273 |
| | | F2:3-JH(2021) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 37.04 | 12.09 | 9.231 |
| | | F2:3-XY(2021) | hDN125239_c1_g4_1476 | 75.5 | yDN127251_c0_g1_756 | 76.5 | 54.25 | 20.22 | 16.479 |
FC | 1 | qFC1.1 | F2(2020) | yDN145946_c2_g3_400 | 107.5 | yDN125063_c0_g1_87 | 108.5 | 21.37 | 9.44 | 2.049 |
| | qFC1.2 | F2:3-JH(2021) | dDN53089_c3_g1_46 | 109.5 | yDN129665_c0_g3_238 | 110.5 | 14.85 | 6.26 | 1.379 |
| 3 | qFC3.1 | F2:3-XY(2021) | yDN148417_c0_g1_302 | 48.5 | hDN142257_c0_g1_1594 | 49.5 | 15.43 | 5.97 | 1.057 |
| | qFC3.2 | F2:3-XY(2021) | yDN119514_c0_g1_312 | 166.5 | hDN145176_c0_g1_497 | 167.5 | 13.65 | 5.32 | -0.962 |
| 4 | qFC4.1 | F2:3-JH(2021) | hDN122239_c0_g2_1049 | 98.5 | hDN154311_c1_g1_631 | 99.5 | 10.16 | 4.97 | 1.008 |
| 6 | qFC6.1 | F2:3-XY(2021) | yDN145987_c0_g1_369 | 67.5 | yDN138086_c0_g1_82 | 71.5 | 10.82 | 4.65 | 0.686 |
TC | 1 | qTC1.1 | F2(2020) | hDN124375_c0_g1_451 | 255.5 | dDN47789_c0_g1_281 | 256.5 | 22.70 | 6.46 | -0.061 |
| | qTC1.2 | F2:3-JH(2021) | hDN122621_c4_g3_51 | 249.5 | yDN154539_c0_g2_558 | 251.5 | 17.61 | 5.17 | -0.034 |
StC | 1 | qStC1.1 | F2(2020) | dDN40232_c0_g1_396 | 162.5 | yDN134012_c0_g1_800 | 163.5 | 13.77 | 8.19 | -1.306 |
| | qStC1.2 | F2:3-XY(2021) | hDN148143_c3_g2_213 | 34.5 | yDN141447_c5_g1_240 | 35.5 | 18.44 | 5.53 | 1.041 |
| 5 | qStC5.1 | F2(2020) | hDN148575_c1_g1_723 | 7.5 | yDN151173_c1_g2_935 | 9.5 | 11.38 | 6.64 | -1.192 |
PC | 1 | qPC1.1 | F2(2020) | dDN40232_c0_g1_396 | 162.5 | yDN134012_c0_g1_800 | 163.5 | 17.32 | 6.12 | 1.070 |
| 5 | qPC5.1 | F2(2020) | dDN41265_c0_g1_1106 | 0 | dDN41265_c0_g1_1104 | 0.5 | 13.65 | 4.99 | 0.875 |
LC | 1 | qLC1.1 | F2(2020) | hDN155223_c0_g1_2012 | 86.5 | hDN146106_c2_g1_2134 | 88.5 | 20.78 | 5.91 | -0.141 |
| 3 | qLC3.1 | F2:3-XY(2021) | yDN119514_c0_g1_312 | 166.5 | hDN145176_c0_g1_497 | 167.5 | 21.35 | 12.72 | -0.130 |
The QTL with underlines means stable QTL for each trait. |
Seed phenotypic traits
Three QTLs of HSW were detected and had peak LOD scores of 4.64–17.81, which explained 7.26–38.88% of the HSW variation. One was located on LG4, and two were mapped to LG5 (Table 3). QTLs detected more than two times among F2, F3-XY and F3-JH were considered environmentally stable. qHSW5.1 was detected in F2 and F3-JH (Table 3; Table S10).
A total of 28 QTLs were found for several seed shape traits, and 6 were regarded as stable (Table 3; Table S10). Five QTLs were detected on LG5 with a peak LOD score of 4.69–24.81, and they explained 4.90–51.51% of the SA variation. qSA5.1 was detected in F2 and F3-XY. Only one environmentally stable QTL (qSP5.1) of SP was identified on LG5 with a peak LOD score ranging from 14.82–23.55, and it explained 40.13–55.47% of the SP variation. Four QTLs associated with SL had peak LOD scores ranging 4.73–28.30, which explained 5.50–48.32% of the SL variation and were located on LG1, LG3, LG5, and LG6. According to the results, qSL5.1 was a stable QTL, which detected in F2, F3-XY and F3-JH.
Five QTLs explained 6.64–42.93% of the SW variance, with peak LOD scores ranging from 4.48–22.55, which were identified in linkage groups LG2 (1), LG3 (1), LG5 (1), and LG6 (2). An environmentally stable QTL (qSW5.1) was also identified. Five QTLs explained 9.59–25.02% of the SLWR variance, and the peak LOD scores varied from 5.33–11.11, and qSW6.1 was stable.
For ST, eight QTLs were detected in LG4 (3), LG5 (3), and LG6 (2), with LOD scores ranging from 4.48–12.25, and they explained 6.65–24.78% of the total phenotypic variation. qST5.1 was detected in F2, F3-XY and F3-JH. Among these QTLs, four were overlapping for seed shape traits.
For seed coat color traits, 12 QTLs were detected, including 3, 5, and 4 QTLs for R, G, and B, respectively. The phenotypic variation explained by each individual QTL ranged from 5.00–73.99%, with a peak LOD of 4.53–35.25 (Table 3). Three were overlapping QTLs and one was a stable QTL, both located in linkage group LG1 (Table S10).
Nutritional quality traits
The results from the QTL analysis identified 22 QTLs associated with nutritional quality traits (Table 3 and Table S10), 7 QTLs explained 9.25–21.35% of the FC variance, 2 QTLs explained 22.70–17.61% of the TC variance, 7 QTLs explained 7.09–18.44% of the StC variance, 2 QTLs explained 13.65–17.32% of the PC variance, and 4 QTLs explained 6.74–21.35% of LC variance. qFC3. 3 was considered stable.
Analysis of candidate genes
The genes in the QTL intervals were screened using the Faba_bean_ 130 K SNP TNGS genotyping platform (Table 4). The results showed that 333 genes (Table S11) and 610 SNPs were detected at 65 QTL intervals. Among the 333 genes, HSW, seed shape, seed coat color, and nutritional quality traits contained 8, 117, 100, and 109 genes, respectively, and 173 genes were functionally annotated by database comparison (Table S11). The results showed that 213 genes in 41 QTLs explained > 10% of the observed phenotypic variance, and they were further assessed. There were 6 genes related to HSW within these QTL intervals, and 5 were annotated, including the CCCH-type zinc finger protein and calcium-binding protein. There were 53 seed shape-related genes and 30 genes were annotated, including serine/threonine phosphatase, bHLH transcription factor, calcium-binding protein Ca2+/H+-exchanging protein, and other functional genes. Seed color-related genes included 39 and 19 genes that were annotated, including ubiquitin-like protein, the WD40 family, and transcription factors. There were 79 genes associated with nutritional quality traits, and 41 genes were annotated, including numerous genes encoding enzymes, functional genes, and some transcription factors.
Table 4
Details of genes and SNPs of 15 seed-related traits in QTL interval based on 130K TNGS
Trait | Total QTL number | SNP number | Gene number | VG/VP > 10 QTLs interval Gene number | Stable QTLs interval gene number |
HSW | 3 | 15 | 8 | 6 | 5 |
SA | 5 | 23 | 13 | 9 | 2 |
SP | 1 | 4 | 2 | 2 | 2 |
SL | 4 | 37 | 20 | 2 | 2 |
SW | 5 | 31 | 22 | 9 | 2 |
SLWR | 5 | 38 | 20 | 15 | 2 |
ST | 8 | 61 | 40 | 23 | 10 |
SC-R | 3 | 74 | 30 | 25 | 13 |
SC-G | 5 | 88 | 42 | 14 | 13 |
SC-B | 4 | 63 | 28 | 21 | 13 |
FC | 7 | 28 | 20 | 17 | 3 |
TC | 2 | 91 | 49 | 49 | 0 |
StC | 7 | 30 | 21 | 10 | 0 |
PC | 2 | 5 | 4 | 4 | 0 |
LC | 4 | 22 | 15 | 7 | 0 |
Total | 65 | 610 | 333 | 213 | 67 |