Phenotypic variation and correlation of the traits relating to fiber quality and yield
A RIL population developed between the two parents STS and DYM was planted in three places (HB, NX and GS). The phenotypic variation ranges of the six traits PAL, STE OLE, LIN, LNA, and OIL for parents and the RIL population are summarized in Table 1 and Fig. 1. All the traits showed approximately normal distributions with an absolute skewness value of less than one and were characterized by transgressive segregations with respective to their parental performances during the evaluations.
Table 1
Statistics of the six seed quality traits in the parents and the RIL population
Trait | Environment | Parents | Population |
DYM | STS | Range | Average | Standard error | standard deviation | variance | kurtosis | skewness | minimum | Maximum |
PAL | GS | 6.46 | 5.58 | 0.88 | 6.03 | 0.03 | 0.45 | 0.20 | -0.71 | -0.34 | 4.90 | 7.04 |
| NX | 6.87 | 5.54 | 1.33 | 6.26 | 0.03 | 0.48 | 0.23 | 0.17 | 0.14 | 5.13 | 8.15 |
| HB | 6.48 | 6.12 | 0.36 | 5.85 | 0.03 | 0.43 | 0.19 | 0.11 | 0.04 | 4.76 | 7.37 |
STE | GS | 4.83 | 3.78 | 1.05 | 4.27 | 0.05 | 0.73 | 0.53 | -0.78 | 0.20 | 2.89 | 6.18 |
| NX | 4.71 | 3.60 | 1.11 | 3.90 | 0.06 | 0.87 | 0.75 | 1.53 | -0.37 | 0.74 | 6.30 |
| HB | 3.62 | 3.45 | 0.17 | 3.24 | 0.05 | 0.71 | 0.50 | -0.16 | 0.40 | 1.37 | 5.19 |
OLE | GS | 21.94 | 24.91 | 2.97 | 23.89 | 0.17 | 2.62 | 6.86 | -0.61 | -0.08 | 17.65 | 31.29 |
| NX | 21.25 | 24.51 | 3.26 | 23.60 | 0.18 | 2.70 | 7.31 | 0.13 | 0.31 | 17.87 | 31.08 |
| HB | 20.19 | 20.00 | 0.19 | 19.34 | 0.14 | 2.14 | 4.56 | 0.27 | 0.20 | 14.36 | 27.03 |
LIN | GS | 27.31 | 13.51 | 13.80 | 19.71 | 0.33 | 5.01 | 25.05 | -1.45 | 0.19 | 11.47 | 30.87 |
| NX | 28.85 | 13.39 | 15.46 | 19.68 | 0.32 | 4.84 | 23.46 | -1.35 | 0.20 | 11.70 | 29.93 |
| HB | 25.41 | 24.54 | 0.87 | 20.10 | 0.30 | 4.63 | 21.45 | -1.34 | 0.16 | 8.37 | 29.36 |
LNA | GS | 39.47 | 52.23 | 12.76 | 46.10 | 0.32 | 4.81 | 23.12 | -0.89 | -0.01 | 35.56 | 58.04 |
| NX | 38.32 | 52.95 | 14.64 | 46.56 | 0.32 | 4.91 | 24.08 | -0.80 | -0.15 | 33.51 | 56.39 |
| HB | 44.30 | 45.90 | 1.59 | 51.34 | 0.30 | 4.60 | 21.18 | -0.76 | -0.27 | 36.12 | 61.08 |
OIL | GS | 40.89 | 42.90 | 2.01 | 41.38 | 0.14 | 2.09 | 4.36 | 0.41 | -0.44 | 34.56 | 47.50 |
| NX | 42.21 | 43.99 | 1.78 | 42.07 | 0.13 | 1.94 | 3.78 | 0.71 | -0.46 | 34.61 | 46.60 |
| HB | 41.29 | 40.92 | 0.36 | 43.20 | 0.11 | 1.74 | 3.01 | 0.90 | -0.62 | 35.83 | 46.81 |
Table 2
The correlation analysis between the six traits
Trait | PAL | STE | OLE | LIN | LNA | OIL |
PAL | 1 | -0.150* | 0.034 | 0.169** | -0.276** | -0.091 |
STE | -0.150* | 1 | 0.137* | -0.075 | -0.127 | 0.021 |
OLE | 0.033 | 0.137* | 1 | -0.355** | -0.183** | -0.224** |
LIN | 0.169** | -0.075 | -0.355** | 1 | − .831** | -0.039 |
LNA | -0.276** | -0.127 | -0.183** | -0.831** | 1 | 0.160* |
OIL | -0.091 | 0.021 | -0.224** | -0.039 | 0.160* | 1 |
** indicates the significant level of 0.01. |
* indicates the significant level of 0.05. |
For the correlation analysis, the result showed that the PAL showed significant negative correlation to STE and LNA and showed significant positive correlation to LIN; the STE showed significant positive to OLE; the OLE showed significant negative correlation to LIN, LNA and OIL; the LIN showed significant negative correlation to LNA and OIL; the LNA showed significant positive correlation to OIL.
Analysis of Gbs Data and Snp Marker Identification
Genomic DNA from STS, DYM and 232 RIL individuals was double digested by MseI and EcoRI to construct libraries and sequencing by Illumina HiSeq2500. Totally,we obtained 2,372,287,104 bp and 2,345,804,352 bp clean data from STS and DYM separately, and 105.6 Gb total raw data from 232 RIL individuals with 451 Mb raw data of each individual. Raw data from both parent and RILs was high quanlity with Q20 ≥ 95%༌Q30%≥89% of parents and Q20 ≥ 90%༌Q30%≥85% of 232 progenies (Table S2). To further assess data quality, we analysis the average of MseI catch ratio was 98.37% and MseI or EcoRI enzyme cut completely ratio was 87.2% which indicated a high effective enzyme production sequenced a depth of approximately 69 × and high quality 25.9 Gb flax genome sequences (Table S3). We used BWA to map the parents and 232 progenies sequencing data to the published flax genome and obtained a high depth and wide coverage mapping results. In STS and DYM, mapping rate was more than 98% and average depth was more than 34 and in 232 RILs, mapping rate was more than 98% and coverage at least 1X was more than 10% (Table S4).
BWA alignment results of parent plants and RILs were further analyzed to identify SNP. As a result, for the parent STS, a total of 45,720 SNPs including 37,925 homozygous SNPs and 7,795 heterozygosis SNPs were identified. For DYM, 51,666 SNPs including 45,231 homozygous SNPs and 6,435 heterozygosis SNPs were identified. Subsequently, we chose 24,641 markers with the genotype of aa × bb and the polymorphism between parents (Table S5 and Table S6) for genetic map construction.
Genetic Map Construction and Structure Analysis
Of the 24,641 markers with aa × bb genotype in progeny, 10,045 with coverage more than 75% were screened and applied to construct the genetic map. The genetic map contains 15 linikage maps ranging from 178.28 cM to 28.14 cM in length. The genetic map contains 2,239 SNP markers, spanned total genetic distance of 1032.90 cM. The largest linkage group (LG) was LG1 that contains 528 SNPs in178.279 cM, while the smallest LG was LG13 that contains 51 SNPs in 28.14 cM. LG4 contains the lowest 43 SNP number. The average density of the SNP marker in the map was 0.46 marker per cM. In this map, there were two gaps, one was in the group 6 (21.48 cM) and the other was in the group 12 (31.927 cM) (Table 3 and Fig. 2).
Table 3
The detail information of the high density genetic map
Linkage Group Number | No. Marker | Genetic Distance (cM) | Average Distance (cM) | Max Gap (cM) |
LG 1 | 528 | 178.28 | 0.34 | 19.1 |
LG 2 | 410 | 105.11 | 0.26 | 10.92 |
LG 3 | 175 | 68.02 | 0.39 | 10.58 |
LG 4 | 43 | 50.42 | 1.17 | 23.18 |
LG 5 | 63 | 45.71 | 0.73 | 16.3 |
LG 6 | 87 | 37.06 | 0.43 | 21.48 |
LG 7 | 93 | 55.75 | 0.60 | 7.7 |
LG 8 | 53 | 65.86 | 1.24 | 12.18 |
LG 9 | 82 | 49.52 | 0.60 | 7.13 |
LG 10 | 79 | 43.21 | 0.55 | 19.16 |
LG 11 | 163 | 41.23 | 0.25 | 3.84 |
LG 12 | 83 | 107.25 | 1.29 | 31.93 |
LG 13 | 51 | 28.14 | 0.55 | 6.16 |
LG 14 | 269 | 122.35 | 0.45 | 7.01 |
LG 15 | 60 | 35 | 0.58 | 6.2 |
Total | 2239 | 1032.9 | 0.46 | 31.93 |
QTL location of seed quality related traits in three environments
Based on the phenotypic data of the population and the genetic map, we determined 21 QTLs of six traits two for PAL, four for STE, two for OLE, five for LIN, five for LAN three for OIL. Among them, one for LIN and one for LAN could be identify in multiple environments and the other could be only identifiy in a single environment.
For PAL, there were total two QTLs have been identified. On group two, there were one QTL. the qPAL-Group2-1 could be detect in the environment HB, located on the position of 27.11 cM, explained 5.69% of the observed PV, and the direction was negative; on group 14, there were one QTL. The qPAL-Group14-1 could be detected in the environment HB, located on the position of 6.41 cM, explained 5.24% of the observed PV, and the direction was positive.
For STE, there were four QTLs have been identified. On group one, there were three The qSTE-Group1-1, qSTE-Group1-2 and qSTE-Group1-3 could be detect in the environment GS, GS and NX respectively, located on the position of 23.61 cM, 70.21 cM and 95.71 cM respectively, explained 5.76%, 5.15% and 5.32% respectively; and the direction of them were negative, negative and positive respectively; on group eight, there were one QTL, qSTE-Group8-1 could be detected in the environment GS, located on the position of 39.31 cM, explained 7.93% of the observed PV, and the direction of it was positive.
For OLE, there were total two QTLs have been identified. On group one, there were two QTLs. The qOLE-Group1-1 and qOLE-Group1-2 could be both detected in the environment GS, located on the position of 15.31 cM and 94.41 cM respectively, explained 7.61% and 5.64% of the observed PV respectively, and their direction were negative and positive respectively.
For LIN, there were five QTLs have been detected. On group one, there were one QTL. The qLIN-Group1-1 could be detected in the environment HB, located on the position on 16.71 cM, explained 5.43% of the observed PV, and its direction was positive; on group 11, there were one QTL. The qLIN-Group11-1 could be detected in the environment HB, located on the position on 8.51 cM, explained 4.00% of the observed PV, and the direction were positive; on group 12, there were two QTLs. The qLIN-Group12-1 could be detected in the environment NX, located on the position of 34.21 cM, explained 6.83% of the observed PV, and the direction was positive; The qLIN-Group12-2 could be detected in all the three environments, located on the position interval of 46.81–48.81 cM, explained 76.09%-78.05% of the observed PV and the direction was positive; on group 14, there were one QTL. The qLIN-Group14-1 could be detected in the environment HB, located on the position of 38.41 cM, explained 5.20% of the observed PV, and the direction was negative.
For LNA, there were total five QTLs have been identified. On group one, there were one QTL. The qLNA-Group1-1 could be detected in the environment HB, located on the position of 144.31 cM, explained 6.62% of the observed PV, and the direction was positive; On group 4, there were one QTL. The qLNA-Group4-1 could be detected in the environment NX, located on the position of 42.91 cM, explained 5.40% of the observed PV, and the direction was negative; On group 11, there were one QTL. the qLNA-Group11-1 could be detected in the environment HB, located on the position of 8.51 cM, explained 4.69% of the observed PV, and the direction was negative; On group 12, there were two QTLs, the qLNA-Group12-1 could be detected in the environment NX, located on the position of 32.21 cM, explained 8.00% of the observed PV and the direction was negative; the qLNA-Group12-2 could be detected in the environment NX and HB, located on the position interval 46.81–48.81 cM, explained 57.33%-62.78% of the observed PV, and the direction was negative;
For, there were total two QTLs have been identified. On group three, there were two QTLs qOIL-Group3-1 and qOIL-Group3-2, they both could be detected in the environment NX, located on the position of 39.31 cM and 58.71 cM respectively, explained 7.43% and 6.00% of the observed PV respectively and their direction were positive and negative respectively. On group 13, there were one QTL. The qOIL-Group13-1 could be detected in the environment NX, located on the position of 26.11 cM, explained 4.00% of the observed PV, and its direction was positive (Table 4 and Fig. 3).
Table 4
The detail information of the QTLs for the six traits
QTL Name | Environment | Trait | Group Number | Position (cM) | LOD | Additive | R2 (%) | Left CI (cM) | Right CI (cM) |
qPAL-Group2-1 | HB | PAL | 2 | 27.11 | 2.62 | -0.44 | 5.69 | 26.70 | 27.70 |
qPAL-Group14-1 | HB | PAL | 14 | 6.41 | 2.50 | 0.13 | 5.24 | 2.30 | 11.90 |
qSTE-Group1-1 | GS | STE | 1 | 23.61 | 2.85 | -0.25 | 5.76 | 22.00 | 24.80 |
qSTE-Group1-2 | GS | STE | 1 | 70.21 | 2.68 | -0.27 | 5.15 | 68.80 | 71.40 |
qSTE-Group1-3 | NX | STE | 1 | 95.71 | 2.82 | 0.25 | 5.32 | 92.50 | 97.90 |
qSTE-Group8-1 | GS | STE | 8 | 39.31 | 3.69 | 1.00 | 7.93 | 38.70 | 39.60 |
qOLE-Group1-1 | GS | OLE | 1 | 15.31 | 3.87 | -2.06 | 7.61 | 14.70 | 15.60 |
qOLE-Group1-2 | GS | OLE | 1 | 94.41 | 3.14 | 1.00 | 5.64 | 91.40 | 97.70 |
qLIN-Group1-1 | HB | LIN | 1 | 16.71 | 2.67 | 2.89 | 5.43 | 15.90 | 18.10 |
qLIN-Group11-1 | HB | LIN | 11 | 8.51 | 2.00 | 1.45 | 4.00 | 7.30 | 9.70 |
qLIN-Group12-1 | NX | LIN | 12 | 34.21 | 3.51 | 1.66 | 6.83 | 28.30 | 34.80 |
qLIN-Group12-2 | NX | LIN | 12 | 46.81 | 18.85 | 4.30 | 76.10 | 44.00 | 50.90 |
| GS | | | 48.81 | 24.34 | 4.55 | 80.28 | 45.30 | 52.00 |
| HB | | | 48.81 | 16.57 | 4.15 | 78.05 | 45.10 | 52.00 |
qLIN-Group14-1 | HB | LIN | 14 | 38.41 | 2.72 | -2.64 | 5.20 | 38.20 | 39.80 |
qLNA-Group1-1 | HB | LNA | 1 | 144.31 | 3.22 | 1.94 | 6.62 | 143.50 | 144.60 |
qLNA-Group4-1 | NZ | LNA | 4 | 42.91 | 2.90 | -1.33 | 5.40 | 42.70 | 45.90 |
qLNA-Group11-1 | HB | LNA | 11 | 8.51 | 2.49 | -2.15 | 4.69 | 7.30 | 9.30 |
qLNA-Group12-1 | NZ | LNA | 12 | 32.21 | 4.13 | -1.96 | 8.00 | 30.70 | 39.90 |
qLNA-Group12-2 | NZ | LNA | 12 | 46.81 | 3.45 | -3.80 | 57.33 | 41.80 | 51.20 |
| HB | | | 48.81 | 5.46 | -3.73 | 62.78 | 44.90 | 53.00 |
qOIL-Group3-1 | NZ | OIL | 3 | 39.31 | 3.31 | 1.14 | 7.43 | 37.80 | 40.30 |
qOIL-Group3-2 | NZ | OIL | 3 | 58.71 | 2.93 | -1.0149 | 6.0012 | 57.5 | 59.2 |
qOIL-Group13-1 | NZ | OIL | 13 | 26.11 | 1.81 | 0.8202 | 4.0007 | 28.1 | 28.1 |