2. Discussion
Genotyping by sequencing allows analysis of genome-wide sequence variation among individuals, which enables accurate and efficient identification of genes controlling important agronomic traits [
31].
We observed a large variation in SNP coverage between the chromosomes of the parental lines, from 574 on chromosome A08 to 15,152 on chromosome C07. Whilst A08 has the lowest number of SNPs, the short length of this chromosome makes it appears more extreme. The low marker density on chromosome A08 has already been observed in our previous study [
32], and it might be due to the missing read coverage in both of the parental lines. Missing read coverage could be caused by differences between the genome of the parental lines and the Darmor reference that we used to align the reads or due to the genomic differences between those lines. The low SNP density on chromosome A08 may also be a result of a relatively low level of genetic diversity between the parental lines as they both represent low erucic acid and low glucosinolates (double zero) winter-type oilseed rape [
33]. Intensive breeding of double zero oilseed rape led to a restricted gene pool, which reduces its genetic variation. Chromosome A08 could also represent a genomic region of identity-by-descent that was not efficiently disrupted by recombination during selection [
34].
Seed fibre and glucosinolates considerably reduce the value of
B. napus meal, especially for poultry; therefore, the identification of functional candidate genes related to these traits is of importance [
35,
36,
37,
38]. In the studied population, derived from the yellow-seeded ‘Z114’ and black-seeded ‘M305’ DH lines, the highest phenotypic variation was found for fibre (ADF/NDF) and SCC. These traits were correlated with glucosinolates and protein content in previous studies [
39]. Decreased amounts of cell wall polysaccharides in seeds containing less fibre can cause increased carbon availability for protein deposition [
40].
The correlation found between ADF/NDF/SCC/GLS/SPC can be reflected in the identification of common QTL for the studied traits (e.g., qSCC/ADF/NDF/GLS) and many interesting candidate genes related to plant cell wall, lignin biosynthesis underlying SPC and GLS-QTLs. The QTL for these traits was found on chromosomes A02, A04, A06, C02, C06, C07, and C08, with PVE ranging between 21.52% and 31.65%. The regions of A02, A04, A06, C02, C06, and C08 were also found to be correlated with seed fibre by Miao [
11]. It is difficult to compare the positions of the identified QTL regions due to different reference genomes used by Miao (ZS11) and here in this study (Darmor). QTL for ADF and GLS were found previously on chromosome C02 [
10], whereas A06, C08, and A09 regions were repeatedly detected for SCC in a GWAS study performed by Wang
4. Here we found a similar region of A06 (20.5 Mbp) and C08 (distal end) to that found in the Wang GWAS study. Interestingly, the distal region of chromosome C08 correlated with SCC, NDF, and GLS in our study showed high homology with a region of chromosome A09 [
4,
13], detected as a major QTL for seed fibre and seed colour in various genetic backgrounds [
2,
5,
6,
7,
8,
9,
17]. Major QTL for seed colour were also detected on A09 or C08 chromosomes, depending on the genetic background [
41], which also indicates that different black-seeded forms may possess different seed colour genes. Previous studies also showed a correlation with seed fibre on chromosomes A05 and C05; however, here, we did not detect any QTL on these chromosomes [
3,
10].
A number of candidate genes associated with seed fibre deposition, seed coat development, flavonoid, and anthocyanin biosynthesis were identified in previous studies [
1,
4,
16]. Strong candidates include cinnamoyl-CoA reductase 1 (
CCR1) and cinnamyl alcohol dehydrogenase (
CAD2/CAD3),
SEC8,
PAL4,
CESA3, and
GPAT5 [
2,
3,
5,
7,
8]. Some of the candidate genes identified in this study belong to the same gene family but are located on a different chromosome. The most interesting candidate genes
BnaC02g38340D and
BnaC02g38710D were identified here for seed fibre, and SCC was located on chromosome C02 and encoded transparent testa 10/laccase-like 15
(TT10/LAC15) and transparent testa 4 (
TT4), respectively. In other studies,
TT4 was found to be associated with ADL (acid detergent lignin) on chromosome C09 (
BnaC09g43250) [
3], and
TT10 was detected as a major gene for SCC and fibre on A09 [
5,
9]. Transparent testa (
TT) are key enzymes in proanthocyanidins and lignin biosynthesis pathways [
9,
25,
28,
42].
Since the highest phenotypic variation in the Z114 × M305 mapping population was found for ADF/NDF and SCC, the strongest emphasis on the identification of candidate genes was made for genes known to be involved in carbohydrate metabolism and flavonoid biosynthesis. In the
qGLS-A02 region, one of the identified genes, Korrigan2 (
KOR2), encodes endo-1,4-β-d-glucanase, known to be involved in cellulose synthesis [
18,
43]. Interestingly, the same region of chromosome A02 was found to be correlated with seed fibre in a study performed by Miao [
11]. The interesting candidate genes underlying QTL regions on chromosome A04 include pectin methylesterase (
BnaA04g27070D) and pectin lyase-like protein (
BnaA04g25420D). The presence of SNP variation in these genes and their relatively close physical locations from the QTL (100–500 kbp) indicates that they are very likely regulators of fibre composition in
B. napus seeds. Another gene,
BnaA04g03060D, located 10 kbp from
qGLS-A04, encodes β-1,3-glucanase 3, glycoside hydrolase, which functions in cell wall degradation [
19]. Other interesting genes underlying QTL on chromosome C08 include cellulase, glycosyl hydrolase family 5
GH5, and UDP-glycosyl transferase
UGT73C7, which are known to be involved in carbohydrate metabolic process and cell wall lignification [
27,
30]. Another strong candidate is peroxidase 64 (
PRX64) (
BnaC07g05860D), located 173 kbp from the
qSCC/ADF-C07, the major oxidase enzyme known to play a role in proanthocyanidins and lignin biosynthesis [
9,
25,
28,
43]. RING-type E3 ubiquitin transferase (
CMPG1) (
BnaC07g05860D) with two SNPs located 16,6 kbp from the QTL. These genes are known to play a role in lignin biosynthesis and response to chitin [
21,
29,
44]. A key gene found to be correlated with seed colour in previous studies, namely transparent testa 12 (
TT12), was not identified in this study.
TT12 encodes a multidrug and toxic compound extrusion (MATE) secondary transporter that is specifically expressed in the developing seed coat and is involved in the transportation of proanthocyanidin precursors into the vacuole [
45]. It was found that the
BnaC06g17050D gene, which is orthologous to Arabidopsis
TT12, is associated with seed coat colour in oilseed rape [
4]. However, we could not find any association between this gene and SCC in the present study.
3. Conclusions
In conclusion, a QTL genetic mapping study using an NGS SkimGBS approach allowed us to identify several promising genes, including PE, PLL, TT10/LAC15, SUS2, and GH5, which provides insight into the complex genetic architecture of seed fibre and colour biosynthesis in B. napus. Understanding the mechanism of action and causal polymorphisms of these genes will provide a better understanding of the role of those genes in the regulation of complex traits affecting RSM quality.