Influence of Genotype on High Glucosinolate Synthesis Lines of Brassica rapa.

This study was conducted to investigate doubled haploid (DH) lines produced between high GSL (HGSL) Brassica rapa ssp. trilocularis (yellow sarson) and low GSL (LGSL) B. rapa ssp. chinensis (pak choi) parents. In total, 161 DH lines were generated. GSL content of HGSL DH lines ranged from 44.12 to 57.04 μmol·g-1·dry weight (dw), which is within the level of high GSL B. rapa ssp. trilocularis (47.46 to 59.56 μmol g-1 dw). We resequenced five of the HGSL DH lines and three of the LGSL DH lines. Recombination blocks were formed between the parental and DH lines with 108,328 single-nucleotide polymorphisms in all chromosomes. In the measured GSL, gluconapin occurred as the major substrate in HGSL DH lines. Among the HGSL DH lines, BrYSP_DH005 had glucoraphanin levels approximately 12-fold higher than those of the HGSL mother plant. The hydrolysis capacity of GSL was analyzed in HGSL DH lines with a Korean pak choi cultivar as a control. Bioactive compounds, such as 3-butenyl isothiocyanate, 4-pentenyl isothiocyanate, 2-phenethyl isothiocyanate, and sulforaphane, were present in the HGSL DH lines at 3-fold to 6.3-fold higher levels compared to the commercial cultivar. The selected HGSL DH lines, resequencing data, and SNP identification were utilized for genome-assisted selection to develop elite GSL-enriched cultivars and the industrial production of potential anti-cancerous metabolites such as gluconapin and glucoraphanin.

1. Introduction

There is experimental evidence supporting the idea that compounds in Cruciferous plants are effective against cancer and heart ailments [1,2,3]. Strong anti-carcinogenic effects of members of the family Brassicaceae are attributed to their glucosinolate (GSL) content [4,5]. For consumption, some GSLs such as glucoraphanin (GRA), glucoalyssin (GAL), gluconapin (GNA), neoglucobrassicin (NGBS), and gluconasturtiin (GNT) are considerably beneficial whereas hydrolysis products from progoitrin (PRO), epiprogoitrin (epiPRO), and gluconapoleiferin (GNL) can cause goiter in animals [6]. GSLs are not actual bioactive agents, but rather their hydrolysis products such as isothiocyanates (ITCs), nitriles, epithionitriles, thiocyanates, and indoles [7]. Some of the important bioactive compounds are butenyl sulforaphane (SFN), isothiocyanate (BITC) and pentenyl isothiocyanate (PEITC) (Figure 1) [6,7].

Figure 1

Chemical structure of glucosinolates and hydrolyzed bioactive compounds. Structure was drawn in ChemSketch software using smile notation from ChemSpider (www.chemspider.com (accessed on 7 June 2021)) database.

The biosynthesis of GSL is one of the more complex processes and more than 130 GSLs have been identified so far [8]. The existence of the different GSL structures is controlled by variability in genes on individual genotypes, especially at loci involved in initial elongation and side-chain modification reactions [9]. In addition, the molecular function of a gene can be altered depending on the plant species, allelic condition, and polymorphic state of the regulatory network controlling it [10]. According to previous works, four major loci, GS-ELONG, GS-OX, GS-AOP (GS-ALK and GS-OHP), and GS-OH, control the difference in the accumulation of aliphatic GSLs [6,11,12]. GS-ELONG consists of genes methylthioalkylmalate synthase 1 (MAM1), MAM2, and MAM3 to regulate the chain length of GSLs [13,14]. The GS-OX loci contain flavin monooxygenase (FMO) and GS-AOP two tightly linked loci, GS-ALK for 2-oxoglutarate-dependent dioxygenases (AOP2) and GS-OHP for AOP3 involved in the side-chain modification reaction determines the type of GSL products [15,16,17]. AOP2 genes are responsible for the conversion of GRA to GNA. In most B rapa, GNA makes up the major proportion of total GSL. However, the presence of stop codons in B. oleracea leads BoAOP2.2 and BoAOP2.3 into non-functional genes. This results in the major GSL content in B. oleracea being GRA instead of GNA [18,19,20]. GS-OH locus controls oxidation of 3-butenyl GSL to alkenyl GSL [6,21]. Most importantly, the accumulation and content of GSL are strongly influenced by R2R3-myeloblastosis (MYB) transcription factors (TFs) [22]. MYB28, MYB29, and MYB76 regulate the aliphatic GSL genes while MYB34, MYB51, and MYB122 regulate the indolic GSL genes [23]. For fine mapping and selection of individual genotypes, it is necessary to identify allelic discrepancies on key loci and for different TFs.

Resequencing provides the opportunity to develop a vast amount of novel markers. Identification of genes related to important agronomic traits, genetic diversity analysis, characterize and environmental factors influences are mandatory in genome-assisted breeding for crop improvement [24]. Combining quantitative trait loci (QTL) mapping with whole-genome sequencing helps in the precise detection of functional loci for traits of interest and their candidate genes. Major advantages of resequencing are functional allele mining and single-nucleotide polymorphism (SNP) discovery between large populations. The identification of trait loci in biparental cross populations by high-resolution linkage map and functional allele mining utilizing SNPs and Insertions–Deletions (InDels) as markers provides a powerful complementary strategy to genome-wide association studies (GWAS) [25]. Recently, Chen et al. (2016) resequenced 199 and 119 accessions representing 12 and nine morphotypes of B. rapa and B. oleracea, respectively. This resequencing data aided in the identification of leaf-heading and tuberous morphotypes associated with sub-genome parallel selection during diversification and domestication of B. rapa and B. oleracea [26]. Meanwhile, the resequencing of 588 B. napus accessions led to the identification of A and C sub-genome origins [27]. Similarly, genome mapping helped to characterize genes responsible for club root resistance [28], blackleg resistance [29], black rot resistance [30], and flowering time [31], etc.

B. rapa is one of the model plants for GSL metabolism and polyploid species with phenotypically diverse cultivated subspecies. In our previous study, we determined that eight subspecies of B. rapa have different GSL levels ranging from 4.42 μmol g−1 dry weight (dw) in B. rapa ssp. narinosa to 53.51 μmol·g−1·dw in B. rapa ssp. trilocularis [32]. The current study was conducted to identity SNPs based on resequencing between high GSL (HGSL) and low GSL (LGSL) content doubled haploid (DH) lines generated from two different parents, B. rapa ssp. trilocularis (yellow sarson) and B. rapa ssp. chinensis (pak choi). Yellow sarson is an oil plant with relatively high amounts of several beneficial GSLs, whereas pak choi is one of the major leafy vegetables in Korean and Chinese diets but contains lesser amounts of GSL. The aim of this work is to produce edible DH lines with high beneficial GSLs and low toxic substances like PRO. Detailed analysis of recombinant blocks between the HGSL and LGSL lines was carried out based on GSL biosynthetic pathways. The results from this study could be useful for precise genome-assisted selection in the development of elite B. rapa cultivars with enriched GSL content for commercial purposes.

2. Results

2.1. Generation of BrYSP DH Lines and GSL Content Profiles

Accessions YS-033 (CGN06835) and PC-099 (CGN132924) [33] were maintained in our greenhouse. As neither of these accessions were perfect inbred genotypes, we performed selfing for four generations (S4) of YS-033 and named LP08. DH plant of PC-099 was named LP21. LP08 and LP21 were used as the set of homozygous parents. The F1 plant was developed using a female of LP08 and male of LP21 (Figure 2). Microspore cultures were followed according to our previous method [34,35]. The leaf edge shapes were segregated in female and male parent’s phenotype boundary (Figure 2, middle). Phenotype classification of the leaf edge shape consisted of (a) entire, (b) slightly serrated, (c) intermediately serrated, and (d) very serrated. Our population was named “BrYSP_DH000” in accordance with B. rapa (Br), yellow sarson + pak choi (YSP), doubled haploid (DH), and a unique three-digit number for each line. The regulation of GSL biosynthesis was then examined in Chiffu, LP08, LP21, F1, and 161 BrYSP_DH plants.

Figure 2

Development strategy of high glucosinolate synthesis lines using hybrid plants from different B. rapa subspecies.

We selected five HGSL lines and three LGSL lines among the 161 BrYSP_DH plants for further studies, including resequencing and GSL analysis (Table 1, Figure 2). Total GSL amounts were detected from 44.12 ± 2.86 μmol·g−1·dw (BrYSP_DH005) to 57.04 ± 1.54 μmol·g−1·dw (BrYSP_DH026). It was consistent with the range of total GSL for B. rapa ssp. trilocularis, a higher GSL content subspecies in our previous study (47.46 to 59.56 μmol g−1 dw) [32]. The most abundant GSL in the HGSL lines was GNA, which was 80 to 91% of the total GSL. We selected the HGSL lines based on lesser PRO content (<0.6 μmol g−1 dw) as PRO is known to cause harmful effects on human health. As shown in Table 1, the BrYSP_DH005 line exhibited a highly unique profile with enriched aliphatic GSLs GRA (1.23 ± 0.18 μmol g−1 dw) and GAL (3.47 ± 0.18 μmol g−1 dw), indolic GSL NGBS (2.03 ± 0.20 μmol g−1 dw), and aromatic GSL GNT (0.68 ± 0.34 μmol g−1 dw). Significance difference between the DH lines was observed in all GSLs (Table 1).

Table 1

Total and individual GSL content (µmol·g−1·dw) in leaves of Chiffu, a parent of LP08 and LP21, F1, and eight BrYSP_DH lines of Brassica rapa.

Genotype		Aliphatic							Indolic			Aromatic
		GRA	GAL	SNG	GNA	GBN	GNL	PRO	GBS	4-MOGBS	NGBS	GNT	Totalμmol g−1 dw
	Chiffu	ND	ND	ND	0.00 e	0.00 f	0.00 c	0.38 ± 0.00 def	4.00 ± 0.26 a	1.09 ± 0.04 a	3.16 ± 0.52 b	0.14 ± 0.09 de	8.77 ± 0.69 ef
	LP08(♀)	0.11 ± 0.05 b	0.55 ± 0.05 g	0.17 ± 0.00 a	43.40 ± 3.63 b	0.66 ± 0.06 e	0.00c	0.71 ± 0.06 cd	0.40 ± 0.01 d	0.31 ± 0.04 c	0.76 ± 0.06 d	0.47 ± 0.14 bc	47.55 ± 3.66 bc
	LP21(♂)	0.00b	0.89 ± 0.10 def	0.00e	1.82 ± 0.66 d	0.00f	0.27 ± 0.03 a	0.88 ± 0.09 c	2.03 ± 0.02 b	0.63 ± 0.03 b	3.44 ± 0.24 b	0.81 ± 0.09 a	10.76 ± 0.36 e
	F1	0.10 ± 0.01 b	0.85 ± 0.12 ef	0.00e	1.97 ± 0.16 d	1.68 ± 0.14 d	0.34 ± 0.03 a	3.86 ± 0.29 a	0.59 ± 0.02 c	0.64 ± 0.03 b	4.58 ± 0.16 a	0.47 ± 0.05 bc	15.07 ± 0.79 d
High GSL lines	BrYSP_DH005	1.23 ± 0.18 a	3.47 ± 0.18 a	0.05 ± 0.01 cde	35.51 ± 2.84 c	0.63 ± 0.04 e	0.10 ± 0.04 b	0.22 ± 0.03 ef	0.14 ± 0.00 e	0.06 ± 0.00 f	2.03 ± 0.20 c	0.68 ± 0.34 ab	44.12 ± 2.86 c
	BrYSP_DH014	0.07 ± 0.02 b	2.16 ± 0.05 b	0.05 ± 0.01 cde	45.19 ± 2.69 ab	7.13 ± 0.62 a	0.02 ± 0.01 bc	0.06 ± 0.02 f	0.11 ± 0.01 e	0.10 ± 0.01 ef	0.77 ± 0.04 d	0.39 ± 0.04 cd	56.06 ± 3.28 a
	BrYSP_DH016	0.12 ± 0.07 b	0.43 ± 0.05 g	0.11 ± 0.07 b	44.42 ± 1.34 b	1.68 ± 0.07 d	ND	0.57 ± 0.37 cde	0.20 ± 0.01 e	0.16 ± 0.01 d	0.76 ± 0.02 d	0.24 ± 0.03 cde	48.69 ± 0.95 b
	BrYSP_DH017	0.07 ± 0.01 b	0.62 ± 0.17 fg	0.08 ± 0.02 bc	45.66 ± 1.25 ab	2.70 ± 0.09 c	0.10 ± 0.11 b	0.32 ± 0.03 ef	0.19 ± 0.01 e	0.19 ± 0.02 d	0.50 ± 0.02 def	0.20 ± 0.01 cde	50.62 ± 1.51 b
	BrYSP_DH026	0.09 ± 0.02 b	2.05 ± 0.04 b	0.07 ± 0.04 bcd	48.23 ± 1.31 a	5.39 ± 0.25 b	0.03 ± 0.02 bc	0.05 ± 0.00 f	0.08 ± 0.00 e	0.11 ± 0.00 e	0.57 ± 0.05 de	0.39 ± 0.03 cd	57.04 ± 1.54 a
Low GSL lines	BrYSP_DH009	0.04 ± 0.03 b	1.16 ± 0.10 d	0.00 ± 0.00 e	0.25 ± 0.05 d	0.26 ± 0.07 ef	0.09 ± 0.03 b	0.82 ± 0.26 c	0.12 ± 0.02 e	0.10 ± 0.01 ef	0.33 ± 0.03 ef	0.08 ± 0.03 e	3.26 ± 0.62 g
	BrYSP_DH059	0.06 ± 0.01 b	1.44 ± 0.19 c	0.01 ± 0.01 de	0.32 ± 0.06 d	0.52 ± 0.08 ef	0.30 ± 0.03 a	1.97 ± 0.23 b	0.10 ± 0.02 e	0.07 ± 0.01 ef	0.38 ± 0.03 def	0.15 ± 0.02 de	5.31 ± 0.67 fg
	BrYSP_DH061	ND	1.13 ± 0.26 de	0.01 ± 0.01 de	2.35 ± 0.81 d	1.70 ± 0.64 d	0.00 ± 0.00 c	0.05 ± 0.01 f	0.05 ± 0.01 e	0.01 ± 0.00 g	0.11 ± 0.02 f	0.23 ± 0.07 cde	5.63 ± 1.76 fg
ANOVA	Sum of square	3.852	29.573	0.091	16,645	165.68	0.525	39.6	46.36	3.601	72.88	1.669	16,984
	Mean sum of square	0.350	2.688	0.008	1513.2	15.061	0.047	3.6	4.215	0.327	6.626	0.152	1544
	F value	68.22	103.25	9.09	348.84	132.28	22.81	80.11	471.38	494.16	129.53	7.5	286.69
	p	****	****	****	****	****	****	****	****	****	****	****	****

GRA, Glucoraphanin; GAL, Glucoalyssin; SNG, Sinigrin; GNA, Gluconapin; GBN, Glucobrassicanapin; GNL, Gluconapoleiferin; PRO, Progoitrin; GBS, Glucobrassicin; 4-MOGBS, 4-Methoxyglucobrassicin; NGBS, Neoglucobrassicin; GNT, Gluconasturtiin; ND, Not detected. Different letters indicate significant difference between the genotypes under Duncan’s test (p ≤ 0.05) for three individual biological replicates. F-test are significant at **** p ≤ 0.0001 or Nonsignificant, respectively.

2.2. Resequencing of Parents, HGSL Lines, and LGSL Lines

Sequencing reads of 150 bp were generated using 10 paired-end sequencing libraries of 350 bp insert size. Resequencing of LP08 and LP21 produced around 775 and 704 million raw reads, respectively. The total bases of sequence obtained were 78.2 Gb for LP08 and 71.1 Gb for LP21. The sequences of the DH lines ranged from around 125.5 to 192.4 million clean reads. These reads sequenced were mapped to the B. rapa v3.0 reference genome [36]. Mapping reads (%) between parents differed with 90.65% (LP08) and 93.19% (LP21). The DH lines had slightly higher and more similar mapping read percentages, ranging from 94.8% to 95.98%. The genome coverages for the two parent sequences had a 157.05× depth for LP08 and 156.07× depth for LP21. Nine DH line were mapped from 46.89× (BrYSP_DH059) to 70.80× (BrYSP_DH026) (Table 2).

Table 2

Summary of re-sequencing and alignment result for DH lines of Brassica rapa.

Sample ID	No. of Reads	No. of Bases	No. of Clean Reads	Clean Reads (%)	No. of Clean Bases	Clean Bases (%)	De-Duplicated Reads	De-Duplicated Reads (%)	MappedReads	Mapped Reads (%)	Ave. Coverage (x)
LP08(♀)	775,036,332	78,278,669,532	748,357,766	96.56	75,085,735,554	95.92	613,028,554	81.92	555,700,094	90.65	157.05
LP21(♂)	704,684,562	71,173,140,762	676,201,118	95.96	67,699,611,150	95.12	592,219,743	87.58	551,893,412	93.19	156.07
BrYSP_DH005	135,586,708	20,473,592,908	132,434,320	97.68	19,674,335,422	96.10	121,145,704	91.48	116,215,959	95.93	48.48
BrYSP_DH009	130,566,110	19,715,482,610	128,070,232	98.09	19,054,916,880	96.65	120,327,985	93.95	115,494,194	95.98	48.06
BrYSP_DH014	143,278,512	21,635,055,312	141,074,216	98.46	21,128,122,142	97.66	133,925,200	94.93	127,330,977	95.08	53.5
BrYSP_DH016	134,052,434	20,241,917,534	132,201,870	98.62	19,784,969,829	97.74	123,574,666	93.47	117,994,498	95.48	49.38
BrYSP_DH017	145,561,748	21,979,823,948	144,029,952	98.95	21,590,666,966	98.23	133,547,331	92.72	126,848,299	94.98	53.23
BrYSP_DH026	194,633,904	29,389,719,504	192,401,316	98.85	28,832,473,186	98.1	176,347,873	91.66	168,647,644	95.63	70.80
BrYSP_DH059	126,815,042	19,149,071,342	125,484,032	98.95	18,829,119,983	98.33	117,116,774	93.33	111,549,893	95.25	46.89
BrYSP_DH061	138,437,794	20,904,106,894	137,010,406	98.97	20,534,954,754	98.23	127,865,424	93.33	122,305,767	95.65	51.45

2.3. SNP Genotyping and InDels

Approximately ~3.5 million and ~2.5 million SNPs were predicted in LP08 and LP21 lines in reference to B. rapa v3.0 genome, respectively [36]. SNP density was calculated as 27.5 per 1-kb in LP08 and 22.2 per 1-kb in LP21. The maximum number of InDels were identified in LP08, i.e., 724,760. Comparatively, a lesser number of InDels were detected in LP21, i.e., 552,220. On average, 2,707,400 high-quality SNPs and 617,408 InDels were identified in the DH lines. Details regarding the SNPs and InDels of the parents and DH lines are provided in Table S1.

2.4. Identification of GSL Biosynthesis-Specific Recombinant Blocks

Overall, 108,328 variants were extracted as allelic differences between LP08 and LP21 in the recombinant block search of 342 recombinant blocks among the genotypes (Table S2, Figure S1). Out of 110 GSL biosynthetic genes, 75 were identified in recombinant blocks and mapped to their respective chromosomes (Figure 3). Uniformly, the recombinant blocks between the region of 12,929,867–23,248,122 bp of A03 with 3716 SNPs were discovered and identified to be present in only the HGSL lines (Table 3). The ten GSL synthesis genes positioned between 12.9 Mb and 23.2 Mb of A03 commonly differed between all resequenced HGSL and LGSL lines. In detail, TFs MYB28.1 of aliphatic GSLs and MYB34.2 of indolic GSLs of the HGSL lines were LP08 types. Similarly, for the chain elongation step, branched-chain amino acid aminotransferase 4 (BCAT4; Bra001761), MAM1 (Bra013007), MAM3 (Bra013009 and Bra013011), and bile acid transporter 5 (BAT5; Bra000760) of the HGSL lines belong to LP08. AOP1 (Bra000847) and AOP2 (Bra000848) were the two keys genes derived from LP08 in high GSL lines for the side-chain modification process. One of the genes involved in the sulfur donation from chloroplast to sulfotransferase for production of desulfo GSL, APS kinase (APK)1 (Bra013120) is the LP08 type in all high GSL lines (Table 3).

Figure 3

Glucosinolate (GSL) biosynthesis genes identified from recombinant blocks between LP08 and LP21. Key genes uniformly present as LP08 type in high GSL lines are indicated in red. Chromosomal regions are highlighted in the green boxes.

Table 3

Gene blocks identified as high (LP08) and low (LP21) GSL parents with the DH lines.

							♀	HGSL Line					LGSL Lines			♂
Chrom	Start	Stop	Name	Gene ID V1.5	Gene ID V3.0	Stages	LP08	DH005	DH014	DH016	DH017	DH026	DH009	DH059	DH061	LP21
A03	21326869	21328218	MYB28.1	Bra012961	BraA03g044440.3C	Transcription factors—Aliphatic	LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A09	3469113	3470483	MYB28.2	Bra035929	BraA09g007000.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A02	25403875	25405492	MYB28.3	Bra029311	BraA02g043310.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP21	LP21
A03	1364309	1365719	MYB29.1	Bra005949	BraA03g003070.3C		LP08	LP08	LP08	LP21	LP21	LP08	LP21	LP21	LP08	LP21
A09	3356433	3357581	MYB34.1	Bra035954	BraA09g006760.3C	Transcription factors—Indolic	LP08	LP21	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A03	21129279	21130669	MYB34.2	Bra013000	BraA03g043850.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A02	25183826	25184998	MYB34.3	Bra029349	BraA02g042890.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP21	LP21
A02	25172906	25179858	MYB34.4	Bra029350	BraA02g042880.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP21	LP21
A08	18248352	18249890	MYB51.2	Bra016553	BraA08g028300.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21
A06	6841688	6842966	MYB51.3	Bra025666	BraA06g013940.3C		LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP08	LP21	LP21
A07	23411313	23412768	MYB122.1	Bra015939	BraA07g037950.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A02	12327349	12329278	MYB122.2	Bra008131	BraA02g022140.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP08	LP21
A03	18302919	18305368	BCAT-4	Bra001761	BraA03g039030.3C	Side chain elongation	LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A05	18305152	18307632	BCAT-4	Bra022448	BraA05g027600.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A06	8849237	8851230	BCAT-3	Bra017964	BraA06g017190.3C		LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP08	LP21	LP21
A01	14814889	14817183	BCAT-3	Bra029966	BraA01g023550.3C		LP08	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21
A02	2391691	2393547	IPMDH1	Bra023450	BraA02g005400.3C		LP08	LP21	LP21	LP21	LP21	LP21	LP08	LP21	LP08	LP21
A03	21085392	21093219	MAM1	Bra013007	BraA03g043770.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A03	21061112	21065885	MAM3	Bra013009	BraA03g043760.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A03	21054537	21056963	MAM3	Bra013011	BraA03g043750.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A02	15934744	15944413	MAM1	Bra018524			LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP21	LP21
A02	25115507	25119702	MAM1	Bra029355	BraA02g042820.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP21	LP21
A02	25103449	25106649	MAM3	Bra029356	BraA02g042810.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP21	LP21
A04	5653874	5657118	IPMI LSU1	Bra032708	BraA04g008730.3C		LP08	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21
A08	5964064	5967370	IPMI LSU1	Bra040341	BraA08g006340.3C		LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP21	LP21	LP21
A05	1608381	1609157	IPMI SSU2	Bra004744	BraA05g003360.3C		LP08	LP21	LP21	LP08	LP08	LP21	LP21	LP08	LP21	LP21
A03	12943140	12945015	BAT5	Bra000760	BraA03g027810.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A09	17204468	17206516	BAT5	Bra029434	BraA09g026220.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21
A06	6007926	6010112	CYP79F1	Bra026058	BraA06g012170.3C	Core structure synthesis—Aliphatic	LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP08	LP21	LP21
A04	5460393	5462018	CYP83A1	Bra032734	BraA04g008410.3C		LP08	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21
A02	768271	770517	CYP79A2–Aromatic	Bra028764	BraA02g001710.3C		LP08	LP21	LP21	LP21	LP21	LP21	LP08	LP21	LP08	LP21
A05	24811698	24812553	GSTF11	Bra032010	BraA05g041750.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP08	LP21	LP08	LP21
A07	17498418	17499341	GSTU20	Bra003645	BraA07g026570.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A01	3359620	3360837	GGP1	Bra011201	BraA01g007200.3C		LP08	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP08	LP21
A03	27901998	27903448	GGP1	Bra024068	BraA03g029390.3C		LP08	LP08	LP08	LP21	LP21	LP08	LP21	LP21	LP21	LP21
A08	12833451	12834884	GGP1	Bra010282			LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP21	LP21	LP21
A08	12835830	12837549	GGP1	Bra010283	BraA08g017720.3C		LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP21	LP21	LP21
A09	5949618	5952361	SUR1	Bra036703	BraA09g011980.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP08	LP21	LP08	LP21
A09	25123658	25125223	UGT74B1	Bra024634	BraA09g038870.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21
A01	16964878	16965828	ST5b	Bra031476	BraA01g028650.3C		LP08	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21	LP21
A07	18479546	18480592	ST5b	Bra003817			LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A07	18481748	18482782	ST5b	Bra003818	BraA07g028360.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A07	18002729	18003723	ST5b	Bra003726	BraA07g027400.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A07	23419048	23420082	ST5b	Bra015938	BraA07g037960.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A07	23426595	23427635	ST5b	Bra015936	BraA07g037980.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A09	7726767	7727825	ST5b	Bra027623	BraA09g012830.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A09	9019311	9020375	ST5b	Bra027117	BraA09g016490.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A09	9021641	9022747	ST5b	Bra027118	BraA09g016500.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A09	9977892	9978911	ST5b	Bra027880	BraA09g017830.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21
A06	6850053	6851066	ST5c	Bra025668	BraA06g013960.3C		LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP08	LP21	LP21
A01	374137	375874	CYP79B2	Bra011821	BraA01g000840.3C	Core structure synthesis—Indolic	LP08	LP21	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A03	31203169	31205210	CYP79B2	Bra017871	BraA03g061480.3C		LP08	LP08	LP08	LP21	LP21	LP08	LP21	LP21	LP21	LP21
A08	14973444	14975600	CYP79B2	Bra010644	BraA08g021670.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21
A08	5058097	5059748	CYP83B1	Bra034941	BraA08g007380.3C		LP08	LP21	LP21	LP08	LP08	LP21	LP08	LP21	LP21	LP21
A03	7230400	7231268	GSTF9	Bra022815	BraA03g016240.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A04	13901442	13902299	GSTF9	Bra021673	BraA04g022220.3C		LP08	LP21	LP08	LP21	LP21	LP08	LP21	LP21	LP21	LP21
A03	7232844	7233982	GSTF10	Bra022816	BraA03g016250.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A02	12337180	12338199	ST5a	Bra008132	BraA02g022150.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP08	LP21
A07	23427909	23428469	ST5a	Bra015935			LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A08	19203810	19205763	FMOGS-OX5	Bra016787	BraA08g030720.3C	Side chain modification—Aliphatic	LP08	LP21	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21
A09	8217824	8219479	FMOGS-OX2	Bra027035	BraA09g015600.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP08	LP21
A02	15973741	15976278	AOP2	Bra018521	BraA02g027430.3C		LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP21	LP21
A03	13492936	13494533	AOP1	Bra000847	BraA03g028760.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A03	13498815	13503183	AOP2	Bra000848	BraA03g028770.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A09	1170626	1172022	AOP1	Bra034182	BraA09g001250.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A09	1168503	1169786	AOP1	Bra034181	BraA09g001260.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A09	1165028	1166807	AOP2	Bra034180			LP08	LP21	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A03	7768293	7769612	GSL-OH	Bra022920	BraA03g017350.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A04	13852551	13853809	GSL-OH	Bra021670	BraA04g022180.3C		LP08	LP21	LP08	LP21	LP21	LP08	LP21	LP21	LP21	LP21
A04	13877397	13878646	GSL-OH	Bra021671	BraA04g022190.3C		LP08	LP21	LP08	LP21	LP21	LP08	LP21	LP21	LP21	LP21
A02	5846392	5848174	CYP81F2	Bra020459	BraA02g012540.3C	Side chain modification—Indolic	LP08	LP08	LP21	LP21	LP21	LP21	LP08	LP21	LP08	LP21
A03	5233924	5236233	CYP81F2	Bra006830	BraA03g012390.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A03	20376817	20378290	APK1	Bra013120	BraA03g042630.3C		LP08	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21	LP21
A01	361733	363288	APK2	Bra011822	BraA01g000800.3C	Sulphur supplementation	LP08	LP21	LP08	LP08	LP08	LP08	LP21	LP21	LP08	LP21
A03	31218969	31220359	APK2	Bra017872	BraA03g061500.3C		LP08	LP08	LP08	LP21	LP21	LP08	LP21	LP21	LP21	LP21
A08	14980248	14981406	APK2	Bra010645	BraA08g021680.3C		LP08	LP21	LP08	LP08	LP08	LP08	LP08	LP21	LP21	LP21

Bold letters are indicated when all HGSL lines had the LP08 genotypes whereas LGSL lines had the LP21 genotypes. Pink (LP08) and sky-blue (LP21).

Though all MAM genes are LP08 type in BrYSP_DH005, only three MAM genes are LP21 types in other high DH lines. In a similar way, all MAM genes were LP21 in BrYSP_DH059 and BrYSP_DH061 but three genes were LP08 type in the BrYSP_DH009 line. Even though in the core structure synthesis phase of BrYSP_DH005 most of the genes were identified as LP21 type, other HGSL lines showed about 50% of genes were LP08 type. It is noteworthy that BrYSP_DH014, BrYSP_DH026 and BrYSP_DH016, BrYSP_DH017 shared similar parental type recombinant blocks of GSL biosynthetic genes.

2.5. Comparative Analysis of GSL Pathway between Individual Genotypes

Stepwise comparative analysis on the GSL biosynthetic pathway between the HGSL line BrYSP_DH005 and LGSL line BrYSP_DH059 was performed based on the recombinant blocks of parents LP08 and LP21. Key differences were observed between BrYSP_DH005 and BrYSP_DH059 in amino acid elongation and side-chain modification step TFs such as MYB28 and MYB29. Entire MAM1, AOP1, AOP2, and GSL-OH genes of BrYSP_DH059 were present as LP21 recombinant blocks. In contrast, complete MAM1, one AOP1 (Bra000847), two AOP2 (Bra018521 and Bra000848), and one GSL-OH (Bra022920) were LP08 blocks in BrYSP_DH005. Interestingly, all genes of indolic and aromatic pathways in BrYSP_DH059, except ST5a (Bra024634) and UGT74B1 (Bra024634), were LP21 recombination blocks. Contrastingly, critical genes for the synthesis of glucobrassicin (GBA) in BrYSP_DH005, such as ST5a (Bra008132 and Bra015935), and CYP81F2 (Bra020459 and Bra006830), were LP08 blocks. Except for MYB28.2 (Bra035929), TFs involved in the biosynthesis of the aliphatic GSL were LP08 blocks in BrYSP_DH005, whereas, aliphatic TFs of BrYSP_DH059 were LP21 blocks. A similar trend was observed in MYB34 TFs. Other than MYB34.2 (Bra013000), all MYB34 homologues in BrYSP_DH005 were LP08 types. MYB34 TFs in BrYSP_DH059 remained as the LP21 genotype (Figure 4).

Figure 4

Metabolic pathway profiling of genes derived from recombinant blocks between high and low GSL parents of doubled haploid lines BrYSP_DH005 (A) and BrYSP_DH059 (B). The pathway and product structures are adapted from previously published reports [6,18,21,37] and the Brassica database BRAD. Genes not identified in recombinant blocks are not highlighted. Structure of all products were drawn using ChemSketch software.

2.6. GSL Hydrolysis Products

Main hydrolysis products of GSL such as BITC, 4-PEITC, 2-PEITC, and SFN of high GSL lines were compared with the commercial pak choi cultivar used in South Korea. All five of the representative HGSL DH lines possessed increased amounts of hydrolysis products. Overall, BrYSP_DH014 had the highest level of hydrolysis products (870.29 µg∙g−1 dw). It is about 6.3-fold higher levels than that of the commercial cultivar. The HGSL line with the lowest level of hydrolysis products was the BrYSP_DH017 line which is about 417.5 µg∙g−1 dw. Still, it had a 3.0-fold high-level hydrolysis product than that of the control pak choi. Most importantly, the amount of anti-carcinogenic agent SFN in BrYSP_DH005 was 20.2 µg∙g−1 dw. This was 7–10-fold higher than that in the other HGSL DH lines and nearly 35-times more than that of the commercial cultivar (Table 4).

Table 4

Hydrolysis products of high glucosinolate doubled haploid (DH) lines.

Accessions	BITC	4-PEITC	2-PEITC	SFN	Total	Fold
BrYSP_DH005	601.0 ± 16.8	47.7 ± 1.3	40.8 ± 0.8	20.24 ± 1.33	709.8 ± 16.7	5.1
BrYSP_DH014	778.8 ± 32.9	64.5 ± 3.9	25.0 ± 1.2	2.03 ± 0.15	870.3 ± 37.6	6.3
BrYSP_DH016	425.1 ± 10.0	100.1 ± 2.5	18.5 ± 0.7	2.90 ± 0.13	546.6 ± 12.5	3.9
BrYSP_DH017	281.7 ± 15.3	120.7 ± 5.3	12.7 ± 0.8	2.35 ± 0.21	417.5 ± 21.5	3.0
BrYSP_DH026	653.0 ± 20.8	94.0 ± 2.3	34.5 ± 0.5	2.39 ± 0.15	783.9 ± 23.5	5.6
pak choi©	108.9 ± 2.7	21.6 ± 0.6	8.1 ± 0.2	0.57 ± 0.04	139.2 ± 3.6	Control

pak choi©, Commercial cultivar in South Korea. BITC, 3-Butenyl isothiocyanate; 4-PEITC, 4-pentenyl isothiocyanate (brassicanapin); 2-PEITC, 2-Phenethyl isothiocyanate; and SFN, Sulforaphane. Unit, µg∙g−1 dw.

2.7. Nitrile Formation

Among the five HGSL DH lines tested, BrYSP_DH005 showed significantly lower nitrile formation compared to that of the other HGSL DH lines, from both SNG and GNT substrate-based assays (Figure 5). The lower SNG substrate-based nitrile formation may explain why BrYSP_DH005 generated higher concentrations of SFN compared to other HGSL DH lines (Table 4). Although a decrease in GNT-based nitrile formation was observed in BrYSP_DH005 compared with other HGSL DH lines, it was more increased than the commercial pak choi.

Figure 5

Percentage of nitrile formation in high GSL DH lines using sinigrin (A) and gluconasturtiin (B) as substrate. Lowercase letters above the error bar indicate significant differences among the accessions as determined by Tukey’s HSD test at p < 0.05.

3. Discussion

In this study, the representative lines were selected from previously developed 161 DH lines with HGSL and LGSL content for metabolite, resequencing, SNP mapping, and GSL biosynthetic pathway analysis. Total GSL content ranged from 44 μmol·g−1·dw to 57 μmol·g−1·dw in HGSL DH lines (Table 1). Amounts of GSL were significantly higher than those in the inbred line Brassicaraphanus “BB1” [7] and broccoli (B. oleracea var. italica) [38]. Glucoraphasatin is the major GSL of Raphanus sativus [39] and Brassicoraphanus “BB1” [7], SNG in mustard [1] and horseradish [40] cultivars. Similar to our study, GNA was found to be the predominant GSL in B. rapa “Chinese cabbage”. However, the range is around 54% only [41], but in our study, GNA is 80% to 91% of total GSL. Though GNA is the major GSL present in HGSL DH lines, BrYSP_DH005 possessed a considerable amount of other important metabolites such as GRA, GAL, NGBS, and GNT (Table 1). Due to its higher content of several beneficial GSLs, BrYSP_DH005 was selected as a vital genotype and detailed analysis on the GSL pathway was carried out in comparison with the representative LGSL line BrYSP_DH059 (Table 1).

For any hybrid studies, SNPs and InDels are valuable for developing linkage maps of trait loci in the cross-population lines. High-density SNPs and InDels polymorphism markers could be a valuable resource for genetic-linkage studies and precise QTL mapping of desirable traits [24,42] in Brassica (Table S1, Figure S1). The predicted recombinant block between 12.9–23.2 bp of A03 chromosome in the GSL-rich parent, all five HGSL lines, and three LGSL lines indicate that it is a key region that should be focused on GWAS (Table 3, Figure 3). GWAS analysis in combination with metabolite profiling has gained widespread acceptance to assess natural variations between populations [43].

Sønderby et al. (2010) noted that decreases in a few transcripts can have a major impact on the accumulation of GSL [22]. Epistatic effects of transcript expression levels are highly complex to mirrored with the GSL content. Resequencing followed by recombinant block analysis showed a clear picture of genes derived from HGSL and LGSL parents (Table S2, Figure 3). The results of this study indicate that integration of critical recombinant blocks from parents LP08 and LP21 triggered to turn on the genes involved in biosynthesis, transportation and regulation in the GSL metabolic pathway (Figure 2, Figure 3 and Figure 4).

Transcriptional activation of aliphatic and indolic genes by MYB factors are as follows: MYB28, MYB29 and MYB76 activate MAM, CYP79F1/F2, CYP83A1, GGP1, SUR1, UGT74B1/C1, and STb/c of the aliphatic pathway and MYB34, MYB51, and MYB 122 activate CYP79B2/B3, CYP83B1, YGT74B1, and STa of the indolic pathway [23]. MYB factors also modulate genes involved in the sulfur supplementation route such as ATPS1/S3 and APK1/2 [44]. For instance, MYB28.2, MYB 34.1 and MYB51.2 are LP08 derived in all HGSL DH lines except BrYSP_DH005. Contrastingly, MYB28.3, MYB34.3, MYB34.4 and MYB122.2 were LP08 types in BrYSP_DH005 whereas they are LP21-derived in other HGSL lines. This could be correlated to the excess amounts of GRA, GAL, NGBS, and GNT and also to the lesser content of GNA, GBN, and 4-MOGBS in BrYSP_DH005 than other HGSL lines. Similarly, the LP08 type of MYB29.1 in BrYSP_DH005, BrYSP_DH014, and BrYSP_DH026 can be associated with an appreciable amount of GAL (Table 1 and Table 3). Induction of glucoraphasatin biosynthesis genes by MBY29 in radish (R. sativus L.) root was reported [39]. LP08-type recombinant block with MYB29.1 could also be related to a higher level of GSL hydrolysis products (Table 3 and Table 4). Though MYBs involved in aliphatic and indolic pathways are close homologs to each other, they have distinct functional roles [45]. However, their functions overlap and they are able to substitute for each other in cases of mutants, decreased expression, and overexpression [22,45,46,47].

The first chain elongation process catalyzed by BCAT4 deaminates Met and homoMet to corresponding 2-oxoacids. Two copies of BAT5 serve as the importer of 2-oxo acids into plastid. Out of two copies of BCAT4 and one copy (Bra001761) present in all HGSL lines belonged to the LP08 genotype. The other copy (Bra022448) was also LP08-type in all HGSL DH lines except BrYSP_DH005. Both copies of BAT5 were LP08-type in BrYSP_DH005. Individual mutants of bcat4 and bat5 exhibit approximately a 50% reduction in the level of aliphatic GSLs [48]. As the GS-ELONG locus contains MAM genes, the number of side chains of GSL is decided by the elongation cycles it undergoes the initial steps [14]. Gene duplication, neo-functionalization, and polymorphism of MAM1 lead to the diversification of GSL profiles [49]. Following isomerization by isopropylmalate (IPM) isomerase and oxidative decarboxylation by IPM dehydrogenase (IPM-DH), the 2-oxo acid yields homoMet and chain-elongated derivatives of Met to enter the core GSL structure pathway [6].

There are five genes present in the GS-OX locus of A. thaliana (FMO), but only one copy of FMO and two copies of FMO have been identified in B. rapa. This locus is responsible for the oxygenation of glucoibervirin, glucoerucin, and glucoberteroin based on the co-expression of TFs for aliphatic GSLs [15,16]. AOP2 belongs to GS-ALK for conversion of S-oxygenated GSLs and AOP3 belongs to GS-OHP for conversion of hydroxyl GSLs [6]. Our fine-mapping on the key locus regions such as BCAT4, MAM1, BAT5, AOP2, and GS-OH in recombinant inbred lines showed the high production of GNA with less conversion of PRO (Figure 4, Table 1, Table 3 and Table S2).

Cytochrome P450 (CYP) CYP79F1 catalyzes the conversion of all chain-elongated Met derivatives. CYP79F1 mainly for long-chained Met derivatives, but no copies of CYP79F2 have been found in B. rapa [50]. CYP79B2 and CYP79B3 are identified for indolic whereas CYP79A2 for aromatic derivatives [51]. The resulting aldoximes are then converted into nitrile oxides or aci-nitro compounds by CYP83A1 for Met derivatives and CYP83B1 for Trp and Phe derivatives [52]. Glutathione-S-transferase (GSTF) is involved in the catalysis of nitrile into S-alkyl-thiohydroximate. In this step, sulfur is supplied to alkyl-thiohydroximate as glutathione (GSH) [53]. GSH is a tripeptide with a gamma peptide linkage between glutamate and cysteine with glycine. S-alkyl thiohydroximates are converted into thiohydroximates by four copies of Y-glutamyl peptidase 1 (GGP1) and two copies of superroot1 (SUR1) [54]. S-glucosylated is catalyzed by glucotransferase. UDP-glucosyl transferase 74C1 (UGT74C1) and UGT74B1 metabolize Met-derived and Phe-derived compounds of thiohydroximates into desulfo GSL [55]. In the final step of core structure biosynthesis, the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) is produced by two steps. First, ATP sulfurylase (ATPS) produces the intermediate adenosine-5′-phosphosulfate (APS). APS kinase then catalyzes APS to Cys. Of four copies of APK1, Bra013120 present in all the HGSL were LP08 type. The other three copies varied among the HGSL DH lines (Table 3). All APK1 and APK2 genes were LP21 type in BrYSP_DH059 (Figure 4b). Double mutants of apk1 and apk2 reduce total GSL in A. thaliana by almost 80%. In addition, the accumulation of desulfoGSL is noticeable in mutants, but present at undetectable levels in wild-type plants [56]. Core structures of GSLs depend on sulfur assimilation, especially sulfotransferase (ST) reaction [9,55]. Two copies of ST5a catalyze both Trp and Phe desulfoGSL. About ten copies of ST5b and one copy of ST5c are involved in the synthesis of aliphatic GSLs, such as glucoibervirin, glucoerucin, and glucoberteroin [57].

Hydrolysis of GSLs by endogenous myrosinase (β-D-thioglucosidase) produces active compounds, such as ITCs, nitriles, and indoles [3]. An important ITC, SFN is reported to be a natural inducer of phase II detoxification enzymes, including glutathione-S-transferase and quinone reductase (QR). SFN triggers cytostasis and apoptosis and also detoxifies xenobiotics [7]. BITC and PEITC have proved to induce apoptosis in cancer cell lines [8,9]. Nitriles have a weaker chemopreventive effect than ITCs [7]. Although broccoli is a well-known health-promoting vegetable due to high SFN concentrations, it has a wide range of ESP activity from 17.1% to 46% among 20 commercial broccoli cultivars [58]. In contrast, the HGSL DH lines of the current study exhibited much lower nitrile formation, and increasing GRA levels may have directly contributed to the induction of phase II detoxification enzymes (Figure 6). Nitrile formation using SNG as a substrate in the HGSL DH lines ranged from 0.44% to 2.66%, which was considerably lower than nitrile formation compared with 11 commercial mustard cultivars (7.4–62.4%) or USDA fancy horseradish (average, 7.1%) [1,40]. Although nitrile formation based on GNT of BrYSP_DH005 was significantly higher than commercial pak choi, it was still lower than the recent report on broccoli [38]. In this study, important ITCs including SFN, BITC, 4-PEITC, and 2-PEITC were the major hydrolysis compounds along with some nitrile hydrolysis products from GNA (1-cyano-3,4-epithiobutane) and GNT (benzenepropanenitrile) (Table S3). Our current multilayered analysis of resequencing and the revelation of SNP-based recombinant block discovery results will be helpful for further fine QTL mapping.

Figure 6

Comparison of cancer-preventive effect of high glucosinolate DH_line with low nitrile formation ability with broccoli as well-known cancer fighting vegetable as model. Genome-assisted precision breeding in B. rapa achieved high GSLs DH line that directly contribute to high ITCs-mediated restoration of Nrf2/ARE signaling.

This information will be beneficial to the production of elite GSL-enriched cultivars for commercialization of potential anti-cancerous metabolites, such as GRA, GLA, NGBS, and GNT, with higher SFN activity.

4. Materials and Methods

4.1. Plant Materials

Accessions YS-033 (CGN06835) and PC-099 (CGN132924) [33] were provided by professor Guusje Bonnema. We performed selfing for four generations (S4) of YS-033 and named LP08. DH plant of PC-099 was named LP21. LP08 and LP21 were used as the set of homozygous parents. The F1 plant was developed using an LP08 as female and LP21 as male parent (Figure 1). Microspores were collected and cultures were followed according to our previous method [34,35]. B. rapa ssp. perkinensis “Chiffu-401-42” was used as the reference.

4.2. HPLC Analysis for Identification of GSL Content

GSL content was estimated according to Seo et al. [32]. Fresh leaves of 6-weeks old plants were freeze-dried and 100 mg samples were used for protein extraction by boiling with 1.5 mL of 70% (v/v) methanol in a 10 mL test tube for 10 min at 95 °C. Extracts were loaded on Sephadex A25 columns and desulfation was conducted with aryl sulfatase (EC 3.1.6.1) before HPLC. Desulfated GSLs were quantified in Agilent 1200 Series HPLC System (Agilent Technologies, Santa Clara, CA, USA) equipped with an Inertsil ODS-3 column (150 × 3.0 mm inner diameter, particle size 3 µm; GL Science, Tokyo, Japan). Analysis was done using a flow rate of 0.4 mL·min−1 at a column over temperature of 35 °C and a wavelength of 227 nm. Sinigrin (SNG) was used as an external standard for quantification. Total and individual GSL content was calculated as means of three biological replicates.

4.3. Resequencing

Genomic DNA was extracted from fresh leaves as previously described [59]. In liquid nitrogen, 5 g of samples were finely ground and put in 50 mL falcon tube. It is mixed with the pre-warmed 15 mL of DNA extraction buffer (500 mM NaCl, 100 mM Tris-HCL, pH 8.0, 50 mM EDTA, pH 8.0, 1.25% SDS, and add 0.38% sodium bisulfite before use with adjust pH 8.0 with 0.2 N, NaOH). Incubate for 30 min, and invert gently every ten min at 65 °C. Add 5 mL of aquaphenol and rotate at 14 rpm for 10 min at room temperature (RT). Add to an equal volume of Chloroform: Isoamyalchole (24:1) and rotate at 20 rpm for 15 min at RT. Centrifuge for 15 min at 15 °C at 10,000 rpm. Supernatant was transferred to a new 50 mL falcon tube. Add 10 µL of RNaseA (20 mg/mL) and incubate at 37 °C in 10 min. An equal proportion of isopropanol was added and gently mix by inverting. Carefully pull out the DNA pellet with the closed sterile glass tube. DAN pellet was washed with 70% ethanol several times. Completely dry the pellet under vacuum pressure. Finally, dissolve the pellet in 0.1× TE buffer. Libraries with an average insert size of 350 bp were constructed using the genomic DNA TruSeq Nano DNA Sample Prep Kit according to the manufacturer’s protocol. Sequencing was performed with 150 bp paired-end sequencing using the NovaSeq6000 platform. Reads were converted from the binary base call (BCL) format file using bcl2fastq V2.20 software with parameter “–barcode-mismatches 0”.

4.4. Variant Calling

To remove low-quality bases, clean reads were trimmed using sickle v1.33 with a Phred quality score threshold of 20 (accuracy 99%) to derive high-quality reads. The mapped reads were aligned against the B. rapa reference genome sequence available in the Brassica database (BRAD V3.0, http://brassicadb.org/brad/ (accessed on 1 December 2020) using mem algorithm in Burrows–Wheeler Aligner (BWA) V0.7.17 software “-Am”, “-k 45”. After alignment, duplicates were marked using MarkDuplicates tools in GATK V4.0.2.1. Multi-sample variant calling of SNPs and InDels were performed using Haplotype Caller in GATK V4.0.2.1.

4.5. Identification of Recombinant Blocks

For recombinant block search, extraction of variants with allelic differences between LP08 and LP21 was performed. Recombinant blocks for each sample were defined as LP08, LP21, or hetero based on SNPs and InDels using in-house scripts. GSL biosynthetic genes were identified using BLASTp with E-value of 1 × 10−5 against the B. rapa reference genome [36]. BioSample accession number of DH lines deposited in NCBI are as follows: BrYSP_DH005, SAMN18818129; BrYSP_DH009, SAMN18818130; BrYSP_DH014, SAMN18818131; BrYSP_DH016, SAMN18818133; BrYSP_DH017, SAMN18818134; BrYSP_DH026, SAMN18818135; BrYSP_DH059, SAMN18818139; BrYSP_DH061, SAMN18818140.

4.6. Quantification of Glucosinolate Hydrolysis Products

Freeze-dried sample powder (50 mg) was suspended in 2 mL micro-centrifuge tube (Fisher Scientific, Waltham, MA, USA) with 1 mL distilled water. Under darkness for 24 h, hydrolysis products were generated naturally by endogenous myrosinase. Samples were added with 1 mL of dichloromethane and centrifuged at 12,000× g for 2 min. Lower organic layer was carefully collected. To quantify the GSL hydrolysis products, gas chromatograph (GC) (6890N, Agilent Technologies) coupled to an MS detector (5975B, Agilent Technologies) equipped with an auto sampler (7683B, Agilent Technologies) and a capillary column (30 m × 0.32 mm × 0.25 µm J&W HP-5, Agilent Technologies) was used. From extract, 1 µL was injected in GC-MS with split ratio of 1:1. Initial temperature was set to 40 °C for 2 min, then the oven temperature was increased to 260 °C at 10 °C/min and hold for 10 min. Temperature of injector and detector were set at 200 °C and 280 °C, respectively, with the flow rate in the helium carrier at 1.1 mL/min. Peaks were identified using the respective standards [40,41].

4.7. Measurement of Nitrile Formation

Nitrile formation (%) was measured to estimate the epithiospecifier protein (ESP) activity as ESP enhances the formation of nitriles over isothiocyanates. Nitrile formation in each sample was determined by incubating concentrated horseradish root extract with crude protein extract of the sample and analyzed using gas chromatography–mass spectrometry (GC-MS). Firstly, concentrated horseradish extract was prepared by powdered 10 g root samples were mixed in 100 mL of 70% methanol. After centrifuging at 4000× g for 5 min, supernatant was boiled in the beaker until all solvent was evaporated and reconstituted in 50 mL of deionized water. Freeze-dried powder (75 mg) of B. rapa DH leaves sample were mixed with 1.5 mL of concentrated “1091” horseradish root extract in 2 mL microcentrifuge. Centrifugation was carried out at 12,000× g for 2 min. Supernatant (0.6 mL) was transferred to 1.5 mL Teflon centrifuge tube (Savillex Corporation, Eden Prairie, MN, USA) and mixed with 0.6 mL of dichloromethane. Samples were incubated in RT for 1 h upside down to minimize volatile compounds loss. Vortexed tubes were centrifuged at 12,000× g for 2 min. Bottom organic layer was injected to GC-MS (Trace 1310 GC, Thermo Fisher Scientific, Waltham, MA, USA) coupled to a MS detector system (ISQ QD, Thermo Fisher Scientific, Waltham, MA, USA) and an auto sampler (Triplus RSH, Thermo Fisher ScientificA capillary column (DB-5MS, Agilent Technologies; 30 m × 0.25 mm × 0.25 µm capillary column). The sample was held at 40 °C for 2 min. Oven temperature was increased to 320 °C at 15 °C/min and held for 4 min. Injector and detector temperature were set at 270 °C and 275 °C, respectively. Flow rate of helium carrier gas was 1.2 mL/min. Standard curve was used to quantify hydrolysis rate of nitriles [40,41].

4.8. Statistical Analysis

Analysis of variance (ANOVA) was performed using SAS Enterprise Guide 7.1 (SAS Institute Inc., Carrey, NC, USA). Tukey’s honest significant difference (HSD) test was performed using Prism 5 software (GraphPad, San Diego, CA, USA).

5. Conclusions

The present study advances our knowledge regarding the inheritance of GSL biosynthetic genes in B. rapa for high GSL synthesis and increased ITCs with low concentrations of nitriles. The result of this work will be useful for genome-assisted precise breeding in B. rapa. Metabolite profiling in various DH lines and its recombinant block predicted in A03 (12.9 Mb-23.2 Mb) chromosome based on SNPs and InDels broadens our understanding of GSL biosynthesis and the key genes responsible for the production of beneficial GSL. The integrative genetic-linkage map brings detailed knowledge of variants in GSL biosynthetic genes in the A03 chromosome region. Further recombinant blocks responsible for GSL biosynthesis can be used for the selection and development of GSL-rich edible cultivars of B. rapa.

6. Patents

High GSL content DH lines have been patented with “variety protection right” under Korean Patent Law and Seed Industry Law. Patent IDs are as follows: BrYSP_DH005, 10-2020-0130241; BrYSP_DH014, 10-2020-0130238; BrYSP_DH016, 10-2020-0130239; BrYSP_DH017, 10-2020-0130240; and BrYSP_DH026, 10-2020-0130242.