Transferring clubroot resistance from Chinese cabbage (Brassica rapa) to canola (B. napus)

Figures & data

Fig. 1 (Colour online) Schematic diagram of interspecific backcross breeding strategy to introgress clubroot resistance gene from Brassica rapa (Chinese cabbage) to Brassica napus (canola).

Table 1. Sequence information for simple sequence repeats (SSR) markers used in marker-assisted selection to transfer clubroot resistance from Brassica rapa to Brassica napus.

SSR marker	Primer	Sequence information
SScr045b	*Forward	5ʹ-GCTATTAAAGTTAGGAATGCT-3’
	Reverse	5ʹ-GCGCTTAATGAATACTCTCT-3’
SScr062	*Forward	5ʹ-GGTTCTGAGGAGATGGAGAT-3’
	Reverse	5ʹ-TACAGATTTTAACGAGTTTAACT-3’
SScr071	*Forward	5ʹ-AATAGAAGTTATAACGTACGAT-3’
	Reverse	5ʹ-CTAGTCTGTTACAGAACGTGT-3’
SScr072	*Forward	5ʹ-CTAGTTTTGGAAGGTATTGT-3’
	Reverse	5ʹ-TACGTACTTTAACAGTCTCT-3’
SScr079b	*Forward	5ʹ-CTTCATAGAGCTAAGCATGT-3’
	Reverse	5ʹ-ATGTACTGAACCAAGATCCT-3’
SScr082b	*Forward	5ʹ-TAGTTTGACCCAAACGCAT-3’
	Reverse	5ʹ-CACTATGGTTGGGCATTGGT-3’
SScr077	*Forward	5ʹ-CACAAATTCAATGCAACGCT-3’
	Reverse	5ʹ-TTTGACTCACATCTAATCGT-3’
SScr070	*Forward	5ʹ-GTCAACGAACCACTAGACT-3’
	Reverse	5ʹ-GTTCTAAACCACATGAATGCT-3’

*M13-CACGACGTTGTAAAACGAC tail was added at the 5ʹ of forward primer sequence to detect polymorphism on the ABI genetic analyser using labelled M13 primer.

Table 2. Chinese cabbage (Brassica rapa) hybrid cultivars and canola (Brassica napus) cultivar ‘Topas’ evaluated for clubroot disease reaction by inoculation with a Canadian field population of P. brassicae under controlled environmental conditions.

Cultivars	Score of clubroot disease symptoms
	0	1	2	3
Qulihuang	12	0	0	0
Sijihuang	12	0	0	0
Daifeng1	10	0	0	0
Nongke1	14	0	0	0
Huxinbaicai	10	0	0	0
Topas	0	0	0	18

Clubroot disease symptom scores: 0 for plants without galls on the roots, 1 for plants with a few small galls on secondary roots; 2 for plants with a few small galls on both primary and secondary roots; and 3 for plants with many large galls on both primary and secondary roots.

Table 3. Evaluation of clubroot reaction phenotypes in BC₃ and in BC₃S₂ homozygous individuals selected using marker-assisted selection in a Brassica rapa cross between Qulihuang × Yellow Sarson.

Populations	Clubroot phenotypes			χ^2	P†
	CR Resistance	CR Susceptible	Total
TQ1BC3F1_1	29	20	49	1.65	0.198543
TQ1BC3F1_2	19	22	41	0.22	0.639412
TQ1BC3F3_1*	17	0	17	-	-
TQ1BC3F3_2*	37	0	37	-	-
Qulihuang	25	0	25	-	-
Topas	0	23	23	-	-

† Expected segregation ratio of 1:1 in BC₃ population of a cross between Qulihuang × Yellow Sarson in Brassica rapa.

* Represent all progenies resistant to clubroot disease in the BC₃S₂ that possess homozygous clubroot resistance alleles for SScr071, SScr072, SScr045b and SScr079b molecular markers.

Table 4. Analysis of molecular markers for clubroot resistance gene introgression in canola using marker-assisted selection in a cross between the clubroot resistant Brassica rapa cv. Qulihuang and the susceptible B. napus cv. Topas. The BC₁, BC₂, BC₃ and BC₃S₁ populations developed using ‘Topas’ as the recurrent parent and screened using a set of SSR markers linked with a clubroot resistance allele.

Populations	Name of markers	Total*	CR resistance allele	Susceptible allele	χ^2	P^†
BC1	SScr062	69	41	28	2.45	0.12
	SScr045b	69	40	29	1.74	0.19
	SScr079b	69	31	38	0.71	0.4
BC2	SScr062	27	15	12	0.33	0.56
	SScr045b	30	18	12	1.2	0.27
	SScr079b	29	18	11	1.69	0.19
BC3	SScr071	87	38	49	1.39	0.24
	SScr072	87	44	43	0.01	0.91
	SScr082b	86	45	41	0.19	0.67
	SScr062	82	42	40	0.05	0.83
	SScr045b	82	43	39	0.2	0.66
	SScr079b	83	41	42	0.01	0.91
		AA	AB	BB	χ^2	P^€
BC3S1	SScr071	7	16	9	0.25	0.88
	SScr072	7	15	7	0.31	0.86
	SScr045b	7	13	7	0.81	0.67
	SScr079b	7	16	8	0.13	0.94

^† Expected segregation ratio was 1:1.

^€ Expected segregation ratio was 1:2:1 in BC₃S₁.

* Indicates total number of individual plants excluding missing values.

Fig. 2 (Colour online) Reliability of the SSR markers based on dominant polymorphism between clubroot resistance alleles and susceptible alleles in canola/rapeseed quality Brassica napus and Brassica rapa cultivars and inbred lines.

Table 5. Advanced backcrossed lines (BC₃S₂) carrying homozygous clubroot resistance alleles selected using marker-assisted selection evaluated under field conditions in Henan, China.

Line name	Generations	Clubroot disease scores^†				Total
		0	1	2	3
TQ2-2-1-1	BC3S2	11				11
TQ2-4-5-5	BC3S2	13				13
TQ2-4-5-23	BC3S2	6				6
TQ2-5-3-32	BC3S2	14		1		15
TQ2-5-3-6	BC3S2	13			1	14
TQ3-2-2-7	BC3S2	12	1			13
TQ3-2-2-18	BC3S2	14				14
TQ3-2-2-9	BC3S2	7				7
YS	Susceptible CK	0	0	4	15	19
Total	-	90	1	5	16	112

^† Clubroot disease rating scores: 0 for plants without galls on the roots, 1 for plants with a few small galls on secondary roots; 2 for plants with a few small galls on both primary and secondary roots; and 3 for plants with many large galls on both primary and secondary roots.

Fig. 3 (Colour online) Clubroot symptoms for advanced backcross families inoculated with Plasmodiophora brassicae. (a) Segregation of clubroot susceptible and resistant plants in the BC₃ family, TQ1BC3F1-1; (b) Segregation of clubroot susceptible and resistant plants in the BC₃ family, TQ1BC3F1-2; (c) BC₃S₂ family, TQ1BC3F3-1 displayed all the plants with clubroot resistance phenotypes that carried clubroot resistance locus in a homozygous dominant state; (d) BC₃S₂ family, TQ1BC3F3-2 displayed all the plants with clubroot resistance phenotypes that carried homozygous dominant clubroot resistance; (e) All the plants with clubroot susceptible phenotypes that carried homozygous susceptible alleles; (f) The clubroot susceptible parent ‘Topas’ used in backcross breeding as female.