Resynthesis of Brassica juncea for resistance to Plasmodiophora brassicae pathotype 3.

The oilseed crop Brassica juncea carries many desirable traits; however, resistance to clubroot disease, caused by Plasmodiophora brassicae, is not available in this species. We are the first to report the clubroot resistant resynthesized B. juncea lines, developed through interspecific crosses between a clubroot resistant B. rapa ssp. rapifera and two susceptible B. nigra lines, and the stability of the resistance in self-pollinated generations. The interspecific nature of the resynthesized B. juncea plants was confirmed by using A- and B-genome specific SSR markers, and flow cytometric analysis of nuclear DNA content. Self-pollinated progeny (S1 and S2) of the resynthesized B. juncea plants were evaluated for resistance to P. brassicae pathotype 3. The S1 and S2 progenies of one of the resynthesized B. juncea lines were resistant to this pathotype. However, resistance was lost in 6 to 13% plants of the S2 progenies derived from the second resynthesized B. juncea line; this apparently resulted from the loss of the genomic region carrying resistance due to meiotic anomalies.

Introduction

The oilseed crop species Brassica juncea (AABB; 2n = 36) carry many desired traits, such as tolerance to heat (Gunasekera ), drought (Wright ) and silique shatter (Wang ), and resistance to blackleg (Roy 1978) and leaf blight disease (Wechter ). This crop species yields greater than B. napus in heat- and drought-prone areas as well as in short growing season areas (Burton , Potts ). Although, B. juncea possess all these desired properties, resistance to Plasmodiophora brassicae Woronin, causing clubroot disease, is not available in this species (Hasan ). Clubroot disease can result in up to 90% yield loss and about 4–6% decrease in seed oil content (Pageau ). Resistance to this disease can be found in the two parental species of B. juncea, viz., B rapa (AA; 2n = 20) and B. nigra (BB; 2n = 16) (Buczacki , Hasan ). At least eight clubroot resistance genes have been mapped to date in B. rapa (reviewed by Piao ), and this species has been used widely in the breeding of clubroot resistant B. napus lines and cultivars (Diederichsen and Sacristan 1996). The objective of the present study was to develop a clubroot resistant B. juncea line using a resistant B. rapa line, and to investigate the stability of this resistance in the resynthesized B. juncea line.

Materials and Methods

Plant materials

A B. rapa line, homozygous for resistance to P. brassicae pathotype 3, was used as female in the interspecific crosses with two susceptible B. nigra (BB, 2n = 16) accessions CR 2136 and CR 2137 as male. The B. rapa line was developed through self-pollination of the B. rapa ssp. rapifera cv. Gelria (AA; 2n = 20). The cv. Gelria carries the clubroot resistance gene CRa (Matsumoto , Ueno ) and CRb (Piao , 2009); however, recent studies have showed that the CRa and CRb to be the same locus and located on the chromosome A3 (Hatakeyama , Kato ). Seeds of Gelria were obtained from the Green Gene International, Hill Castles, United Kingdom, and the B. nigra accessions were obtained from the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany. The interspecific cross-derived hybrid ovules were cultured in vitro following the technique described by Bennett . After about 14 days in culture, the visible embryos were transferred to solid B5 medium containing 0.1 mg/L GA3, 20 g/L sucrose and 8 g/L agar (Coventry ) for 3–4 weeks until roots and shoots are developed. The plantlets were then planted in six-inch pots containing soil-free growth medium.

Interspecific nature of the plantlets was confirmed by the use of A- and B-genome specific simple sequence repeat (SSR or microsatellite) markers. For this, a total of 29 SSR markers specific to the 10 A-genome linkage groups (A1 to A10) and 36 markers from the eight B-genome linkage groups (B1 to B8) were used. Details of DNA extraction and PCR amplification of the SSR markers is described elsewhere (Hasan and Rahman 2016).

Chromosome doubling and generation of resynthesized B. juncea lines

The S0 plantlets identified as B. rapa × B. nigra interspecific hybrid were treated with 0.34% (w/v) aqueous solution of colchicine for chromosome doubling. The chromosome-doubled fertile S0 plants were self-pollinated using 5% NaCl solution (Tingdong ) for S1 seeds. The S1 families were grown in a glasshouse and were self-pollinated by bag isolation for S2 seeds.

Assessment of the ploidy level

The ploidy level of the S2 generation resynthesized B. juncea lines and their diploid parents were determined through flow cytometric analysis of nuclear DNA content using a Partec CyFlow® Ploidy Analyzer (www.partec.com). The ultra-violet (UV) light of the instrument was set at 365 nm wavelength and the samples were run at a rate of 20 to 50 nuclei/sec. Data was acquired for 1500 to 2500 nuclei per sample. One canola quality B. juncea breeding line from the Canola Breeding Program of the University of Alberta was used as the reference. The ploidy level of the samples was calculated by using the following equation (Dolezel );

Evaluation for clubroot resistance

The S1 and S2 generation resynthesized B. juncea lines and their diploid parents were evaluated for resistance to the single-spore derived P. brassicae isolate, classified as pathotype 3 based on Williams (1966) differentials. Resting spore suspension (inoculums) was prepared from preserved galls following the protocol described by Strelkov . The details of inoculation and screening of the inoculated plants for resistance is described in Hasan and Rahman (2016).

Results

Production of resynthesized Brassica juncea

A total of 43 interspecific crosses were made which gave 14 silique carrying fertilized ovules (developed to normal size) (Table 1). Five siliques of the B. rapa cv. Gelria × B. nigra CR 2136 cross yielded 34 fertilized ovules, and this translated to 6.8 fertilized ovules/silique. Fifteen (44.1%) of the 34 cultured ovules yielded zygotic embryos of which 13 (38.2%) grew into plantlets. On the other hand, nine siliques of the B. rapa cv. Gelria × B. nigra CR 2137 cross yielded 56 ovules translating to 6.2 fertilized ovules/silique; only 21 (37.5%) of the 56 ovules yielded zygotic embryos of which 17 (30.4%) developed into plant. All 30 plantlets obtained from the two crosses were treated with colchicine, however, only two (6.67%) plants of B. rapa cv. Gelria × B. nigra CR 2137 became amphidiploid (AABB). These plants produced fertile pollen and viable seed under self-pollination (Table 1). Single silique from each of the two S0 plants, 1578.001 and 1578.002, produced 13 and seven S1 seeds, respectively. A total of eight and seven S1 plants, respectively, of 1578.001 and 1578.002 were grown in a glasshouse of which three of 1578.001 and four of 1578.002 were self-pollinated by bag isolation for S2 seeds.

Table 1

Resynthesis of Brassica juncea (AABB, 2n = 36) though in vitro culture of ovules of Brassica rapa (AA, 2n = 20) × Brassica nigra (BB, 2n = 16) interspecific cross

Female	Male	No. pollination	No. silique formed	No. ovule cultured	No. zygotic embryo developed	No. plants transferred	No. resynthesized plants obtained	Plant ID
B. rapa ssp. rapifera cv. Gelria, p1	B. nigra (CR 2136), p1	9	3	20	9	9	0
B. rapa ssp. rapifera cv. Gelria, p3	B. nigra (CR 2136), p3	8	2	14	6	4	0
B. rapa ssp. rapifera cv. Gelria, p1	B. nigra (CR 2137), p1	11	5	26	13	11	1	1578.001
B. rapa ssp. rapifera cv. Gelria, p1	B. nigra (CR 2137), p2	15	4	30	8	6	1	1578.002
Total		43	14	90	36	30	2

Molecular characterization of the resynthesized B. juncea lines

Interspecific nature of the S0 plants was confirmed using SSR markers. For this, 190 SSR markers from the 10 A-genome chromosomes (A1 to A10) were screened of which 29 showed clear polymorphism between the A and B genome parental species (Table 2). Of the 29 polymorphic markers, 15 amplified the expected alleles in B. rapa but showed no amplification product in B. nigra; these 15 markers also amplified similar size alleles in the resynthesized B. juncea plants. The other 14 markers amplified alleles both in B. rapa and B. nigra, and similar size alleles were also detected in the resynthesized B. juncea plants. Based on this marker analysis, it can be anticipated that all 10 A-genome chromosomes of B. rapa were present in the resynthesized B. juncea plants.

Table 2

Evaluation of the resynthesized Brassica juncea lines by SSR (microsatellite) markers from the ten A genome linkage groups including those are specific to the A genome of Brassica rapa

Linkage group (LG)	Total no. marker tested	No. marker poly-morphic between diploid parents	Primer name	Amplified allele size (bp)
				B. rapa ssp. rapifera cv. Gelria (AA genome)	B. nigra (CR2137) (BB genome)	Resynthesized B. juncea (AABB genome)
A1	22	4	sNRA51nm	198	–	198
			sS2136b	123	138	123, 138
			sN11665	276	272	272, 276
			sN11824 (aNP)	384	–	384
A2	24	3	sR12095	349	–	351
			sORE27 (aNP)	213, 239	239	213, 239
			BrSTS-78	158	162	158, 162
A3	15	3	sNRA85	133	162	133, 162
			sN1087(cNP)	471	–	471
			BoGMS1587	282	–	288
A4	31	2	sN2025	155	138	138, 155
			Na12-A01C	135	–	135
A5	15	3	Na10E02	155	–	155
			CB10080	133, 140	146	140, 146
			CB10545	96	–	96
A6	26	4	sN12508II	324	334	324, 334
			sR12156	198	–	198
			sN1958 (bNM)	365	361	365
			sN0904 (a)	234, 247, 255	255	234, 247, 255
A7	14	3	BRAS023	207, 217	–	207, 217
			BnGMS608	158	–	156
			BRMS129	276, 295	276, 284	276, 295
A8	12	2	Na12B05a	191	–	191
			BRMS185	254	–	254
A9	14	3	CB10373A	245	257	245, 257
			Ni4-D09	209	203	203, 209
			BnGMS81	397	–	397
A10	17	2	CB10524	239	–	239
			BRMS244	268	252	252, 268
Total	190	29

A total of 48 B-genome specific (chromosome B1 to B8) SSR markers were tested on the two parents; 36 of them amplified alleles only in B. nigra (Table 3) and these alleles were also detected in the resynthesized B. juncea lines. This marker analysis confirmed the presence of all eight B genome chromosomes of B. nigra in the resynthesized B. juncea lines.

Table 3

Evaluation of the resynthesized Brassica juncea plants by SSR (microsatellite) markers from the eight B-genome linkage groups

Linkage group (LG)	No. marker tested	No. markers poly-morphic between parents	Primer name	Allele size (bp) in
				B. rapa ssp. rapifera cv. Gelria (AA genome)	B. nigra (CR2137) (BB genome)	Resynthesized B. juncea (AABB genome)
B1	6	6	sJ3838F	–	289	289
			sJ4933	–	360	360
			sJ84165	–	307	307
			sJ0644	–	457	457
			sJ3891	–	123	123
			sB0563I	–	459	459
B2	6	3	sJ3302RI	–	433	420
			sJ03104	–	405	405
			sB4817R	–	270	270
B3	6	6	sJ3627R	–	308	308
			sB1822	–	282	282
			sB1672	–	208	208
			sJ7046	–	304	304
			sB1990F	–	511	511
			sB1752	–	450	450
B4	6	5	sA0306	–	382	351, 382
			sB0372	–	255	255
			sB2141AI	–	401	401
			sB1935A	–	275	275
			sJ8033	–	167	167
B5	6	5	sB3140	–	231	231
			sJ3874I	–	184	184
			sJ6842	–	355	355
			sB2556	–	268	268
			sB3872	–	197	197
B6	6	3	sJ7104	–	346	346
			sJ0338	–	359	359
			sJ0502	–	268	268
B7	6	5	sJ39119I	–	366	366
			sJ13133	–	317	317
			sJ1536	–	231	231
			sB1937	–	280	280
			sJ4633	–	328	328
B8	6	3	sJ34121	–	359	359
			sJ1668I	–	325	325
			sB3739	–	397	397
Total	48	36

Ploidy assessment of the resynthesized B. juncea plants

A total of 36 plants belonging to seven S2 families were analyzed for nuclear DNA content to determine their ploidy level. Of the seven S2 families, three derived from the S1 line 1578.001 showed a mean ploidy level of 4.10 ± 0.218, which is similar to the natural B. juncea (Table 4). On the other hand, mean ploidy level of the four S2 families, derived from the S1 line 1578.002, was 4.44 ± 0.119 indicating the occurrence of plants with greater chromosome number in this population.

Table 4

Ploidy level of the 36 S2 generation resynthesized Brassica juncea plants measured through estimation of nuclear DNA content using a flow cytometer

Family ID	Generation	No. plants tested	Ploidy (Mean ± SE)
Brassica junceaa	Inbred	5	4.00 ± 0.083
S2 derived from 1578.001 (S1)
1578.003	S2	5	4.86 ± 0.254
1578.005	S2	4	3.41 ± 0.185
1578.008	S2	4	3.85 ± 0.222
Sub total		13	4.10 ± 0.218
S2 derived from 1578.002 (S1)
1578.004	S2	6	4.46 ± 0.069
1578.006	S2	6	3.93 ± 0.372
1578.007	S2	4	4.77 ± 0.059
1578.009	S2	7	4.69 ± 0.097
Sub total		23	4.44 ± 0.119

Canola quality Brassica juncea breeding line from University of Alberta Canola breeding program.

Resistance to Plasmodiophora brassicae

A total of 15 S1 plants derived from the two resynthesized B. juncea lines (1578.001 and 1578.002) were evaluated for resistance to P. brassicae pathotype 3. All 15 plants were completely resistant to this pathotype (disease score 0). Seven of the 15 S1 plants were self-pollinated by bag isolation for S2 seeds. The S1 plants showed wide variation for seed set—ranging from as low as 30 seeds per plant to as high as 515 seeds per plant.

A total of 103 plants belonging to seven S2 families were evaluated for resistance to pathotype 3. All S2 plants belonging to three S2 families, 1578.003, 1578.005 and 1578.008 which derived from the S1 family 1578.001, were resistant. On the other hand, 87 to 94% S2 plants belonging to four S2 families, 1578.004, 1578.006, 1578.007 and 1578.009 which derived from the S1 family 1578.002, were resistant to this pathotype; thus, resistance was lost in about 6 to 13% of the S2 plants of these four families during their development through self-pollination (Table 5). No significant correlation between seed set on the S1 plants and clubroot resistance in the S2 families could be found (r = −0.523, R = 0.274; df = 5, p < 0.05).

Table 5

Resistance in S1 and S2 generation plants of resynthesized Brassica juncea to Plasmodiophora brassicae pathotype 3

Family ID	Generation	No. selfed seed produced	No. plants tested	No. R plant (Score 0)	Number of S plant				Percent resistant plant
					Score 1	Score 2	Score 3	Total S plant
1578.001	S1	13	8	8	0	0	0	0	100.0
1578.002	S1	7	7	7	0	0	0	0	100.0
Sub total			15	15				0	100.0
S2 derived from 1578.001
1578.003	S2	42	9	9	0	0	0	0	100.0
1578.005	S2	208	8	8	0	0	0	0	100.0
1578.008	S2	54	29	29	0	0	0	0	100.0
Sub total			46	46				0	100.0
S2 derived from 1578.002
1578.004	S2	118	18	17	0	0	1	1	94.4
1578.006	S2	215	15	13	0	0	2	2	86.7
1578.007	S2	30	9	8	0	0	1	1	88.9
1578.009	S2	515	15	13	0	0	2	2	86.7
Sub total			57	51				6	89.5
Grand Total	S2		103	97				6	94.2

Note: R = Resistant; S = Susceptible.

Discussion

The present study demonstrated that a clubroot resistant B. juncea line in the S2 generation could be achieved through resynthesis of this species by exploiting the resistance available in one of the parental species, B. rapa. The allopolyploid resynthesized B. juncea lines, theoretically, were assumed to be homozygous and the resistance was expected to be inherited in a stable manner through the self-pollinated generation; however, loss of resistance occurred in some of the S2 plants that obtained from these experiments (Table 5). Several researchers have reported that chromosomes in the resynthesized Brassica allopolyploids can undergo meiotic anomalies and homoeologous pairing in their early generations, and this can result in some structural rearrangements including loss or gain of chromosomes (Gaeta , Gaeta and Pires 2010, Szadkowski , Udall , Xiong ). The mechanisms driving the change in chromosome number and structure in the newly formed polyploid is not well understood; this may result from downsizing of nuclear DNA content, inter- and intra-genomic rearrangements, chromosome breakage and fusion, rDNA change, and loss of repeat sequences (Han , Leitch and Bennett 2004, Liu , Renny-Byfield , Xiong , for review, see Renny-Byfield and Wendel 2014). According to Xiong , chromosome number in self-pollinated progeny of a resynthesized B. napus (2n = 38) plant can vary from 2n = 36 to 42; in this regard, the occurrence of greater nuclear DNA content in S2 progeny of the resynthesized B. juncea plant 1578.002 agree with the result reported by Xiong . In addition to chromosomal change, allopolyploids can also exhibit a change in gene expression (reviewed by Adams and Wendel 2005, Chen and Ni 2006) which can cause a change in the phenotype. Salmon found DNA methylation in about 30% of the parental fragments in the allopolyploids of Spartina spp. Structural rearrangement of chromosomes in resynthesized B. napus can also contribute to the variation of a quantitative trait, such as flowering time (Pires ). In case of qualitative traits, such as self-incompatibility (Rahman 2005) and clubroot resistance (Diederichsen and Sacristan 1996), stability of the trait has often been seen in self-pollinated progeny of a resynthesized B. napus plant. The clubroot resistance in the resynthesized B. juncea lines developed in this research is derived from the B. rapa cv. Gelria. This cultivar reported to carry the major clubroot resistance gene CRa/CRb; however, the reason of the loss of resistance in some of the S2 plants was beyond the scope of the present study. The loss of resistance might have resulted from the loss of the genomic region carrying the resistance; further investigation would be needed to resolve this.

The resynthesized B. juncea lines obtained in this study showed wide variation for seed set under self-pollination. Poor seed set in a resynthesized allopolyploid is a common phenomenon, especially in their early generations, as reported by Srivastava in B. juncea. Meiotic anomalies in the resynthesized Brassica allopolyploids, as discussed above, can result in reduced pollen viability and thus poor seed set (Ramsey and Schemske 2002). Xiong found an inverse correlation of seed yield and pollen viability with the increased aneuploidy; they observed the highest fertility in the resynthesized B. napus lines carrying the parental chromosomes with least change. Self-incompatibility of the parental species may also have contributed to this reduced seed set under self-pollination in the resynthesized B. juncea lines developed in this study. Rahman (2005) also reported the effect of the self-incompatibility genes on reduced seed set in resynthesized B. napus.