Breeding Science
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Resynthesis of Brassica juncea for resistance to Plasmodiophora brassicae pathotype 3
Muhammad Jakir HasanHabibur Rahman
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2018 Volume 68 Issue 3 Pages 385-391

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Abstract

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 et al. 2006), drought (Wright et al. 1995) and silique shatter (Wang et al. 2007), and resistance to blackleg (Roy 1978) and leaf blight disease (Wechter et al. 2007). This crop species yields greater than B. napus in heat- and drought-prone areas as well as in short growing season areas (Burton et al. 1999, Potts et al. 1999). Although, B. juncea possess all these desired properties, resistance to Plasmodiophora brassicae Woronin, causing clubroot disease, is not available in this species (Hasan et al. 2012). Clubroot disease can result in up to 90% yield loss and about 4–6% decrease in seed oil content (Pageau et al. 2006). 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 et al. 1975, Hasan et al. 2012). At least eight clubroot resistance genes have been mapped to date in B. rapa (reviewed by Piao et al. 2009), 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 et al. 1998, Ueno et al. 2012) and CRb (Piao et al. 2004, 2009); however, recent studies have showed that the CRa and CRb to be the same locus and located on the chromosome A3 (Hatakeyama et al. 2017, Kato et al. 2013). 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 et al. (2008). 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 et al. 1988) 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 et al. 1992) 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 et al. 2007);

  
Sample ploidy  ( integer ) = ( Mean position of the G 1 sample peak ) ( Mean position of the G 1 reference peak ) × Reference ploidy

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 et al. (2007). 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
a  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, R2 = 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 et al. 2007, Gaeta and Pires 2010, Szadkowski et al. 2010, Udall et al. 2005, Xiong et al. 2011). 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 et al. 2005, Leitch and Bennett 2004, Liu et al. 1998, Renny-Byfield et al. 2013, Xiong et al. 2011, for review, see Renny-Byfield and Wendel 2014). According to Xiong et al. (2011), 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 et al. (2011). 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 et al. (2005) 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 et al. 2004). 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 et al. (2004) 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 et al. (2011) 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.

Acknowledgements

H.R. would like to thank Alberta Crop Industry Development Fund (ACIDF), Alberta Canola Producers Commission (ACPC), Agriculture and Agri-Food Canada (AAFC) and Natural Sciences and Engineering Research Council (NSERC) for financial support to this project. The authors would also like to thank Dr. S. Strelkov for providing the P. brassicae isolate, and personnel’s from the canola program for help in different routine operations.

Literature Cited
 
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