Abstract

Three pairs of sequence characterized amplified region (SCAR) primers designated SCAC-3, SCX-7 and SCAC-20, 21 to 24 bp in length were designed based on the sequences of RAPD markers RAC-3, RX-7 and SAC-20 respectively. Only the SCX-7 primer produced polymorphic markers corresponding to RX-7 amplified from ‘shatter-resistant’ DNA individuals. SCAC-3 and SCAC-20 produced amplified products corresponding to RAC-3 and SAC-20 to both shatter-resistant and shatter-susceptible DNA individuals. SCAR SCX-7 was subsequently tested with other Brassica populations including B. napus and BC₁F₇ of B. napus introgressed with B. rapa to investigate the presence of the sh2 gene. All lines from the backcross populations possessed the expected SCX-7 marker with the exception of one line where the marker was absent. This indicated that shatter-resistant gene sh2 was present in all these populations except for the one line.

INTRODUCTION

Seed loss from siliqua shattering is a major problem of canola production worldwide and especially in Australia because the crop matures in summer under hot and windy condition. Little variation for shatter resistance is available in Brassica napus, which is the major canola species. However, considerable variation for shatter resistance is present in B. rapa, which is a relatively minor oilseed species. Shatter resistance has been transferred from B. rapa into B. napus by backcrossing. Screening for shatter resistance in canola breeding programs is currently based on mechanical tests that measure individual siliqua strength. These tests, which are time consuming, can only take place with completely dry and mature siliquae. This study aimed to identify molecular markers linked to shatter resistance in a population segregating for this trait, and to develop specific and robust primers to be used in a routine test for screening for shatter resistance in canola breeding programs.

The phenotypic segregation in the F₂ population of a cross between the shatter-susceptible Canadian cv. Torch of B. rapa L. ssp. oleifera (Metzg.) Sinsk and a shatter-resistant Indian line, DS-17-D of B. rapa L. ssp. oleifera var. Brown Sarson (Singh) Prakash fitted a Mendelian ratio of 12: 3: 1 for shatter-susceptible (S), intermediate-shattering (M) and shatter-resistant (R) respectively. Phenotypic segregation in 19 F₃ families also supported the F₂ ratio of 12: 3: 1 (Mongkolporn et al., 1999). These results indicated that shatter resistance was a recessive trait controlled by two major genes designated sh1 and sh2.

Bulked segregant analysis coupled with random amplified polymorphic DNA (RAPD) analysis was applied to the entire F₃ population of the above cross. Three RAPD markers, designated RAC-3, RX-7 and SAC-20, were identified from the F₃ population. RAC-3 and RX-7 appeared to be linked in coupling to sh1 and sh2 at approximate distances of 13 cM and 20 cM respectively, whereas SAC-20 appeared to be linked in repulsion to both of these alleles at approximately 20 cM (Mongkolporn et al., 1999).

This paper reports the evaluation of SCAR markers developed from the three RAPD markers (RAC-3, RX-7 and SAC-20) linked to shatter resistance genes (sh1 and sh2) in the F₂ and F₃ populations of the cross Torch x DS-17-D, other B. rapa accessions, backcross populations and some B. napus accessions.

MATERIALS AND METHODS

Cloning and sequencing of RAC-3, RX-7 and SAC-20 RAPD markers

Three RAPD markers RAC-3, RX-7 and SAC-20 were amplified using the original bulked DNA from the F₃ population of a cross between Torch x DS-17-D as templates and the primers OPAC-3, OPX-7 and OPAC-20 (Operon Technologies, USA) respectively (Mongkolporn et al., 1999). The DNA fragments of the three markers were isolated from the agarose gel and purified using GenElute^TM Minus EtBr spin columns (Supelco Inc., USA). The purified RAC-3, RX-7 and SAC-20 fragments were cloned using the pGEM^®-T vector system (Promega Corporation, USA). Subsequently, the cloned products were each sequenced in both forward and reverse orientations using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corporation, USA).

Designing of SCAR primers

Pairs of SCAR primers were designed based on the forward and reverse sequences of each RAPD marker. Each primer pair contained the original 10 bases of the RAPD primer and 11 to 14 bases of adjacent sequence resulting in primers 21 to 24 oligonucleotides in length. The designed SCAR primers were analyzed using the computer program 'Primer Premier Mini' (Premier Biosoft International, CA, USA), for their potential not to produce dimers and hairpins by self-binding. Three pairs of SCAR primers SCAC-3, SCX-7 and SCAC-20 were subsequently synthesized from Gibco-BRL (Australia).

PCR conditions for SCAR primers

Genomic DNA templates from individuals classed as 'shatter-resistant' and ‘shatter-susceptible’ from the F₃ population of the cross between Torch and DS-17-D were used as templates to optimize annealing temperature. Annealing temperatures between 60 and 80°C were varied in a PCR thermal program: initial denaturation at 94°C for 1 min followed by 35 cycles of 94°C for 1 min, 60-80°C for 1 min and 72°C for 2 min with a final extension at 72°C for 5 min. Each PCR reaction contained 40 ng of genomic DNA template, 0.75 unit of Taq DNA polymerase (Gibco-BRL, Australia), 0.24 mM each of dATP, dCTP, dGTP and dTTP (Gibco-BRL, Australia), 0.2 μM each of forward and reverse SCAR primers, and PCR buffer with a final concentration of 0.01 M Tris-HCl, 2.5 mM MgCl₂, 0.05 M KCl, 0.1 mg/ml gelatin (pH 8.3) in a final volume of 25 μl.

Testing the SCX-7 SCAR primers on cultivars of B. rapa and B. napus; and backcross populations

The SCAR primer SCX-7 was tested on two Indian B. rapa lines var. Yellow Sarson, ‘B-46’ and ‘IB-5’; seven backcross populations; and three cultivars of B. napus. The backcross populations were BC₁F₇ derived from crosses between either B-46 or IB-5 (B. rapa), and one of the two B. napus accessions, 76-407R5*1-13-5 and 76-497R5*10-10-7, with subsequent backcrosses to the B. napus parent (Kadkol, 1985). The seven BC₁F₇ backcross lines selected for shatter resistance testing, YF33-1, YG16-1, YG32-1, YH35-1, YI18-1, YI26-1 and YN5-1 (Table 1) exhibited high levels of shatter resistance after measuring siliqua rupture energy using the pendulum test. Of the three cultivars of B. napus, var Excel was shatter-susceptible, and Oscar and Eureka were considered as tolerant to field shattering (Kadkol GP, pers. comm.).

Table 1 Parents of the seven backcross lines

RESULTS AND DISCUSSION

Evaluation of the SCAR primers SCAC-3, SCX-7 and SCAC-20

A pair of SCAR primers for each RAPD marker was designed based on the first 200 nucleotides of both forward and reverse sequences (data not shown). At the annealing temperatures of 70 and 80 ^oC, the SCAR primers SCAC-3 and SCAC-20 amplified single products, at approximately 900 and 1300 bp respectively, from both ‘shatter-susceptible’ and ‘shatter-resistant’ DNA. The loss of polymorphism between the resistant and susceptible lines with the two SCAR primers SCAC-3 and SCAC-20 could be because the original polymorphisms with the 10-base RAPD primers were due to small mismatches at the priming sites (Paran and Michelmore, 1993) resulting in no amplification product at this site. Whereas, the longer SCAR primers (21 to 24 bases) were not affected by small mismatches at the priming sites hence, the amplification products were produced at both sites on the resistant and susceptible lines. The SCX-7 SCAR primers amplified a single product at approximately 1000 bp from only ‘shatter-resistant’ DNA (Fig 1). The SCAR amplification product corresponded to the polymorphic band produced by the RAPD primer RX-7 in resistant lines (Fig 1b).

In Figure 1 there was no polymorphic marker in resistant plant 2 and a polymorphic marker was present in susceptible plant 19. These ‘incorrect’ markers may represent recombination that occurred between the marker and the gene loci thus indicating that this marker is not closely linked to the gene loci. To determine more precisely the linkage of the marker to the loci a larger population would need to be assessed.

Testing the SCX-7 primers on cultivars of B. rapa and B. napus, and backcross populations

A single amplification product of 1000 bp was produced with the SCX-7 primers from both accessions of Yellow Sarson, six of the seven backcross lines, and also from all cultivars of B. napus. No amplification product was observed in the line 'YN5-1' of the backcross population.

The presence of the PCR products amplified from SCX-7 primer indicated the presence of the sh2 allele, one of the alleles conferring shatter resistance. Therefore, it was likely that both B. rapa accessions B-46 and IB-5, three B. napus cultivars and six backcross lines (except 'YN5-1') may possess the sh2 allele. The Yellow Sarson B-46 and IB-5 were shatter-resistant with multivalved siliqua and yellow seed coat. The inheritance of shatter resistance in both the Yellow Sarson accessions was found to be controlled by three recessive genes (Kadkol, 1985; Kadkol et al., 1986). The presence of PCR products amplified from SCX-7 indicated that sh2 could have been one of the three recessive alleles in both genomes. The results also indicated that these backcross materials (except 'YN5-1') contained an sh2 allele. Since the background genome of these backcross lines was B. napus (76-407R5*1-13-5 and 76-497R5*10-10-7 accessions), the presence of the SCAR SCX-7 product in both these B. napus parents should be tested. However, these materials were not available for testing.

Figure 1 a) SCAR products amplified using SCX-7 primers with 10 'shatter-resistant' (1 to 10) and 10 'shatter-susceptible' (11 to 20) DNA samples from the F₃ population of the cross between Torch and DS-17-D, corresponding to b) the RAPD marker RX-7 amplified with the same DNA samples as those of SCAR products, which were members of the 'R' and 'S' DNA bulks in BSA. The DNA fragment sizes were compared with 100-bp molecular weight marker (M) and Lamda DNA/EcoR I + Hind III (m)

The backcross line 'YN5-1' showed the absence of PCR product amplified from SCX-7 indicating that the sh2 allele was absent in this line. The shatter-resistant phenotype of this line may be due to the effect of the presence of the other recessive loci from the Yellow Sarson genomes. Markers linked to those loci have not yet been identified. Alternatively, the absence of SCX-7 PCR product in 'YN5-1' could have resulted from the loss of the marker due to a recombination between the marker and the gene. Loss of the marker due to recombination was possible since the SCX-7 marker was found to be greater than 10 cM from the sh2 locus.

The three B. napus cultivars (Excel, Eureka and Oscar) also possessed the SCX-7 marker. This indicated that these cultivars possessed at least one copy of the sh2 allele. Eureka and Oscar have been reported to be tolerant to siliqua shattering (Mongkolporn et al. 1999). To express intermediate shattering in B. rapa, two copies of sh2 are required. However, as the B. napus genome is comprised of the B. rapa and B. oleracea genomes, the expression of partial shatter resistance in B. napus may require at least four copies of sh2. In the present study, the investigation of the copy number was not attempted, therefore, it would be interesting to determine the copy number of the resistance genes in further work. In a recent study using the SCX-7 primers, Kahlon et al. (1999) found that the sh2 gene was absent in a range of B. napus cultivars confirming the variation in the distribution of this resistance gene in the Brassica genome.

Shatter resistance has not been found in B. oleracea (Kadkol GP, pers. comm.). Kahlon et al. (1999) also could not find the sh2 gene present in B. oleracea after screening lines with the SCX-7 primers. B. rapa and B. oleracea, the progenitors of B. napus, are believed to originate from a common ancestor with a diploid chromosome number of six (Röbbelen, 1960; Prakash and Hinata, 1980). Molecular studies based on linkage maps showed that these two species were closely related and shared some chromosome homology (Truco et al., 1996). Mongkolporn et al., (1999) identified RAPD markers linked to the two dominant alleles Sh1 and Sh2 that confer shattering and concluded that these genes were likely to reside on duplicated chromosomes. B. oleracea may also contain at least two copies of the dominant alleles that confer shattering, thus as a consequence, complete shatter resistance does not occur in B. napus. Therefore, B. napus may still be shatter-susceptible, even though it contains the sh2 allele originating from B. rapa and/or B. oleracea.

To properly identify the introgression of genes conferring shatter resistance, it is necessary to develop two SCAR markers linked to the two recessive genes, sh1 and sh2, and also two SCAR markers linked to the two dominant alleles, Sh1 and Sh2. For developing SCAR markers from SCAC-3 and SCAC-20, more primers need to be designed or the SCAR bands amplified from resistant and susceptible lines need to be digested to reveal possible restriction site differences between the marker sequences of these lines. Ultimately, more molecular markers, produced from RAPD, AFLP or microsatellite analysis, might need to be screened on the bulked DNA samples using BSA to identify polymorphic bands that are more closely linked to shatter resistance. These bands would then need to be cloned, sequenced, and new SCAR primers designed.

Conclusion

The conversion of three RAPD markers, RAC-3, RX-7 and SAC-20, into SCAR markers was partially successful. Two approaches, based on more primer designing and digestion with restriction enzymes, may lead to the identification of polymorphisms for the PCR products amplified from the SCAR primers SCAC-3 and SCAC-20. New SCAR primers could be designed from the sequence data of the RAPD markers and then tested for discriminating the shatter-resistant from shatter-susceptible genotypes.

ACKNOWLEDGEMENTS

This project was partly funded by Ag-Seed Reseach Pty Ltd.

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