Phil Salisbury

The importance of the type and time of inoculation and assessment in the determination of resistance in Brassia napus and B. juncea to Sclerotinia sclerotiorum

Sclerotinia stem rot (SSR) is a significant agricultural problem worldwide. Finding sources of resistance is crucial to the ongoing search for better management of this disease. Brassica germplasm from Australia, China and India was screened for resistance to SSR under Western Australian field conditions following stem inoculation, application of a spray of mycelial suspension, or as a consequence of myceliogenic germination originating from sclerotia resident in soil. Significant differences in response were observed among 53 genotypes using each of the three screening methods. There was a variable impact of the time of inoculation on the disease level depending upon time of assessment post-stem inoculation. However, this impact could be reduced to an insignificant level provided the assessment after stem inoculation was delayed until 3 weeks post-inoculation. The results of these studies indicate that the use of appropriate inoculation and assessment methods could significantly reduce variability in the responses commonly observed in screening for resistance in crop plants against Sclerotinia sclerotiorum.

Genome-wide Association Study Identifies New Loci for Resistance to Leptosphaeria maculans in Canola

Key message “We identified both quantitative and quantitative resistance loci to Leptosphaeria maculans, a fungal pathogen, causing blackleg disease in canola. Several genome-wide significant associations were detected at known and new loci for blackleg resistance. We further validated statistically significant associations in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance in canola.” Blackleg, caused by Leptosphaeria maculans, is a significant disease which affects the sustainable production of canola (Brassica napus). This study reports a genome-wide association study based on 18,804 polymorphic SNPs to identify loci associated with qualitative and quantitative resistance to L. maculans. Genomic regions delimited with 694 significant SNP markers, that are associated with resistance evaluated using 12 single spore isolates and pathotypes from four canola stubble were identified. Several significant associations were detected at known disease resistance loci including in the vicinity of recently cloned Rlm2/LepR3 genes, and at new loci on chromosomes A01/C01, A02/C02, A03/C03, A05/C05, A06, A08, and A09. In addition, we validated statistically significant associations on A01, A07, and A10 in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance and mapped within 13.2 kb from Arabidopsis R gene of TIR-NBS class. We showed that resistance loci are located in the vicinity of R genes of Arabidopsis thaliana and Brassica napus on the sequenced genome of B. napus cv. Darmor-bzh. Significantly associated SNP markers provide a valuable tool to enrich germplasm for favorable alleles in order to improve the level of resistance to L. maculans in canola.

Molecular mapping and validation of Rlm1 gene for resistance to Leptosphaeria maculans in canola (Brassica napus L.)

Abstract. European winter canola (Brassica napus L.) cultivars harbour genes for durable resistance to the fungus Leptosphaeria maculans, which causes blackleg disease under Australian environmental conditions. Previous studies have shown that resistance in winter-type cultivars Maxol and Columbus is controlled by two genes, Rlm1 and Rlm3, which have been mapped using randomly amplified polymorphic DNA markers onto chromosome A7. We mapped a doubled-haploid population that consisted of 101 lines from a cross between Maxol*1 and Westar-10 using diversity arrays technology and simple sequence repeat (SSR)-based markers. Two SSR marker loci, Xol12-e03 and Xra2-a05b, flanked the Rlm1 locus at an interval of 6.7 cM, which corresponds to ∼3.2 Mb of the Brassica rapa genomic sequence; this region contains several genes encoding putative kinase and leucine-rich repeat-type disease-resistance proteins. SSR markers were further tested for their linkage with the Rlm1 locus in an independent population derived from Columbus*3/Westar-10. Our results showed that SSR markers linked to Rlm1 can be useful for monitoring Rlm1 gene introgression in breeding populations derived from Maxol and Columbus.

Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianBrassica juncea lines

In recent years,Brassica species have acquired an important position in the oilseed industry. Even thoughBrassica transformation protocols are well established,there is still a need for the development of new transformation protocols for elite AustralianB. juncea lines,because regeneration inB. juncea is highly genotype-dependent and in addition, their hypocotyl explants are susceptible to necrosis.Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianB. juncea lines is described here. To overcome necrosis, we have adopted 2 strategies: extension of precultivation time of hypocotyl explants, and use of a 2-stage hygromycin selection process.The frequency of recovery of transformants from AustralianB. juncea andBrassica napus lines was 1.7% and 0.9%, respectively. Polymerase chain reaction tests confirmed that allBrassica plants that survived through stringent screening procedures were positive for the inserted hygromycin resistance gene,hph. Progeny from 6Brassica lines tested segregated for thehph gene, and χ2 analysis suggested a 3:1 segregation ratio.This is in line with a tDNA integration into a single locus, which is an important feature of a transformation protocol for subsequent breeding purposes.

Assessing progress in breeding to improve grain yield, quality and blackleg (Leptosphaeria maculans) resistance in selected Australian canola cultivars (1978–2012)

Abstract. Canola breeding in Australia began in the early 1970s with the first cultivars being released in the late 1970s. Thirty-four non-herbicide-tolerant canola cultivars, released in Australia between 1978 and 2012, were evaluated for improvements in yield, quality, blackleg resistance and adaptation to Australian environments. The cultivars were sown at three sites in 2008 and one site in 2014. In addition, blackleg susceptibility was assessed in two independent blackleg experiments in 2008. Yield improvement averaged 21.8 kg ha–1 year–1 (1.25% year–1) but ranged from 8 to 39.1 kg ha–1 year–1 at the lowest to the highest yielding sites, respectively. Although the yield gain shown by our study was for conventional canola only, the different herbicide-tolerant types are derived by incorporating the herbicide tolerance genes into Australian germplasm and so the rate of genetic gain would be expected to be similar for all herbicide tolerance types. Oil and protein concentrations have increased by 0.09% year–1 and 0.05% year–1, respectively, whereas glucosinolate concentration was reduced to between 7 and 16 μmoles per gram of meal by the mid-1990s. Cultivars released before 2002 all had low to moderate resistance to the blackleg isolates present in the fields during the experimental period but more recent releases had improved survival under heavy blackleg pressure due to the incorporation of additional or different resistance genes. The data suggests that at least 25% of the yield improvement achieved by the breeding programs over 30 years was associated with improved blackleg resistance and the remainder with gains in other aspects of potential grain yield. The private breeding companies in Australia will need to continue to produce cultivars with high yield potential and deploy blackleg resistance genes wisely in order to maintain the yield improvements required to remain competitive in global markets.

Pterostilbene Is a Potential Candidate for Control of Blackleg in Canola

Two stilbenes, resveratrol and pterostilbene, exhibit antifungal activity against Leptosphaeria maculans, the fungal pathogen responsible for blackleg (stem canker) in canola (Brassica napus). In vitro studies on the effect of these stilbenes on L. maculans mycelial growth and conidia germination showed that pterostilbene is a potent fungicide and sporicide, but resveratrol only exerted minor inhibition on L. maculans. Cell viability of hyphae cultures was markedly reduced by pterostilbene and SYTOX green staining showed that cell membrane integrity was compromised. We demonstrate that pterostilbene exerts fungicidal activity across 10 different L. maculans isolates and the compound confers protection to the blackleg-susceptible canola cv. Westar seedlings. The potential of pterostilbene as a control agent against blackleg in canola is discussed.

Agricultural selection and presence–absence variation in spring-type canola germplasm

Abstract. Brassica napus (rapeseed, canola) is an important oilseed crop worldwide as well as a recent agricultural hybrid species, resulting from crosses between progenitor B. rapa (turnip) and B. oleracea (cabbage) species in the last few thousand years. No wild form of B. napus is known to exist, making B. napus an interesting model for studies of genetic and genomic evolution in a polyploid under agricultural selective pressure. We generated genotype (Illumina Infinium 60K Brassica array) and phenotype data for elite spring-type B. napus lines from Australia, China and India (only one line). Phenotypically, plant growth, silique development and flowering traits were more likely to differentiate Chinese germplasm, whereas resistance to blackleg disease, secondary branching and seed traits were more likely to differentiate Australian germplasm. Genetic differentiation between the Australian and Chinese populations was low (FST = 0.035). Genetic relationship was not a predictor of similarity in yield traits between lines. Presence–absence variants were detected across the population: variants shared by at least three lines were present in every chromosome in the B. napus genome, and large missing chromosome segments (>1 Mbp) putatively due to A–C genome translocations were observed on chromosomes A7, A10, C1, C2, C6, C8 and C9. Our results highlight that widespread presence–absence variation is usual in B. napus, and may suggest that phenotypic and genetic diversity are not closely linked within spring-type B. napus from Australia and China, although the low sample numbers in our study prevent strong conclusions. We propose that inbreeding and low levels of genetic diversity, coupled with exchanges between the A and C genomes, were major driving forces behind genome evolution in this recent agricultural crop species.

A multiplex PCR for rapid identification of Brassica species in the triangle of U

BackgroundWithin the Brassicaceae, six species from the genus Brassica are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six Brassica species are described by U’s triangle model. Extensive shared traits and diverse morphotypes among Brassica species make identification and classification based on phenotypic data alone challenging and unreliable, especially when dealing with large germplasm collections. Consequently, a major issue for genebank collections is ensuring the correct identification of species. Molecular genotyping based on simple sequence repeat (SSR) marker sequencing or the Illumina Infinium Brassica napus 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of Brassica collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of Brassica accessions for germplasm management. A cheaper, faster and simpler method for Brassica species identification is described here.ResultsA multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the Brassica A, B and C genomes was able to reliably distinguish all six Brassica species in the triangle of U with 16 control samples of known species identity. Further validation against 120 Brassica accessions previously genotyped showed that the MPCR is highly accurate and comparable to more advanced techniques such as SSR marker sequencing or the Illumina Infinium B. napus 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of Brassica accessions.ConclusionA cheap and fast multiplex PCR assay for identification of Brassica species in the triangle of U was developed and validated in this study. The MPCR assay can be readily implemented in any basic molecular laboratory and should prove useful for the management of Brassica germplasm collections in genebanks.