David J. Luckett

Genome-wide delineation of natural variation for pod shatter resistance in Brassica napus

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.

Canola (Brassica napus) germplasm shows variable allelopathic effects against annual ryegrass (Lolium rigidum)

AimsThe allelopathic activity of canola (Brassica napus) germplasm was investigated using the important Australian weed, annual ryegrass (Lolium rigidum) as the target species.MethodsThree different canola plant densities (10, 20, and 30 seedlings/beaker) of each of 70 world-wide genotypes were tested in vitro in close proximity to annual ryegrass seedlings.ResultsThe allelopathic activity of canola, as measured by reduction in annual ryegrass root and shoot growth, increased with canola crop seedling densities. Density did not consistently influence shoot length of annual ryegrass. Greater shoot length suppression was observed in genotype cv. Rivette and BLN3343CO0402. The Australian genotype cv. Av-opal and the breeding line Pak85388-502 suppressed root length of ryegrass more than other genotypes, even at low densities. At the lowest density, the least allelopathic genotypes were cv. Barossa and cv. Cescaljarni-repka, although they became more allelopathic at higher density. An overall inhibition index was calculated to rank each of the canola genotypes. There were significant differences between canola genotypes in their ability to inhibit root and shoot growth in ryegrass.ConclusionConsiderable genetic variation exists among canola genotypes for their allelopathic effects on annual ryegrass. Further investigation is required to determine the allelopathic mechanisms, particularly to identify the responsible allelochemical(s) and the gene(s) controlling the trait. This research suggests that highly allelopathic canola genotypes can be potential for controlling weeds such as annual ryegrass in integrated weed management programs.

A rapid method for the simultaneous quantification of the major tocopherols, carotenoids, free and esterified sterols in canola (Brassica napus) oil using normal phase liquid chromatography

A normal phase high performance liquid chromatography (HPLC) method was developed to simultaneously quantify several prominent bioactive compounds in canola oil vis. α-tocopherol, γ-tocopherol, δ-tocopherol, β-carotene, lutein, β-sitosterol, campesterol and brassicasterol. The use of sequential diode array detection (DAD) and tandem mass spectrometry (MS/MS) allowed direct injection of oils, diluted in hexane without derivatisation or saponification, greatly reducing sample preparation time, and permitting the quantification of both free sterols and intact sterol esters. Further advantages over existing methods included increased analytical selectivity, and a chromatographic run time substantially less than other reported normal phase methods. The HPLC-DAD-MS/MS method was applied to freshly extracted canola oil samples as well as commercially available canola, palm fruit, sunflower and olive oils.

Quantitative genetic analysis of grain yield in an Australian Brassica napus doubled-haploid population

Abstract. High yield is a major objective in canola-breeding programs. We analysed the genetic determinants controlling variation in grain yield in a doubled-haploid (DH) breeding population derived from a single BC1F1 plant from the cross Skipton/Ag-Spectrum//Skipton (designated as the SAgS population). DH lines were evaluated for flowering time and yield in two replicated trials and exhibited significant genetic variation for both traits. Yield showed negative correlation with flowering time; lines that flowered earlier had higher yield than late-flowering lines. A genetic linkage map comprising 7716 DArTseq markers was constructed for the SAgS population, and a ‘bin’ map based on 508 discrete single-position (non-co-segregating) marker loci was used for quantitative trait locus (QTL) analysis. We identified 20 QTLs (LOD ≥2) associated with variation in flowering time and grain yield. Two QTLs (Qy.wwai-A7/Qdtf.wwai-A7/Qfs.wwai-A7 and Qy.wwai-C3a/Qfs.wwai-C3a) appeared repeatedly across experiments, accounting for 4.9–19% of the genotypic variation in flowering time and yield and were located on chromosomes A07 and C03. We identified 22 putative candidate genes for flowering time as well as grain yield, and all were located in a range of 935 bp to 2.97 Mb from markers underlying QTLs. This research provides useful information to be used for breeding high-yielding canola varieties by combining favourable alleles for early flowering and higher grain yield at loci on chromosomes A07, C03 and possibly on A06.

Competitive ability of Australian canola (Brassica napus) genotypes for weed management

Abstract. Canola (Brassica napus L.) is an important break crop in Australian cropping systems but weeds are a major cost to production and herbicide-resistant weeds are spreading. The potential competitive ability of canola genotypes to both suppress weed growth and maintain grain yield and quality in the presence of weeds has not been determined in Australia. Two experiments examined the range in competitive ability of 16 B. napus genotypes against annual ryegrass (Lolium rigidum Gaud.) and volunteer wheat (Triticum aestivum L.) over two contrasting seasons. Weed biomass at flowering was generally reduced 50% more in the presence of the strongly competitive genotypes than the least competitive, and this has significant benefits for lower weed seed production and reduced seedbank replenishment. Suppression of weed growth was negatively correlated with crop biomass. Significant differences in grain yield of canola were recorded between weedy and weed-free plots, depending on crop genotype, presence of weeds and season. Crop yield tolerance (where 0% = no tolerance and 100% = complete tolerance) to ryegrass competition ranged from 0% (e.g. with CB-Argyle) to 30–40% (e.g. with the hybrids 46Y78 and Hyola-50) in the dry season of 2009. Yield tolerance was higher (50–100%) with the lower densities of volunteer wheat and in the 2010 season. The range between genotypes was similar for both conditions. The hybrids and AV-Garnet were higher yielding and more competitive than the triazine-tolerant cultivars. The ranking of genotypes for competitiveness was strongly influenced by seasonal conditions; some genotypes were consistently more competitive than others. Competitive crops are a low-cost tactic for integrated weed management to reduce dependence on herbicides and retard the spread of herbicide-resistant weeds.

Canola cultivar performance in weed-infested field plots confirms allelopathy ranking from in vitro testing

Crop competition and allelopathy are two cultural control options for possible inclusion in cropping systems. This research aimed to identify superior allelopathic canola genotypes through a two-year field study. First year screening results of 312 diverse Brassica genotypes showed genotypes differed significantly in their ability to suppress weed infestations. Crop plant height was correlated with the competitive ability of several genotypes, while other genotypes showed good weed-suppressive ability despite being short. Thirty-six of the genotypes grown in the field had been previously assessed for their allelopathic ability to inhibit the growth of annual ryegrass (Lolium rigidum) seedlings using an in vitro technique. The highly allelopathic genotypes: Av-opal, Sardi603, Rivette and Atr-beacon performed well against annual ryegrass in the laboratory and also against other species, including Capsella bursa-pastoris, Sisymbrium orientale and Hordeum leporinum in the field. The weakly allelopathic Barossa and X-06-6-3725 genotypes performed poorly both in the laboratory studies and in the field. The following year, field testing of selected genotypes at two sowing dates further suggested that the most allelopathic genotypes in the laboratory bioassay were generally those that suppressed weed numbers and their biomass in the field. The late sowing time increased the natural weed pressure leading to a decrease in both canola grain yield and quality. Many of the highly allelopathic canola genotypes, which caused low weed populations in the field, had relatively low grain yield. This suggests that the allelopathic trait is independent of local adaptation and yields potential under weed-free conditions. Ideally, cultivars with both high allelopathy and high competitive ability would be most useful to help farmers maximise yield and control weeds. Selection for allelopathy in canola shows potential as a future non-chemical weed control option and requires further investigation.

Breeding Lupinus albus for resistance to the root pathogen Pleiochaeta setosa

Pleiochaeta root rot (PRR) caused by Pleiochaeta setosa is a serious, widespread fungal disease in lupin crops, especially in Lupinus albus (broad-leaf lupin, or white lupin). PRR resistance is common in the gene pool of L. albus with various landraces from the Mediterranean region being the most resistant, and suitable for use in breeding new cultivars. Heritability of resistance is sufficient to make good gains from selection but only when controlled-environment (CE) screening is used. Field disease nurseries on loamy soil gave much lower heritability of resistance. Field disease nurseries had spatially variable spore counts despite continuous lupin cropping, and this was partly responsible (along with climatic conditions) for their reduced precision compared to tests conducted in a CE. Giving infected L. albus roots a single, most-severe-lesion score on a 0–9 scale was adequate for CE screening but not as precise or discriminating as the more time-consuming method of six scores per root. Replication in CE experiments was reduced to two pots of 16 seedlings each without sacrificing genotype discrimination.

Evidence that Diaporthe toxica infection of Lupinus albus is an emerging concern for the Australian lupin industry

Lupinus albus has been grown commercially in Australia since the 1970s and has long been regarded as resistant to phomopsis caused by Diaporthe toxica. Isolates capable of infecting L. albus were collected following an outbreak of phomopsis in 2004 in southern New South Wales. Glasshouse screening of L. albus cultivars with the pathogen revealed that genotypes differ in their response to D. toxica with some current cultivars having useful resistances. Several breeding lines and old cultivars were susceptible in either vegetative or reproductive tissue or both. The commonly grown commercial cultivar Kiev Mutant was moderately susceptible in all tissues assessed. A principle component analysis gave weak to moderate correlation (r = 0.14 to r = 0.54, depending on the two-way comparison) between vegetative and reproductive tissue, which is suggestive that resistance in different tissues may be under distinct genetic control. The germination of infected seed from the outbreak in 2004 was severely compromised, and much of the seed harboured the pathogen. A survey of 171 commercial seed samples from 2004 to 2006 showed that phomopsis discoloured seed was present in only 20 samples, all of them confined to southern New South Wales. While the pathogen presents a risk to L. albus cropping zones in southern New South Wales, it is not widely spread at this stage. However, vigilance is required to prevent infected seed from being sown so that the disease can be kept in check.

Metabolomics differentiation of canola genotypes: toward an understanding of canola allelochemicals

Allelopathy is one crop attribute that could be incorporated in an integrated weed management system as a supplement to synthetic herbicides. However, the underlying principles of crop allelopathy and secondary metabolite production are still poorly understood including in canola. In this study, an allelopathic bioassay and a metabolomic analysis were conducted to compare three non-allelopathic and three allelopathic canola genotypes. Results from the laboratory bioassay showed that there were significant differences among canola genotypes in their ability to inhibit root and shoot growth of the receiver annual ryegrass; impacts ranged from 14% (cv. Atr-409) to 76% (cv. Pak85388-502) and 0% (cv. Atr-409) to 45% (cv. Pak85388-502) inhibition respectively. The root length of canola also differed significantly between genotypes, there being a non-significant negative interaction (r = -0.71; y = 0.303x + 21.33) between the root length of donor canola and of receiver annual ryegrass. Variation in chemical composition was detected between organs (root extracts, shoot extracts) and root exudates and also between canola genotypes. Root extracts contained more secondary metabolites than shoot extracts while fewer compounds were recorded in the root exudates. Individual compound assessments identified a total of 14 secondary metabolites which were identified from the six tested genotypes. However, only Pak85388-502 and Av-opal exuded sinapyl alcohol, p-hydroxybenzoic acid and 3,5,6,7,8-pentahydroxy flavones in agar growth medium, suggesting that the synergistic effect of these compounds playing a role for canola allelopathy against annual ryegrass in vitro.

Construction of integrated linkage map of a recombinant inbred line population of white lupin (Lupinus albus L.)

We report the development of a Diversity Arrays Technology (DArT) marker panel and its utilisation in the development of an integrated genetic linkage map of white lupin (Lupinus albus L.) using an F8 recombinant inbred line population derived from Kiev Mutant/P27174. One hundred and thirty-six DArT markers were merged into the first genetic linkage map composed of 220 amplified fragment length polymorphisms (AFLPs) and 105 genic markers. The integrated map consists of 38 linkage groups of 441 markers and spans a total length of 2,169 cM, with an average interval size of 4.6 cM. The DArT markers exhibited good genome coverage and were associated with previously identified genic and AFLP markers linked with quantitative trait loci for anthracnose resistance, flowering time and alkaloid content. The improved genetic linkage map of white lupin will aid in the identification of markers for traits of interest and future syntenic studies.