Evolving gene banks: improving diverse populations of crop and exotic germplasm with optimal contribution selection.

We simulated pre-breeding in evolving gene banks - populations of exotic and crop types undergoing optimal contribution selection for long-term genetic gain and management of population genetic diversity. The founder population was based on crosses between elite crop varieties and exotic lines of field pea (Pisum sativum) from the primary genepool, and was subjected to 30 cycles of recurrent selection for an economic index composed of four traits with low heritability: black spot resistance, flowering time and stem strength (measured on single plants), and grain yield (measured on whole plots). We compared a small population with low selection pressure, a large population with high selection pressure, and a large population with moderate selection pressure. Single seed descent was compared with S0-derived recurrent selection. Optimal contribution selection achieved higher index and lower population coancestry than truncation selection, which reached a plateau in index improvement after 40 years in the large population with high selection pressure. With optimal contribution selection, index doubled in 38 years in the small population with low selection pressure and 27-28 years in the large population with moderate selection pressure. Single seed descent increased the rate of improvement in index per cycle but also increased cycle time.

The first genetic map of a synthesized allohexaploid Brassica with A, B and C genomes based on simple sequence repeat markers.

KEY MESSAGE: We present the first genetic map of an allohexaploid Brassica species, based on segregating microsatellite markers in a doubled haploid mapping population generated from a hybrid between two hexaploid parents. This study reports the first genetic map of trigenomic Brassica. A doubled haploid mapping population consisting of 189 lines was obtained via microspore culture from a hybrid H16-1 derived from a cross between two allohexaploid Brassica lines (7H170-1 and Y54-2). Simple sequence repeat primer pairs specific to the A genome (107), B genome (44) and C genome (109) were used to construct a genetic linkage map of the population. Twenty-seven linkage groups were resolved from 274 polymorphic loci on the A genome (109), B genome (49) and C genome (116) covering a total genetic distance of 3178.8 cM with an average distance between markers of 11.60 cM. This is the first genetic framework map for the artificially synthesized Brassica allohexaploids. The linkage groups represent the expected complement of chromosomes in the A, B and C genomes from the original diploid and tetraploid parents. This framework linkage map will be valuable for QTL analysis and future genetic improvement of a new allohexaploid Brassica species, and in improving our understanding of the genetic control of meiosis in new polyploids.

The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1.

LepR3, found in the Brassica napus cv 'Surpass 400', provides race-specific resistance to the fungal pathogen Leptosphaeria maculans, which was overcome after great devastation in Australia in 2004. We investigated the LepR3 locus to identify the genetic basis of this resistance interaction. We employed a map-based cloning strategy, exploiting collinearity with the Arabidopsis thaliana and Brassica rapa genomes to enrich the map and locate a candidate gene. We also investigated the interaction of LepR3 with the L. maculans avirulence gene AvrLm1 using transgenics. LepR3 was found to encode a receptor-like protein (RLP). We also demonstrated that avirulence towards LepR3 is conferred by AvrLm1, which is responsible for both the Rlm1 and LepR3-dependent resistance responses in B. napus. LepR3 is the first functional B. napus disease resistance gene to be cloned. AvrLm1's interaction with two independent resistance loci, Rlm1 and LepR3, highlights the need to consider redundant phenotypes in 'gene-for-gene' interactions and offers an explanation as to why LepR3 was overcome so rapidly in parts of Australia.

Analysis of yield and oil from a series of canola breeding trials. Part I. Fitting factor analytic mixed models with pedigree information.

In this paper multiplicative mixed models have been used for the analysis of multi-environment trial (MET) data for canola oil and grain yield. Information on pedigrees has been included to allow for the modelling of additive and nonadditive genetic effects. The MET data set included a total of 19 trials (synonymous with sites or environments), which were sown across southern Australia in 2007 and 2008. Each trial was designed as a p-rep design using DiGGeR with the default prespecified spatial model. Lines in their first year of testing were unreplicated, whereas there were two or three replications of advanced lines or varieties. Pedigree information on a total of 578 entries was available, and there were 69 entries that had unknown pedigrees. The degree of inbreeding varied from 0 (55 entries) to nearly fully inbred (337 entries). Subsamples of 2 g harvested grain were taken from each plot for determination of seed oil percentage by near infrared reflectance spectroscopy. The MET analysis for both yield and oil modelled genetic effects in different trials using factor analytic models and the residual plot effects for each trial were modelled using spatial techniques. Models in which pedigree information was included provided significantly better fits to both yield and oil data.

Analysis of yield and oil from a series of canola breeding trials. Part II. Exploring variety by environment interaction using factor analysis.

Exploring and exploiting variety by environment (V × E) interaction is one of the major challenges facing plant breeders. In paper I of this series, we presented an approach to modelling V × E interaction in the analysis of complex multi-environment trials using factor analytic models. In this paper, we develop a range of statistical tools which explore V × E interaction in this context. These tools include graphical displays such as heat-maps of genetic correlation matrices as well as so-called E-scaled uniplots that are a more informative alternative to the classical biplot for large plant breeding multi-environment trials. We also present a new approach to prediction for multi-environment trials that include pedigree information. This approach allows meaningful selection indices to be formed either for potential new varieties or potential parents.

An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants.

We present an efficient high-throughput flow cytometric method that builds on previously published methods and permits rapid ploidy discrimination in plants. By using Brassica napus L. microspore-derived plants as an example, we describe how 192 leaf tissue samples may be processed and analyzed comfortably by one operator in 6 h from tissue sampling to ploidy determination. The technique involves placing young leaf samples in two 96-well racks, using a bead-beating procedure to release nuclei into a lysis solution, filtering the samples on 96-well filter plates, staining with propidium iodide, and then rapidly estimating DNA ploidy using a plate loader on a BD FACS-Canto II flow cytometer. Throughout the sample preparation process, multichannel pipetting allows faster and less error-prone sample handling. In two 96-well plates of samples, the histogram peaks of DNA content from flow cytometry were wellresolved in 189 of 192 samples tested (98.4%), with CV values ranging from 2.98% to 6.20% with an average CV of 4.35% (SD = 0.68%). This new method is useful in doubled haploid plant breeding programs where early discrimination of haploid and doubled haploid (i.e., diploid) plantlets can confer significantly improved operational efficiencies. We discuss how this method could be further refined including adapting the method to robotic sample processing.

Divergent patterns of allelic diversity from similar origins: the case of oilseed rape (Brassica napus L.) in China and Australia.

Oilseed rape (Brassica napus) in Australia and China have similar origins, with introductions from Europe, Canada, and Japan in the mid 20th century, and there has been some interchange of germplasm between China and Australia since that time. Allelic diversity of 72 B. napus genotypes representing contemporary germplasm in Australia and China, including samples from India, Europe, and Canada, was characterized by 55 polymorphic simple sequence repeat (SSR) markers spanning the entire B. napus genome. Hierarchical clustering and two-dimensional multidimensional scaling identified a Chinese group (China-1) that was separated from "mixed group" of Australian, Chinese (China-2), European, and Canadian lines. A small group from India was distinctly separated from all other B. napus genotypes. Chinese genotypes, especially in the China-1 group, have inherited unique alleles from interspecific crossing, primarily with B. rapa, and the China-2 group has many alleles in common with Australian genotypes. The concept of "private alleles" is introduced to describe both the greater genetic diversity and the genetic distinctiveness of Chinese germplasm, compared with Australian germplasm, after 50 years of breeding from similar origins.

Tracing B-genome chromatin in Brassica napus x B. juncea interspecific progeny.

We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demonstrate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus x B. juncea interspecific progeny. The B-genome--specific repeat sequence pBNBH35 was used to generate PCR products and FISH probes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspecific hybrid and the first backcross. B-genome--positive PCR assays were found in 34/51 fertile F2 progeny (67%), which was more than double the proportion found in fertile BC(1) progeny. Four B-genome--positive F(2)-derived families and 1 BC(1)-derived family were fixed or segregating for B. juncea morphology in the F(4) and BC(1)S(2), respectively, but in only 2 of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISH signals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F(4)-derived line that exhibited isolated and weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome--positive PCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomes tend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines containing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.

A PCR based B-genome-specific marker in Brassica species.

Previous hybridisation studies showed that the repetitive DNA sequence pBNBH35 from Brassica nigra (genome BB, 2n=16) bound specifically to the B-genome and not to the A- or C-genomes of Brassica species. We amplified a sub-fragment of pBNBH35 from B. nigra by PCR, cloned and sequenced this sub-fragment, and confirmed that it was a 329-bp sub-fragment of pBNBH35. PCR and hybridisation techniques were used to confirm that the pBNBH35 sub-fragment was Brassica B-genome-specific. Fluorescence in situ hybridisation (FISH) in B. nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica B-genome chromosomes and absent from the A- and C-genome chromosomes. The pBNBH35 repeat was localised to the centromeric region of each B-genome chromosome. FISH clearly distinguished the B-genome chromosomes from the A-genome chromosomes in the amphidiploid species B. juncea. This is the first known report of a B-genome repetitive marker that is present on all B-genome chromosomes. It will be a useful tool for the detection of B chromosomes in interspecific hybrids and may prove useful for phylogenetic studies in Brassica species.