Colin Morgan

Unraveling the Complex Trait of Crop Yield With Quantitative Trait Loci Mapping in Brassica napus

Yield is the most important and complex trait for the genetic improvement of crops. Although much research into the genetic basis of yield and yield-associated traits has been reported, in each such experiment the genetic architecture and determinants of yield have remained ambiguous. One of the most intractable problems is the interaction between genes and the environment. We identified 85 quantitative trait loci (QTL) for seed yield along with 785 QTL for eight yield-associated traits, from 10 natural environments and two related populations of rapeseed. A trait-by-trait meta-analysis revealed 401 consensus QTL, of which 82.5% were clustered and integrated into 111 pleiotropic unique QTL by meta-analysis, 47 of which were relevant for seed yield. The complexity of the genetic architecture of yield was demonstrated, illustrating the pleiotropy, synthesis, variability, and plasticity of yield QTL. The idea of estimating indicator QTL for yield QTL and identifying potential candidate genes for yield provides an advance in methodology for complex traits.

Dissecting the genome of the polyploid crop oilseed rape by transcriptome sequencing

Polyploidy complicates genomics-based breeding of many crops, including wheat, potato, cotton, oat and sugarcane. To address this challenge, we sequenced leaf transcriptomes across a mapping population of the polyploid crop oilseed rape (Brassica napus) and representative ancestors of the parents of the population. Analysis of sequence variation and transcript abundance enabled us to construct twin single nucleotide polymorphism linkage maps of B. napus, comprising 23,037 markers. We used these to align the B. napus genome with that of a related species, Arabidopsis thaliana, and to genome sequence assemblies of its progenitor species, Brassica rapa and Brassica oleracea. We also developed methods to detect genome rearrangements and track inheritance of genomic segments, including the outcome of an interspecific cross. By revealing the genetic consequences of breeding, cost-effective, high-resolution dissection of crop genomes by transcriptome sequencing will increase the efficiency of predictive breeding even in the absence of a complete genome sequence.

Natural variation for seed oil composition in Arabidopsis thaliana.

The biochemical pathways involved in the biosynthesis and accumulation of storage lipids in seeds have been extensively studied. However, the regulatory mechanisms of those pathways, their environmental interactions and the ecological implications of variation are poorly understood. We have initiated a new approach: the analysis of natural variation in Arabidopsis thaliana. Three hundred and sixty accessions were surveyed for content of oil, very long chain fatty acids (VLCFAs) and polyunsaturated fatty acids (PUFAs) in their seeds. The results revealed extensive natural variation. A core set of accessions, the seeds of which reproducibly contain extreme amounts of oil, VLCFAs and PUFAs have been identified. Reproducible oil content ranged from 34.6 to 46.0% of seed dry weight. VLCFA content ranged from 13.0 to 21.2% of total fatty acids. PUFA content, ranged from 53.3 to 66.1% of total fatty acids. Interactions were also identified for PUFA and VLCFA content of seeds with vernalisation of plants. Mapping of the regions of the genome involved in controlling the traits was conducted in an F(2) population and indicated that natural variation at the loci FAE1 and FAD3 might be involved in the regulation of VLCFA and PUFA content, respectively. A set of accessions, which capture a broad range of the natural variation for these traits available in A. thaliana, has been selected to form a core set which can be used to further dissect the genetics of the regulation of seed lipid traits and to identify the genes involved.

Integration of Brassica A genome genetic linkage map between Brassica napus and B. rapa

An integrated linkage map between B. napus and B. rapa was constructed based on a total of 44 common markers comprising 41 SSR (33 BRMS, 6 Saskatoon, and 2 BBSRC) and 3 SNP/indel markers. Between 3 and 7 common markers were mapped onto each of the linkage groups A1 to A10. The position and order of most common markers revealed a high level of colinearity between species, although two small regions on A4, A5, and A10 revealed apparent local inversions between them. These results indicate that the A genome of Brassica has retained a high degree of colinearity between species, despite each species having evolved independently after the integration of the A and C genomes in the amphidiploid state. Our results provide a genetic integration of the Brassica A genome between B. napus and B. rapa. As the analysis employed sequence-based molecular markers, the information will accelerate the exploitation of the B. rapa genome sequence for the improvement of oilseed rape.

Associative Transcriptomics Study Dissects the Genetic Architecture of Seed Glucosinolate Content in Brassica napus

Breeding new varieties with low seed glucosinolate (GS) concentrations has long been a prime target in Brassica napus. In this study, a novel association mapping methodology termed ‘associative transcriptomics’ (AT) was applied to a panel of 101 B. napus lines to define genetic regions and also candidate genes controlling total seed GS contents. Over 100,000 informative single-nucleotide polymorphisms (SNPs) and gene expression markers (GEMs) were developed for AT analysis, which led to the identification of 10 SNP and 7 GEM association peaks. Within these peaks, 26 genes were inferred to be involved in GS biosynthesis. A weighted gene co-expression network analysis provided additional 40 candidate genes. The transcript abundance in leaves of two candidate genes, BnaA.GTR2a located on chromosome A2 and BnaC.HAG3b on C9, was correlated with seed GS content, explaining 18.8 and 16.8% of phenotypic variation, respectively. Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b. These deletion polymorphisms were then successfully converted into polymerase chain reaction–based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines.

Towards the genetic architecture of seed lipid biosynthesis and accumulation in Arabidopsis thaliana.

We report the quantitative genetic analysis of seed oil quality and quantity in six Arabidopsis thaliana recombinant inbred populations, in which the parent accessions were from diverse geographical origins, and were selected on the basis of variation for seed oil content and lipid composition. Although most of the biochemical steps involved in lipid biosynthesis are known and the key genes have been identified, the regulation of the processes that results in the final oil composition and total amount is not understood. By using physically anchored markers it was possible to compare results across populations. A total of 219 quantitative trait loci (QTLs) were identified, of which 81 were significant at P<0.001. Some of these colocalise with QTLs identified previously, but many novel QTLs were also identified. The results highlight the importance of studying traits in multiple populations, which will lead to a better understanding of the contribution that natural variation makes to the genetic architecture of a phenotype.

An association transcriptomics approach to the prediction of hybrid performance

Many animal and plant species exhibit increased growth rates, reach larger sizes and, in the cases of crops and farm animals, produce higher yields when bred as hybrids between genetically differing strains, a phenomenon known as hybrid vigour or heterosis. Despite the importance of heterosis, and its extensive genetic analysis, little understanding exists of its molecular basis. We aimed to determine whether characteristics of the leaf transcriptome, as an indicator of the innate functional genetic architecture of a plant line, could be used as markers to predict heterosis and the performance of hybrids, a methodology we term Association Transcriptomics. Relationships between transcript abundance of specific genes and the values of heterosis and heterosis-dependent traits were identified and mathematical models were constructed that relate gene expression characteristics in inbred lines of Arabidopsis thaliana and maize with vegetative biomass and grain yield, respectively, in corresponding hybrids. These models were used to predict, using gene expression data, the performance of additional hybrids. The success of the application in a monocot crop of a methodology developed in a dicot model species indicates that transcriptional markers may have widespread applicability in hybrid breeding.

Evaluating the utility of Arabidopsis thaliana as a model for understanding heterosis in hybrid crops

Despite the phenomenon of heterosis having been used to improve crop productivity for almost a century, there is little understanding of the molecular mechanisms involved. Heterosis has been described recently in the widely used plant model Arabidopsis thaliana. To assess the opportunity afforded by this system, we have developed, through pollination to a male sterile female parent (Ler ms1), a range of hybrids of A. thaliana accessions. We tested this method of hybrid production in A. thaliana by comparing seed produced by natural pollination and hand pollination of emasculated plants. Our results demonstrate that using male sterile lines, which more closely represent methods used for hybrid crop production, circumvents the problems associated with the analysis of hybrids produced from emasculated plants. We identified hybrid combinations exhibiting mid-parent heterosis for vegetative fresh weight ranging from −9 to 103% and characterised heterosis in the reciprocal hybrids of one strongly heterotic combination. We found no evidence of a relationship between the extent of genetic relatedness of A. thaliana accessions and the strength of heterosis exhibited by their hybrids.

Sequencing-based variant detection in the polyploid crop oilseed rape

BackgroundThe detection and exploitation of genetic variation underpins crop improvement. However, the polyploid nature of the genomes of many of our most important crops represents a barrier, particularly for the analysis of variation within genes. To overcome this, we aimed to develop methodologies based on amplicon sequencing that involve the incorporation of barcoded amplification tags (BATs) into PCR products.ResultsA protocol was developed to tag PCR products with 5’ 6-base oligonucleotide barcode extensions before pooling for sequencing library production using standard Illumina adapters. A computational method was developed for the de-convolution of products and the robust detection and scoring of sequence variants. Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome. Furthermore, using one-dimensional 8-fold pooling, 4608 lines of a B. napus mutation population were screened for induced mutations in a locus-specific amplicon (an orthologue of GL2.b) and mixed product of three co-amplified loci (orthologues of FAD2), identifying 10 and 41 mutants respectively.ConclusionsThe utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species. Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.

Collinearity analysis of Brassica A and C genomes based on an updated inferred unigene order.

This data article includes SNP scoring across lines of the Brassica napus TNDH population based on Illumina sequencing of mRNA, expanded to 75 lines. The 21, 323 mapped markers defined 887 recombination bins, representing an updated genetic linkage map for the species. Based on this new map, 5 genome sequence scaffolds were split and the order and orientation of scaffolds updated to establish a new pseudomolecule specification. The order of unigenes and SNP array probes within these pseudomolecules was determined. Unigenes were assessed for sequence similarity to the A and C genomes. The 57, 246 that mapped to both enabled the collinearity of the A and C genomes to be illustrated graphically. Although the great majority was in collinear positions, some were not. Analyses of 60 such instances are presented, suggesting that the breakdown in collinearity was largely due to either the absence of the homoeologue on one genome (resulting in sequence match to a paralogue) or multiple similar sequences being present. The mRNAseq datasets for the TNDH lines are available from the SRA repository (ERA283648); the remaining datasets are supplied with this article.