Quantitative trait loci × environment interactions for plant morphology vary over ontogeny in Brassica rapa.

Growth in plants occurs via the addition of repeating modules, suggesting that the genetic architecture of similar subunits may vary between earlier- and later-developing modules. These complex environment × ontogeny interactions are not well elucidated, as studies examining quantitative trait loci (QTLs) expression over ontogeny have not included multiple environments. Here, we characterized the genetic architecture of vegetative traits and onset of reproduction over ontogeny in recombinant inbred lines of Brassica rapa in the field and glasshouse. The magnitude of genetic variation in plasticity of seedling internodes was greater than in those produced later in ontogeny. We correspondingly detected that QTLs for seedling internode length were environment-specific, whereas later in ontogeny the majority of QTLs affected internode lengths in all treatments. The relationship between internode traits and onset of reproduction varied with environment and ontogenetic stage. This relationship was observed only in the glasshouse environment and was largely attributable to one environment-specific QTL. Our results provide the first evidence of a QTL × environment × ontogeny interaction, and provide QTL resolution for differences between early- and later-stage plasticity for stem elongation. These results also suggest potential constraints on morphological evolution in early vs later modules as a result of associations with reproductive timing.

Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP) in Brassica napus L.

Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci -QTL- analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.

Retention of triplicated phytoene synthase (PSY) genes in Brassica napus L. and its diploid progenitors during the evolution of the Brassiceae.

The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSY homologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSY genes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.

Floral genetic architecture: an examination of QTL architecture underlying floral (co)variation across environments.

Genetic correlations are expected to be high among functionally related traits and lower between groups of traits with distinct functions (e.g., reproductive vs. resource-acquisition traits). Here, we explore the quantitative-genetic and QTL architecture of floral organ sizes, vegetative traits, and life history in a set of Brassica rapa recombinant inbred lines within and across field and greenhouse environments. Floral organ lengths were strongly positively correlated within both environments, and analysis of standardized G-matrices indicates that the structure of genetic correlations is ∼80% conserved across environments. Consistent with these correlations, we detected a total of 19 and 21 additive-effect floral QTL in the field and the greenhouse, respectively, and individual QTL typically affected multiple organ types. Interestingly, QTL×QTL epistasis also appeared to contribute to observed genetic correlations; i.e., interactions between two QTL had similar effects on filament length and two estimates of petal size. Although floral and nonfloral traits are hypothesized to be genetically decoupled, correlations between floral organ size and both vegetative and life-history traits were highly significant in the greenhouse; G-matrices of floral and vegetative traits as well as floral and life-history traits differed across environments. Correspondingly, many QTL (45% of those mapped in the greenhouse) showed environmental interactions, including approximately even numbers of floral and nonfloral QTL. Most instances of QTL×QTL epistasis for floral traits were environment dependent.

Development of a set of public SSR markers derived from genomic sequence of a rapid cycling Brassica oleracea L. genotype.

The traditional development of simple sequence repeat (SSR) or microsatellite markers by probe hybridization can be time-consuming and requires the use of specialized laboratory equipment. In this study, probe hybridization was circumvented by using sequence information on 3,500 genomic clones mainly from Brassica oleracea to identify di, tri, tetra and penta-nucleotide repeats. A total of 587 primer pairs flanking SSR were developed using this approach. From these, 420 SSR markers amplified DNA in two parental lines of B. rapa (26% were polymorphic) and 523 in two parental lines of B. oleracea (32% were polymorphic). A diverse array of motif types was identified, characterized and compared with traditional SSR detection methods. The most abundant motifs found were di- (38%) and trinucleotides (33%) followed by penta- (16%) and tetranucleotide (13%) motifs. The type of motif class, motif length and repeat were not indicative of polymorphisms. The frequency of B. oleracea SSRs in genomic shotgun sequence was estimated to be 1 every 4 Kb. In general, the average motif length and repeat numbers were shorter than those obtained previously by probe hybridization, and they contained a more balanced representation of SSR motif types in the genome by identifying those that do not hybridize well to DNA probes. Brassica genomic DNA sequence information is a promising resource for developing a large number of SSR molecular markers in Brassica species.

Spatial and temporal divergence of expression in duplicated barley germin-like protein-encoding genes.

Subfunctionalization is the process by which a pair of duplicated genes, or paralogs, experiences a reduction of individual expression patterns or function while still reproducing the complete expression pattern and function of the ancestral gene. Two germin-like protein (GLP)-encoding genes, GerB and GerF, are paralogs that belong to a small gene family in barley (Hordeum vulgare). Both genes share high nucleotide sequence similarity in coding and noncoding regions and encode identical apoplastic proteins. The use of RNA gel blots, coupled with single-stranded conformation polymorphism (SSCP) analysis of RT-PCR products, elucidated the developmental and tissue-specific expression patterns of each gene. Individual expression patterns provided evidence of both overlapping redundancy and early subfunctionalization. GerB is predominantly expressed in developing shoots, while GerF is predominantly expressed in seedling roots, developing spikes, and pericarp/testa. GerF promoter deletion studies located a region (-356/-97) responsible for high promoter activity and showed the ability of GerB and GerF upstream regions to drive gfp expression in coleoptiles, epicarps, and lemma/palea of developing spikes. The observed expression patterns are consistent with proposed roles in plant development and defense mechanisms for this gene family. These roles may explain why redundancy has been selectively maintained in this duplicate gene pair.