RESUMEN
We present the first genetic map of tedera (Bituminaria bituminosa (L.) C.H. Stirton), a drought-tolerant forage legume from the Canary Islands with useful pharmaceutical properties. It is also the first genetic map for any species in the tribe Psoraleeae (Fabaceae). The map comprises 2042 genotyping-by-sequencing (GBS) markers distributed across 10 linkage groups, consistent with the haploid chromosome count for this species (n = 10). Sequence tags from the markers were used to find homologous matches in the genome sequences of the closely related species in the Phaseoleae tribe: soybean, common bean, and cowpea. No tedera linkage groups align in their entirety to chromosomes in any of these phaseoloid species, but there are long stretches of collinearity that could be used in tedera research for gene discovery purposes using the better-resourced phaseoloid species. Using Ks analysis of a tedera transcriptome against five legume genomes provides an estimated divergence time of 17.4 million years between tedera and soybean. Genomic information and resources developed here will be invaluable for breeding tedera varieties for forage and pharmaceutical purposes.
RESUMEN
Protocols have been proposed for rapid generation turnover of temperate legumes under conditions optimized for day-length, temperature, and light spectra. These conditions act to compress time to flowering and seed development across genotypes. In pea, we have previously demonstrated that embryos do not efficiently germinate without exogenous hormones until physiological maturity is reached at 18 days after pollination (DAP). Sugar metabolism and moisture content have been implicated in the modulation of embryo maturity. However, the role of hormones in regulating seed development is poorly described in legumes. To address this gap, we characterized hormonal profiles (IAA, chlorinated auxin [4-Cl-IAA], GA20, GA1, and abscisic acid [ABA]) of developing seeds (10-22 DAP) from diverse pea genotypes grown under intensive conditions optimized for rapid generation turnover and compared them to profiles of equivalent samples from glasshouse conditions. Growing plants under intensive conditions altered the seed hormone content by advancing the auxin, gibberellins (GAs) and ABA profiles by 4 to 8 days, compared with the glasshouse control. Additionally, we observed a synchronization of the auxin profiles across genotypes. Under intensive conditions, auxin peaks were observed at 10 to 12 DAP and GA20 peaks at 10 to 16 DAP, indicative of the end of embryo morphogenesis and initiation of seed desiccation. GA1 was detected only in seeds harvested in the glasshouse. These results were associated with an acceleration of embryo physiological maturity by up to 4 days in the intensive environment. We propose auxin and GA profiles as reliable indicators of seed maturation. The biological relevance of these hormonal fluctuations to the attainment of physiological maturity, in particular the role of ABA and GA, was investigated through the study of precocious in vitro germination of seeds 12 to 22 DAP, with and without exogenous hormones. The extent of sensitivity of developing seeds to exogenous ABA was strongly genotype-dependent. Concentrations between 5 and 10 µM inhibited germination of seeds 18 DAP. Germination of seeds 12 DAP was enhanced 2.5- to 3-fold with the addition of 125 µM GA3. This study provides further insights into the hormonal regulation of seed development and in vitro precocious germination in legumes and contributes to the design of efficient and reproducible biotechnological tools for rapid genetic gain.
RESUMEN
In alkaline soils in arid and semi-arid areas toxic concentrations of the micronutrient boron (B) are problematic for many cereal and legume crops. Molecular markers have been developed for B toxicity in cereals and Medicago. There is a need for such tools in clovers-Trifolium. To this end, we undertook a genome-wide association study (GWAS) with a diversity panel of subterranean clover (Trifolium subterraneum L.), an established model pasture legume for genetic and genomic analyses for the genus. The panel comprised 124 T. subterraneum genotypes (97 core collection accessions and 27 Australian cultivars). Substantial and useful diversity in B toxicity tolerance was found in T. subterraneum. Such variation was continuously distributed and exhibited a high broad sense heritability H 2 = 0.92. Among the subspecies of T. subterraneum, ssp. brachycalycinum was most susceptible to B toxicity (P < 0.05). From the GWAS, the most important discoveries were single-nucleotide polymorphisms (SNPs) located on Chr 1, 2, and 3, which mapped to haplotype blocks providing potential genes for a B toxicity tolerance assay and meriting further investigation. A SNP identified on Chr 1 aligned with Medicago truncatula respiratory burst oxidase-like protein (TSub_ g2235). This protein is known to respond to abiotic and biotic stimuli. The identification of these novel potential genes and their use to design markers for marker-assisted selection offer a pathway in pasture legumes to manage B toxicity by exploiting B tolerance.
RESUMEN
BACKGROUND: Boron (B) tolerance has been identified as a key target for field pea improvement. Screening for B tolerance in the field is problematic due to variability in space and time, and robust B molecular markers are currently unavailable in field pea. There has been recent progress in developing protocols that can accelerate the life cycle of plants to enable rapid generation turnover in single seed descent breeding programs. A robust B screening protocol that can be fully integrated within an accelerated single seed descent system could lead to rapid identification and introgression of B tolerance into field pea genotypes. Integration with an accelerated single seed descent system requires: (1) screening under artificially lit, temperature-controlled conditions; (2) capacity to use immature precociously germinated seed (PGS); (3) recovery of lines without significant time penalty; and (4) good correlation with results from established screening protocols. RESULTS: We present herein a B toxicity screening system for field pea based on hydroponic growth of PGS in a light and temperature controlled environment that allows recovery of seedlings for rapid seed production. Screening results were compared to traditional methods for B tolerance screening in B-laced soil and with published field tolerance ratings. B tolerance was scored 17 days after sowing using leaf symptoms as a metric. Plants were then transferred to soil with maximum of six days delay in flowering compared to a typical accelerated single seed descent system generation. The use of PGS had minimal impact on B tolerance rankings compared to plants grown from mature seed. The leaf tolerance rankings from hydroponic-grown plants correlated well with those from soil-grown plants, and consistently identified the most tolerant genotypes. CONCLUSIONS: Our 17 day screening protocol represents a major time-saving over previously published B screening protocols for field pea, thereby extending the application of the protocol to traditional single seed descent systems or RIL screening. We anticipate that small modifications to the proposed technique will make it applicable to screen for other individual abiotic stresses, or allow studies of the interactions between B tolerance and stresses such as salinity.
RESUMEN
BACKGROUND: Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species. RESULTS: 432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme. CONCLUSIONS: B. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.
