SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.).

BACKGROUND: Field pea (Pisum sativum L.) is a self-pollinating, diploid, cool-season food legume. Crop production is constrained by multiple biotic and abiotic stress factors, including salinity, that cause reduced growth and yield. Recent advances in genomics have permitted the development of low-cost high-throughput genotyping systems, allowing the construction of saturated genetic linkage maps for identification of quantitative trait loci (QTLs) associated with traits of interest. Genetic markers in close linkage with the relevant genomic regions may then be implemented in varietal improvement programs. RESULTS: In this study, single nucleotide polymorphism (SNP) markers associated with expressed sequence tags (ESTs) were developed and used to generate comprehensive linkage maps for field pea. From a set of 36,188 variant nucleotide positions detected through in silico analysis, 768 were selected for genotyping of a recombinant inbred line (RIL) population. A total of 705 SNPs (91.7%) successfully detected segregating polymorphisms. In addition to SNPs, genomic and EST-derived simple sequence repeats (SSRs) were assigned to the genetic map in order to obtain an evenly distributed genome-wide coverage. Sequences associated with the mapped molecular markers were used for comparative genomic analysis with other legume species. Higher levels of conserved synteny were observed with the genomes of Medicago truncatula Gaertn. and chickpea (Cicer arietinum L.) than with soybean (Glycine max [L.] Merr.), Lotus japonicus L. and pigeon pea (Cajanus cajan [L.] Millsp.). Parents and RIL progeny were screened at the seedling growth stage for responses to salinity stress, imposed by addition of NaCl in the watering solution at a concentration of 18 dS m-1. Salinity-induced symptoms showed normal distribution, and the severity of the symptoms increased over time. QTLs for salinity tolerance were identified on linkage groups Ps III and VII, with flanking SNP markers suitable for selection of resistant cultivars. Comparison of sequences underpinning these SNP markers to the M. truncatula genome defined genomic regions containing candidate genes associated with saline stress tolerance. CONCLUSION: The SNP assays and associated genetic linkage maps developed in this study permitted identification of salinity tolerance QTLs and candidate genes. This constitutes an important set of tools for marker-assisted selection (MAS) programs aimed at performance enhancement of field pea cultivars.

Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil.

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9-11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Quantitation of seven transmembrane proteins from the DHA biosynthesis pathway in genetically engineered canola by targeted mass spectrometry.

Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.

Discrimination of boron tolerance in Pisum sativum L. genotypes using a rapid, high-throughput hydroponic screen and precociously germinated seed grown under far-red enriched light.

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.

Validation of molecular markers associated with boron tolerance, powdery mildew resistance and salinity tolerance in field peas.

Field pea (Pisum sativum L.) is an important grain legume consumed both as human food and animal feed. However, productivity in low rainfall regions can be significantly reduced by inferior soils containing high levels of boron and/or salinity. Furthermore, powdery mildew (PM) (Erysiphe pisi) disease also causes significant yield loss in warmer regions. Breeding for tolerance to these abiotic and biotic stresses are major aims for pea breeding programs and the application of molecular markers for these traits could greatly assist in developing improved germplasm at a faster rate. The current study reports the evaluation of a near diagnostic marker, PsMlo, associated with PM resistance and boron (B) tolerance as well as linked markers associated with salinity tolerance across a diverse set of pea germplasm. The PsMlo1 marker predicted the PM and B phenotypic responses with high levels of accuracy (>80%) across a wide range of field pea genotypes, hence offers the potential to be widely adapted in pea breeding programs. In contrast, linked markers for salinity tolerance were population specific; therefore, application of these markers would be suitable to relevant crosses within the program. Our results also suggest that there are possible new sources of salt tolerance present in field pea germplasm that could be further exploited.