Identification of QTLs associated with seed protein concentration in two diverse recombinant inbred line populations of pea.

Improving the seed protein concentration (SPC) of pea (Pisum sativum L.) has turned into an important breeding objective because of the consumer demand for plant-based protein and demand from protein fractionation industries. To support the marker-assisted selection (MAS) of SPC towards accelerated breeding of improved cultivars, we have explored two diverse recombinant inbred line (RIL) populations to identify the quantitative trait loci (QTLs) associated with SPC. The two RIL populations, MP 1918 × P0540-91 (PR-30) and Ballet × Cameor (PR-31), were derived from crosses between moderate SPC × high SPC accessions. A total of 166 and 159 RILs of PR-30 and PR-31, respectively, were genotyped using an Axiom® 90K SNP array and 13.2K SNP arrays, respectively. The RILs were phenotyped in replicated trials in two and three locations of Saskatchewan, Canada in 2020 and 2021, respectively, for agronomic assessment and SPC. Using composite interval mapping, we identified three QTLs associated with SPC in PR-30 and five QTLs in PR-31, with the LOD value ranging from 3.0 to 11.0. A majority of these QTLs were unique to these populations compared to the previously known QTLs for SPC. The QTL SPC-Ps-5.1 overlapped with the earlier reported SPC associated QTL PC-QTL-3. Three QTLs, SPC-Ps-4.2, SPC-Ps-5.1, and SPC-Ps-7.2 with LOD scores of 7.2, 7.9, and 11.3, and which explained 14.5%, 11.6%, and 11.3% of the phenotypic variance, respectively, can be used for marker-assisted breeding to increase SPC in peas. Eight QTLs associated with the grain yield were identified with LOD scores ranging from 3.1 to 8.2. Two sets of QTLs, SPC-Ps-2.1 and GY-Ps-2.1, and SPC-Ps-5.1 and GY-Ps-5.3, shared the QTL/peak regions. Each set of QTLs contributed to either SPC or grain yield depending on which parent the QTL region is derived from, thus confirming that breeding for SPC should take into consideration the effects on grain yield.

Creating saponin-free yellow pea seeds by CRISPR/Cas9-enabled mutagenesis on ß-amyrin synthase.

Dry pea (Pisum sativum) seeds are valuable sources of plant protein, dietary fiber, and starch, but their uses in food products are restricted to some extent due to several off-flavor compounds. Saponins are glycosylated triterpenoids and are a major source of bitter, astringent, and metallic off-flavors in pea products. ß-amyrin synthase (BAS) is the entry point enzyme for saponin biosynthesis in pea and therefore is an ideal target for knock-out using CRISPR/Cas9 genome editing to produce saponin deficient pea varieties. Here, in an elite yellow pea cultivar (CDC Inca), LC/MS analysis identified embryo tissue, not seed coat, as the main location of saponin storage in pea seeds. Differential expression analysis determined that PsBAS1 was preferentially expressed in embryo tissue relative to seed coat and was selected for CRISPR/Cas9 genome editing. The efficiency of CRISPR/Cas9 genome editing of PsBAS1 was systematically optimized in pea hairy roots. From these optimization procedures, the AtU6-26 promoter was found to be superior to the CaMV35S promoter for gRNA expression, and the use of 37°C was determined to increase the efficiency of CRISPR/Cas9 genome editing. These promoter and culture conditions were then applied to stable transformations. As a result, a bi-allelic mutation (deletion and inversion mutations) was generated in the PsBAS1 coding sequence in a T1 plant, and the segregated psbas1 plants from the T2 population showed a 99.8% reduction of saponins in their seeds. Interestingly, a small but statistically significant increase (~12%) in protein content with a slight decrease (~5%) in starch content was observed in the psbas1 mutants under phytotron growth conditions. This work demonstrated that flavor-improved traits can be readily introduced in any pea cultivar of interest using CRISPR/Cas9. Further field trials and sensory tests for improved flavor are necessary to assess the practical implications of the saponin-free pea seeds in food applications.

Agronomic Performance in Low Phytic Acid Field Peas.

Field pea is a pulse that delivers high protein content, slowly digestible starch and fiber, and many vitamins and minerals, including iron. Naturally occurring plant phytic acid molecules bind iron, lowering its availability for absorption during digestion. Two low phytic acid (lpa) pea lines, 1-2347-144 and 1-150-81, developed by our group had 15% lower yield and 6% lower seed weight relative to their progenitor cultivar. Subsequently, we crossed the two lpa lines and two cultivars, and derived 19 promising lpa pea breeding lines; here we document their agronomic performance based on 10 replicated field trials in Saskatchewan. Seventeen of these lpa lines yielded greater than 95% of the check mean (associated cultivars) and 16 were above 98% of the check mean for 1000 seed weight. The 19 lpa lines showed 27 to 55% lower phytic acid concentration than the check mean. Iron concentrations were similar in all the lpa lines and cultivars, yet the Caco-2 human cell culture assay revealed 14 of the 19 lpa lines had 11 to 55% greater iron bioavailability than check means. Thus, a single round of plant breeding has allowed for closing the gap in performance of low phytic acid pea.

High throughput nutritional profiling of pea seeds using Fourier transform mid-infrared spectroscopy.

Seed samples from 117 genetically diverse pea breeding lines were used to determine the robustness of Fourier transform mid-infrared spectroscopy (FT-MIR) for the rapid nutritional profiling of seeds. The FT-MIR results were compared to wet chemistry methods for assessing the concentrations of total protein, starch, fiber, phytic acid, and carotenoids in pea seed samples. Of the five partial least square regression models (PLSR) developed, protein, fiber and phytic acid concentrations predicted by the models exhibited correlation coefficients greater than 0.83 when compared with data obtained using the wet chemistry methods for both the calibration and validation sets. The starch PLSR model had a correlation greater than 0.75, and carotenoids had correlation of 0.71 for the validation sets. The methods implemented in this research show the novelty and usefulness of FT-MIR as a simple, fast, and cost-effective technique to determine multiple seed constituents simultaneously.

Mapping Quantitative Trait Loci for Carotenoid Concentration in Three F2 Populations of Chickpea.

CORE IDEAS: Quantitative trait locus (QTL) analyses for carotenoids in chickpea were completed for three F2 populations. A moderate number of QTLs and candidate genes associated with carotenoid concentration in chickpea seeds were identified. Green cotyledon color is positively associated with provitamin A carotenoids. Three F2 populations derived from crosses between cultivars with green and yellow cotyledon colors were used to identify quantitative trait loci (QTLs) associated with carotenoid components in chickpea (Cicer arietinum L.) seeds developed by the Crop Development Centre (CDC). Carotenoids including violaxanthin, lutein, zeaxanthin, ß-cryptoxanthin, and ß-carotene were assessed in the F2:3 seeds via high-performance liquid chromatography (HPLC). In the 'CDC Jade' × 'CDC Frontier' population, 1068 bin markers derived from the 50K Axiom CicerSNP array were mapped onto eight linkage groups (LGs). Eight QTLs, including two each for ß-carotene and zeaxanthin and one each for total carotenoids, ß-cryptoxanthin, ß-carotene, and violaxanthin were identified in this population. In the 'CDC Cory' × 'CDC Jade' population, 694 bin markers were mapped onto eight LGs and one partial LG. Quantitative trait loci for ß-cryptoxanthin, ß-carotene, violaxanthin, lutein, and total carotenoids were identified on LG8. A map with eight LGs was developed from 581 bin markers in the third population derived from the 'ICC4475' × 'CDC Jade' cross. One QTL for ß-carotene and four QTLs, one each for ß-cryptoxanthin, ß-carotene, lutein, and total carotenoids, were identified in this population. The highest phenotypic variation explained by the QTLs was for ß-carotene, which ranged from 58 to 70% in all three populations. A major gene for cotyledon color was mapped on LG8 in each population. A significant positive correlation between cotyledon color and carotenoid concentration was observed. Potential candidate genes associated with carotenoid components were obtained and their locations on the chickpea genome are presented.

Construction of high-density linkage maps for mapping quantitative trait loci for multiple traits in field pea (Pisum sativum L.).

BACKGROUND: The objective of this research was to map quantitative trait loci (QTLs) of multiple traits of breeding importance in pea (Pisum sativum L.). Three recombinant inbred line (RIL) populations, PR-02 (Orb x CDC Striker), PR-07 (Carerra x CDC Striker) and PR-15 (1-2347-144 x CDC Meadow) were phenotyped for agronomic and seed quality traits under field conditions over multiple environments in Saskatchewan, Canada. The mapping populations were genotyped using genotyping-by-sequencing (GBS) method for simultaneous single nucleotide polymorphism (SNP) discovery and construction of high-density linkage maps. RESULTS: After filtering for read depth, segregation distortion, and missing values, 2234, 3389 and 3541 single nucleotide polymorphism (SNP) markers identified by GBS in PR-02, PR-07 and PR-15, respectively, were used for construction of genetic linkage maps. Genetic linkage groups were assigned by anchoring to SNP markers previously positioned on these linkage maps. PR-02, PR-07 and PR-15 genetic maps represented 527, 675 and 609 non-redundant loci, and cover map distances of 951.9, 1008.8 and 914.2 cM, respectively. Based on phenotyping of the three mapping populations in multiple environments, 375 QTLs were identified for important traits including days to flowering, days to maturity, lodging resistance, Mycosphaerella blight resistance, seed weight, grain yield, acid and neutral detergent fiber concentration, seed starch concentration, seed shape, seed dimpling, and concentration of seed iron, selenium and zinc. Of all the QTLs identified, the most significant in terms of explained percentage of maximum phenotypic variance (PVmax) and occurrence in multiple environments were the QTLs for days to flowering (PVmax = 47.9%), plant height (PVmax = 65.1%), lodging resistance (PVmax = 35.3%), grain yield (PVmax = 54.2%), seed iron concentration (PVmax = 27.4%), and seed zinc concentration (PVmax = 43.2%). CONCLUSION: We have identified highly significant and reproducible QTLs for several agronomic and seed quality traits of breeding importance in pea. The QTLs identified will be the basis for fine mapping candidate genes, while some of the markers linked to the highly significant QTLs are useful for immediate breeding applications.

Accumulation of Phosphorus-Containing Compounds in Developing Seeds of Low-Phytate Pea (Pisum sativum L.) Mutants.

Low phytic acid (lpa) crops are low in phytic acid and high in inorganic phosphorus (Pi). In this study, two lpa pea genotypes, 1-150-81, 1-2347-144, and their progenitor CDC Bronco were grown in field trials for two years. The lpa genotypes were lower in IP6 and higher in Pi when compared to CDC Bronco. The total P concentration was similar in lpa genotypes and CDC Bronco throughout the seed development. The action of myo-inositol phosphate synthase (MIPS) (EC 5.5.1.4) is the first and rate-limiting step in the phytic acid biosynthesis pathway. Aiming at understanding the genetic basis of the lpa mutation in the pea, a 1530 bp open reading frame of MIPS was amplified from CDC Bronco and the lpa genotypes. Sequencing results showed no difference in coding sequence in MIPS between CDC Bronco and lpa genotypes. Transcription levels of MIPS were relatively lower at 49 days after flowering (DAF) than at 14 DAF for CDC Bronco and lpa lines. This study elucidated the rate and accumulation of phosphorus compounds in lpa genotypes. The data also demonstrated that mutation in MIPS was not responsible for the lpa trait in these pea lines.