Genome-Wide Association Mapping for Heat and Drought Adaptive Traits in Pea.

Heat and drought, individually or in combination, limit pea productivity. Fortunately, substantial genetic diversity exists in pea germplasm for traits related to abiotic stress resistance. Understanding the genetic basis of resistance could accelerate the development of stress-adaptive cultivars. We conducted a genome-wide association study (GWAS) in pea on six stress-adaptive traits with the aim to detect the genetic regions controlling these traits. One hundred and thirty-five genetically diverse pea accessions were phenotyped in field studies across three or five environments under stress and control conditions. To determine marker trait associations (MTAs), a total of 16,877 valuable single nucleotide polymorphisms (SNPs) were used in association analysis. Association mapping detected 15 MTAs that were significantly (p ≤ 0.0005) associated with the six stress-adaptive traits averaged across all environments and consistent in multiple individual environments. The identified MTAs were four for lamina wax, three for petiole wax, three for stem thickness, two for the flowering duration, one for the normalized difference vegetation index (NDVI), and two for the normalized pigment and chlorophyll index (NPCI). Sixteen candidate genes were identified within a 15 kb distance from either side of the markers. The detected MTAs and candidate genes have prospective use towards selecting stress-hardy pea cultivars in marker-assisted selection.

Identification of heat responsive genes in pea stipules and anthers through transcriptional profiling.

Field pea (Pisum sativum L.), a cool-season legume crop, is known for poor heat tolerance. Our previous work identified PR11-2 and PR11-90 as heat tolerant and susceptible lines in a recombinant inbred population. CDC Amarillo, a Canadian elite pea variety, was considered as another heat tolerant variety based on its similar field performance as PR11-2. This study aimed to characterize the differential transcription. Plants of these three varieties were stressed for 3 h at 38°C prior to self-pollination, and RNAs from heat stressed anthers and stipules on the same flowering node were extracted and sequenced via the Illumina NovaSeq platform for the characterization of heat responsive genes. In silico results were further validated by qPCR assay. Differentially expressed genes (DEGs) were identified at log2 |fold change (FC)| ≥ 2 between high temperature and control temperature, the three varieties shared 588 DEGs which were up-regulated and 220 genes which were down-regulated in anthers when subjected to heat treatment. In stipules, 879 DEGs (463/416 upregulation/downregulation) were consistent among varieties. The above heat-induced genes of the two plant organs were related to several biological processes i.e., response to heat, protein folding and DNA templated transcription. Ten gene ontology (GO) terms were over-represented in the consistently down-regulated DEGs of the two organs, and these terms were mainly related to cell wall macromolecule metabolism, lipid transport, lipid localization, and lipid metabolic processes. GO enrichment analysis on distinct DEGs of individual pea varieties suggested that heat affected biological processes were dynamic, and variety distinct responses provide insight into molecular mechanisms of heat-tolerance response. Several biological processes, e.g., cellular response to DNA damage stimulus in stipule, electron transport chain in anther that were only observed in heat induced PR11-2 and CDC Amarillo, and their relevance to field pea heat tolerance is worth further validation.

Genome-Wide Association Mapping for Heat Stress Responsive Traits in Field Pea.

Environmental stress hampers pea productivity. To understand the genetic basis of heat resistance, a genome-wide association study (GWAS) was conducted on six stress responsive traits of physiological and agronomic importance in pea, with an objective to identify the genetic loci associated with these traits. One hundred and thirty-five genetically diverse pea accessions from major pea growing areas of the world were phenotyped in field trials across five environments, under generally ambient (control) and heat stress conditions. Statistical analysis of phenotype indicated significant effects of genotype (G), environment (E), and G × E interaction for all traits. A total of 16,877 known high-quality SNPs were used for association analysis to determine marker-trait associations (MTA). We identified 32 MTAs that were consistent in at least three environments for association with the traits of stress resistance: six for chlorophyll concentration measured by a soil plant analysis development meter; two each for photochemical reflectance index and canopy temperature; seven for reproductive stem length; six for internode length; and nine for pod number. Forty-eight candidate genes were identified within 15 kb distance of these markers. The identified markers and candidate genes have potential for marker-assisted selection towards the development of heat resistant pea cultivars.

Pollen, ovules, and pollination in pea: Success, failure, and resilience in heat.

Field pea (Pisum sativum), a major grain legume crop, is autogamous and adapted to temperate climates. The objectives of this study were to investigate effects of high temperature stress on stamen chemical composition, anther dehiscence, pollen viability, pollen interactions with pistil and ovules, and ovule growth and viability. Two cultivars ("CDC Golden" and "CDC Sage") were exposed to 24/18°C (day/night) continually or to 35/18°C for 4 or 7 days. Heat stress altered stamen chemical composition, with lipid composition of "CDC Sage" being more stable compared with "CDC Golden." Heat stress reduced pollen viability and the proportion of ovules that received a pollen tube. After 4 days at 35°C, pollen viability in flower buds decreased in "CDC Golden," but not in "CDC Sage." After 7 days, partial to full failure of anthers to dehisce resulted in subnormal pollen loads on stigmas. Although growth (ovule size) of fertilized ovules was stimulated by 35°C, heat stress tended to decrease ovule viability. Pollen appears susceptible to stress, but not many grains are needed for successful fertilization. Ovule fertilization and embryos are less susceptible to heat, but further research is warranted to link the exact degree of resilience to stress intensity.

Genotypic and heat stress effects on leaf cuticles of field pea using ATR-FTIR spectroscopy.

MAIN CONCLUSION: ATR-FTIR spectroscopy in combination with uni- and multivariate analysis was used to quantify the spectral-chemical composition of the leaf cuticle of pea, investigating the effects of variety and heat stress. Field pea (Pisum sativum L.) is sensitive to heat stress and our goal was to improve canopy cooling and flower retention by investigating the protective role of lipid-related compounds in leaf cuticle, and to use results in the future to identify heat resistant genotypes. The objective was to use Attenuated Total Reflection (ATR)-Fourier Transform Infrared (FTIR) spectroscopy, a non-invasive technique, to investigate and quantify changes in adaxial cuticles of fresh leaves of pea varieties that were subjected to heat stress. Eleven varieties were grown under control (24/18 °C day/night) and heat stress conditions (35/18 °C day/night, for 5 days at the early flowering stage). These 11 had significant spectral differences in the integrated area of the main lipid region, CH2 region, CH3 peak, asymmetric and symmetric CH2 peaks, ester carbonyl peak, and the peak area ratio of CH2 to CH3 and ester carbonyl to CH2 asymmetric peak, indicating that cuticles had spectral-chemical diversity of waxes, cutin, and polysaccharides. Results indicated considerable diversity in spectral-chemical makeup of leaf cuticles within commercially available field pea varieties and they responded differently to high growth temperature, revealing their diverse potential to resist heat stress. The ATR-FTIR spectral technique can, therefore, be further used as a medium-throughput approach for rapid screening of superior cultivars for heat tolerance.

Symbiosis of selected Rhizobium leguminosarum bv. viciae strains with diverse pea genotypes: effects on biological nitrogen fixation.

Biological nitrogen fixation (BNF) can be improved by optimizing the interaction between the rhizobial inoculant and pea (Pisum sativum L.), leading to increased productivity and reduced nitrogen (N) fertilizer use. Eight Rhizobium leguminosarum bv. viciae strains were used to inoculate the super-nodulating pea mutant Rondo-nod3 (fix+), the hyper-nodulating pea mutant Frisson P88 Sym29, CDC Meadow commercial control, and the non-nodulating mutant Frisson P56 (nod-) to evaluate BNF in a greenhouse assay. Significant differences in strain × cultivar interactions were detected for shoot and root dry masses, which ranged from 1.8 to 4.7 g and from 0.27 to 0.73 g per plant, respectively; for nodule number on lateral roots, which ranged from 25 to 430 per plant; for amount of fixed N2, which ranged from 15 to 67 mg and from 4 to 15 mg per plant for shoot and root tissues, respectively; and for percentage of N derived from atmosphere (%Ndfa), which ranged from 37% to 61% and from 35% to 65% for shoot and root tissue, respectively. Strain × cultivar interactions in this study could contribute to identification of superior strains and pea breeding lines with genetic superiority in BNF. Nodule production in pea plants was not necessarily correlated with the amount of fixed N2, suggesting nodule activity is more important to BNF than is nodule number.

Determination of Photoperiod-Sensitive Phase in Chickpea (Cicer arietinum L.).

Photoperiod is one of the major environmental factors determining time to flower initiation and first flower appearance in plants. In chickpea, photoperiod sensitivity, expressed as delayed to flower under short days (SD) as compared to long days (LD), may change with the growth stage of the crop. Photoperiod-sensitive and -insensitive phases were identified by experiments in which individual plants were reciprocally transferred in a time series from LD to SD and vice versa in growth chambers. Eight chickpea accessions with differing degrees of photoperiod sensitivity were grown in two separate chambers, one of which was adjusted to LD (16 h light/8 h dark) and the other adjusted to SD (10 h light/14 h dark), with temperatures of 22/16°C (12 h light/12 h dark) in both chambers. The accessions included day-neutral (ICCV 96029 and FLIP 98-142C), intermediate (ICC 15294, ICC 8621, ILC 1687, and ICC 8855), and photoperiod-sensitive (CDC Corinne and CDC Frontier) responses. Control plants were grown continuously under the respective photoperiods. Reciprocal transfers of plants between the SD and LD photoperiod treatments were made at seven time points after sowing, customized for each accession based on previous data. Photoperiod sensitivity was detected in intermediate and photoperiod-sensitive accessions. For the day-neutral accession, ICCV 96029, there was no significant difference in the number of days to flowering of the plants grown under SD and LD as well as subsequent transfers. In photoperiod-sensitive accessions, three different phenological phases were identified: a photoperiod-insensitive pre-inductive phase, a photoperiod-sensitive inductive phase, and a photoperiod-insensitive post-inductive phase. The photoperiod-sensitive phase extends after flower initiation to full flower development. Results from this research will help to develop cultivars with shorter pre-inductive photoperiod-insensitive and photoperiod-sensitive phases to fit to regions with short growing seasons.

Seed set, pollen morphology and pollen surface composition response to heat stress in field pea.

Pea (Pisum sativum L.) is a major legume crop grown in a semi-arid climate in Western Canada, where heat stress affects pollination, seed set and yield. Seed set and pod growth characteristics, along with in vitro percentage pollen germination, pollen tube growth and pollen surface composition, were measured in two pea cultivars (CDC Golden and CDC Sage) subjected to five maximum temperature regimes ranging from 24 to 36 °C. Heat stress reduced percentage pollen germination, pollen tube length, pod length, seed number per pod, and the seed-ovule ratio. Percentage pollen germination of CDC Sage was greater than CDC Golden at 36 °C. No visible morphological differences in pollen grains or the pollen surface were observed between the heat and control-treated pea. However, pollen wall (intine) thickness increased due to heat stress. Mid-infrared attenuated total reflectance (MIR-ATR) spectra revealed that the chemical composition (lipid, proteins and carbohydrates) of each cultivar's pollen grains responded differently to heat stress. The lipid region of the pollen coat and exine of CDC Sage was more stable compared with CDC Golden at 36 °C. Secondary derivatives of ATR spectra indicated the presence of two lipid types, with different amounts present in pollen grains from each cultivar.

ATR-FTIR spectroscopy reveals involvement of lipids and proteins of intact pea pollen grains to heat stress tolerance.

With climate change, pea will be more frequently subjected to heat stress in semi-arid regions like Saskatchewan during flowering. The pollen germination percentage of two pea cultivars was reduced by heat stress (36°C) with an important decrease in cultivar 'CDC Golden' compared to 'CDC Sage.' Lipids, protein and other pollen coat compositions of whole intact pollen grains of both pea cultivars were investigated using mid infrared (mid-IR) attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy. Curve fitting of ATR absorbance spectra in the protein region enabled estimation and comparison of different protein secondary structures between the two cultivars. CDC Sage had relatively greater amounts of α-helical structures (48.6-43.6%; band at 1654 cm(-1)) and smaller amounts of ß-sheets (41.3-46%) than CDC Golden. The CDC Golden had higher amounts of ß-sheets (46.3-51.7%) compared to α-helical structures (35.3-36.2%). Further, heat stress resulted in prominent changes in the symmetrical and asymmetrical CH2 bands from lipid acyl chain, ester carbonyl band, and carbohydrate region. The intensity of asymmetric and symmetric CH2 vibration of heat stressed CDC Golden was reduced considerably in comparison to the control and the decrease was higher compared to CDC Sage. In addition, CDC Golden showed an increase in intensity at the oxidative band of 3015 cm(-1). These results reveal that the whole pollen grains of both pea cultivars responded differently to heat stress. The tolerance of CDC Sage to heat stress (expressed as pollen germination percentage) may be due to its protein richness with α-helical structures which would protect against the destructive effects of dehydration due to heat stress. The low pollen germination percentage of CDC Golden after heat stress may be also due to its sensitivity to lipid changes due to heat stress.