The coordinated regulation of early meiotic stages is dominated by non-coding RNAs and stage-specific transcription in wheat.

Reproductive success hinges on precisely coordinated meiosis, yet our understanding of how structural rearrangements of chromatin and phase transitions during meiosis are transcriptionally regulated is limited. In crop plants, detailed analysis of the meiotic transcriptome could identify regulatory genes and epigenetic regulators that can be targeted to increase recombination rates and broaden genetic variation, as well as provide a resource for comparison among eukaryotes of different taxa to answer outstanding questions about meiosis. We conducted a meiotic stage-specific analysis of messenger RNA (mRNA), small non-coding RNA (sncRNA), and long intervening/intergenic non-coding RNA (lincRNA) in wheat (Triticum aestivum L.) and revealed novel mechanisms of meiotic transcriptional regulation and meiosis-specific transcripts. Amidst general repression of mRNA expression, significant enrichment of ncRNAs was identified during prophase I relative to vegetative cells. The core meiotic transcriptome was comprised of 9309 meiosis-specific transcripts, 48 134 previously unannotated meiotic transcripts, and many known and novel ncRNAs differentially expressed at specific stages. The abundant meiotic sncRNAs controlled the reprogramming of central metabolic pathways by targeting genes involved in photosynthesis, glycolysis, hormone biosynthesis, and cellular homeostasis, and lincRNAs enhanced the expression of nearby genes. Alternative splicing was not evident in this polyploid species, but isoforms were switched at phase transitions. The novel, stage-specific regulatory controls uncovered here challenge the conventional understanding of this crucial biological process and provide a new resource of requisite knowledge for those aiming to directly modulate meiosis to improve crop plants. The wheat meiosis transcriptome dataset can be queried for genes of interest using an eFP browser located at https://bar.utoronto.ca/efp_wheat/cgi-bin/efpWeb.cgi?dataSource=Wheat_Meiosis.

Correction to: MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

Following publication of the original article [1], a reader spotted an incorrect citation of the reference 14 [2] in the 'Background'. The male meiocyte isolation work described in this article [2] was carried out in rice and not in Brassica as originally stated in the 'Background' [1]. Thus, the following amendment to the Background section should be noted.

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

BACKGROUND: Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS: We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION: The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.

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.

Gene-based SNP discovery and genetic mapping in pea.

KEY MESSAGE: Gene-based SNPs were identified and mapped in pea using five recombinant inbred line populations segregating for traits of agronomic importance. Pea (Pisum sativum L.) is one of the world's oldest domesticated crops and has been a model system in plant biology and genetics since the work of Gregor Mendel. Pea is the second most widely grown pulse crop in the world following common bean. The importance of pea as a food crop is growing due to its combination of moderate protein concentration, slowly digestible starch, high dietary fiber concentration, and its richness in micronutrients; however, pea has lagged behind other major crops in harnessing recent advances in molecular biology, genomics and bioinformatics, partly due to its large genome size with a large proportion of repetitive sequence, and to the relatively limited investment in research in this crop globally. The objective of this research was the development of a genome-wide transcriptome-based pea single-nucleotide polymorphism (SNP) marker platform using next-generation sequencing technology. A total of 1,536 polymorphic SNP loci selected from over 20,000 non-redundant SNPs identified using deep transcriptome sequencing of eight diverse Pisum accessions were used for genotyping in five RIL populations using an Illumina GoldenGate assay. The first high-density pea SNP map defining all seven linkage groups was generated by integrating with previously published anchor markers. Syntenic relationships of this map with the model legume Medicago truncatula and lentil (Lens culinaris Medik.) maps were established. The genic SNP map establishes a foundation for future molecular breeding efforts by enabling both the identification and tracking of introgression of genomic regions harbouring QTLs related to agronomic and seed quality traits.

Genomic selection for target traits in the Australian lentil breeding program.

Genomic selection (GS) uses associations between markers and phenotypes to predict the breeding values of individuals. It can be applied early in the breeding cycle to reduce the cross-to-cross generation interval and thereby increase genetic gain per unit of time. The development of cost-effective, high-throughput genotyping platforms has revolutionized plant breeding programs by enabling the implementation of GS at the scale required to achieve impact. As a result, GS is becoming routine in plant breeding, even in minor crops such as pulses. Here we examined 2,081 breeding lines from Agriculture Victoria's national lentil breeding program for a range of target traits including grain yield, ascochyta blight resistance, botrytis grey mould resistance, salinity and boron stress tolerance, 100-grain weight, seed size index and protein content. A broad range of narrow-sense heritabilities was observed across these traits (0.24-0.66). Genomic prediction models were developed based on 64,781 genome-wide SNPs using Bayesian methodology and genomic estimated breeding values (GEBVs) were calculated. Forward cross-validation was applied to examine the prediction accuracy of GS for these targeted traits. The accuracy of GEBVs was consistently higher (0.34-0.83) than BLUP estimated breeding values (EBVs) (0.22-0.54), indicating a higher expected rate of genetic gain with GS. GS-led parental selection using early generation breeding materials also resulted in higher genetic gain compared to BLUP-based selection performed using later generation breeding lines. Our results show that implementing GS in lentil breeding will fast track the development of high-yielding cultivars with increased resistance to biotic and abiotic stresses, as well as improved seed quality traits.

Breeding has selected for architectural and photosynthetic traits in lentils.

Genetic progress in seed yield in lentils (Lens culinaris Medik) has increased by 1.1% per year in Australia over the past 27 years. Knowing which plant traits have changed through breeding during this time can give important insights as to how lentil yield has increased. This study aims to identify morphological and physiological traits that were directly or indirectly selected between 1993 and 2020 in the Australian lentil breeding program using 2 years of experimental data. Major changes occurred in plant architecture during this period. Divergent selection has seen the release of varieties that have sprawling to very upright types of canopies. Despite this genetic diversity in recently released varieties, there is an overall tendency of recently released varieties having increased plant height and leaf size with reduced number of branches. Increased light interception was positively correlated with year of release (YOR) and yield, and likely results from indirect selection of yield and taller plant types. There is an indication that recently released varieties have lower CO2 assimilation rate, stomatal conductance and canopy temperature depression (CTD) at high ambient temperatures (~30°C). Understanding lentil physiology will assist in identifying traits to increase yield in a changing climate with extreme weather events.

Identification of agro-physiological traits of lentil that reduce risks of drought.

Ideotype breeding is an essential approach for selection of desired combination of plant traits for testing in crop growth model for potential yield gain in specific environments and management practices. Here we parameterized plant traits for untested lentil cultivars for the APSIM-lentil model in phenology, biomass, and seed yield. We then tested these against independent data and applied the model in an extrapolated analysis (i) to assess the impact of drought on productivity across different rainfall environments; (ii) to identify impactful plant traits and (iii) to design new lentil ideotypes with a combination of desirable traits that mitigate the impact of drought, in the context of various agronomic practices across a wide range of production environments. Desirable phenological and physiological traits related to yield were identified with RUE having the greatest effect on yield followed by HI rate. Leaf size significantly affected seed yield (p< 0.05) more than phenological phases. The physiological traits were integrated into four ideotype designs applied to two baseline cultivars (PBA Hallmark XT and PBA Jumbo2) providing eight ideotypes. We identified a combination of genetic traits that promises a yield advantage of around 10% against our current cultivars PBA Hallmark XT and PBA Jumbo2. Under drought conditions, our ideotypes achieved 5 to 25% yield advantages without stubble and 20 to 40% yield advantages with stubble residues. This shows the importance of genetic screening under realistic production conditions (e.g., stubble retention in particular environments). Such screening is aided by the employment of biophysical models that incorporate both genetic and agronomic variables that focus on successful traits in combination, to reduce the impact of drought in the development of new cultivars for various environments. Stubble retention was found to be a major agronomic contributor to high yield in water-limiting environments and this contribution declined with increasing growing season rainfall. In mid- and high-rainfall environments, the key drivers of yield were time of sowing, physiological traits and soil type. Overall, the agronomic practices, namely, early sowing, residue retention and narrow row spacing deceased the impact of drought when combined with improved physiological traits of the ideotypes based on long term climate data.

Isolating Male Meiocytes from Maize and Wheat for "-Omics" Analyses.

Genome-wide gene expression studies have become a routine approach due to the advances in sequencing technologies, their ease of use, and increasing affordability. Simultaneous investigation of small RNA expression adds further valuable information but is not adopted as widely yet. Both RNA-seq and small RNA-seq benefit from the use of specific cell types. Here, we describe a protocol for the isolation of male meiotic cells from maize or wheat plants, along with the application of downstream RNA sequencing, extendable to other -omics approaches.

Differences in growth and physiological and metabolic responses among Canadian native and hybrid willows (Salix spp.) under salinity stress.

Globally, soil salinization is becoming increasingly prevalent, due to local hydrogeologic phenomena, climate change and anthropogenic activities. This has significantly curtailed current world food production and limits future production potential. In the prairie region of North America, sulfate salts, rather than sodium chloride, are often the predominant cause of soil degradation. In order to amend soil quality, revegetate salt-affected sites and recover economic loss associated with soil salinization, the establishment of short-rotation coppice plantations with willows (Salix spp.) has been suggested as a possible solution. To screen for the best candidates for such an application, 20 hybrid and 16 native willow genotypes were treated with three different salt conditions for 3 months. The treatments were designed to reflect the salt composition and concentrations on North American prairies. Under moderate salinity treatment (7 dS m-1), hybrid willows had better growth, as they established quickly while managing salt transport and mineral nutrition balance. However, native willows showed higher potential for long-term survival under severe salinity treatment (14 dS m-1), showing a lower sodium:potassium ratio in roots and better photosynthetic performance. Two native willow genotypes with high osmotic and salinity tolerance indices, specifically LAR-10 and MJW-9, are expected to show superior potential for remediating salt-affected sites. In addition, we observed significantly higher sulfate/sulfur concentrations in both leaf and root tissues in response to the severe salinity treatment, shedding light on the effect of sulfate salinity on sulfate uptake, and potentially sulfur metabolism in plants.

Physiology Based Approaches for Breeding of Next-Generation Food Legumes.

Plant breeders and agricultural scientists of the 21st century are challenged to increase the yield potentials of crops to feed the growing world population. Climate change, the resultant stresses and increasing nutrient deficiencies are factors that are to be considered in designing modern plant breeding pipelines. Underutilized food legumes have the potential to address these issues and ensure food security in developing nations of the world. Food legumes in the past have drawn limited research funding and technological attention when compared to cereal crops. Physiological breeding strategies that were proven to be successful in cereals are to be adapted to legume crop improvement to realize their potential. The gap between breeders and physiologists should be narrowed by collaborative approaches to understand complex traits in legumes. This review discusses the potential of physiology based approaches in food legume breeding and how they impact yield gains and abiotic stress tolerance in these crops. The influence of roots and root system architectures in food legumes' breeding is also discussed. Molecular breeding to map the relevant physiological traits and the potentials of gene editing those traits are detailed. It is imperative to unlock the potentials of these underutilized crops to attain sustainable environmental and nutritional food security.

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.