Breeding More Crops in Less Time: A Perspective on Speed Breeding.

Breeding crops in a conventional way demands considerable time, space, inputs for selection, and the subsequent crossing of desirable plants. The duration of the seed-to-seed cycle is one of the crucial bottlenecks in the progress of plant research and breeding. In this context, speed breeding (SB), relying mainly on photoperiod extension, temperature control, and early seed harvest, has the potential to accelerate the rate of plant improvement. Well demonstrated in the case of long-day plants, the SB protocols are being extended to short-day plants to reduce the generation interval time. Flexibility in SB protocols allows them to align and integrate with diverse research purposes including population development, genomic selection, phenotyping, and genomic editing. In this review, we discuss the different SB methodologies and their application to hasten future plant improvement. Though SB has been extensively used in plant phenotyping and the pyramiding of multiple traits for the development of new crop varieties, certain challenges and limitations hamper its widespread application across diverse crops. However, the existing constraints can be resolved by further optimization of the SB protocols for critical food crops and their efficient integration in plant breeding pipelines.

Evidence for the Application of Emerging Technologies to Accelerate Crop Improvement - A Collaborative Pipeline to Introgress Herbicide Tolerance Into Chickpea.

Accelerating genetic gain in crop improvement is required to ensure improved yield and yield stability under increasingly challenging climatic conditions. This case study demonstrates the effective confluence of innovative breeding technologies within a collaborative breeding framework to develop and rapidly introgress imidazolinone Group 2 herbicide tolerance into an adapted Australian chickpea genetic background. A well-adapted, high-yielding desi cultivar PBA HatTrick was treated with ethyl methanesulfonate to generate mutations in the ACETOHYDROXYACID SYNTHASE 1 (CaAHAS1) gene. After 2 years of field screening with imidazolinone herbicide across >20 ha and controlled environment progeny screening, two selections were identified which exhibited putative herbicide tolerance. Both selections contained the same single amino acid substitution, from alanine to valine at position 205 (A205V) in the AHAS1 protein, and KASP™ markers were developed to discriminate between tolerant and intolerant genotypes. A pipeline combining conventional crossing and F2 production with accelerated single seed descent from F2:4 and marker-assisted selection at F2 rapidly introgressed the herbicide tolerance trait from one of the mutant selections, D15PAHI002, into PBA Seamer, a desi cultivar adapted to Australian cropping areas. Field evaluation of the derivatives of the D15PAHI002 × PBA Seamer cross was analyzed using a factor analytic mixed model statistical approach designed to accommodate low seed numbers resulting from accelerated single seed descent. To further accelerate trait introgression, field evaluation trials were undertaken concurrent with crop safety testing trials. In 2020, 4 years after the initial cross, an advanced line selection CBA2061, bearing acetohydroxyacid synthase (AHAS) inhibitor tolerance and agronomic and disease resistance traits comparable to parent PBA Seamer, was entered into Australian National Variety Trials as a precursor to cultivar registration. The combination of cross-institutional collaboration and the application of novel pre-breeding platforms and statistical technologies facilitated a 3-year saving compared to a traditional breeding approach. This breeding pipeline can be used as a model to accelerate genetic gain in other self-pollinating species, particularly food legumes.

Lens orientalis Contributes Quantitative Trait Loci and Candidate Genes Associated With Ascochyta Blight Resistance in Lentil.

Australian lentil production is affected by several major biotic constraints including Ascochyta blight (AB), caused by Ascochyta lentis, a devastating fungal disease. Cultivation of AB resistant cultivars, alongside agronomic management including fungicide application, is the current most economically viable control strategy. However, the breakdown of AB resistance in cultivars, such as Northfield and Nipper, suggests the need for introgression of new and diverse resistance genes. Successful introgression entails an understanding of the genetic basis of resistance. In this context, a biparental mapping population derived from a cross between a recently identified AB resistant accession ILWL 180 (Lens orientalis) and a susceptible cultivar ILL 6002 was produced. A genetic linkage map was constructed from single-nucleotide polymorphism markers generated using a genotyping-by-sequencing transcript approach. Genetic dissection of the mapping population revealed a major quantitative trait loci (QTL) region nested with three QTLs on linkage group 5 and explained 9.5-11.5 percent (%) of phenotypic variance for AB resistance. Another QTL was identified on LG2 with phenotypic variance of 9.6%. The identified QTL regions harbored putative candidate genes potentially associated with defense responses to A. lentis infection. The QTL analysis and the candidate gene information are expected to contribute to the development of diagnostic markers and enable marker-assisted resistance selection in lentil breeding programmes.

Fast-forward breeding for a food-secure world.

Crop production systems need to expand their outputs sustainably to feed a burgeoning human population. Advances in genome sequencing technologies combined with efficient trait mapping procedures accelerate the availability of beneficial alleles for breeding and research. Enhanced interoperability between different omics and phenotyping platforms, leveraged by evolving machine learning tools, will help provide mechanistic explanations for complex plant traits. Targeted and rapid assembly of beneficial alleles using optimized breeding strategies and precise genome editing techniques could deliver ideal crops for the future. Realizing desired productivity gains in the field is imperative for securing an adequate future food supply for 10 billion people.

A Trimethylguanosine Synthase1-like (TGS1) homologue is implicated in vernalisation and flowering time control.

KEY MESSAGE: A plant-specific Trimethylguanosine Synthase1-like homologue was identified as a candidate gene for the efl mutation in narrow-leafed lupin, which alters phenology by reducing vernalisation requirement. The vernalisation pathway is a key component of flowering time control in plants from temperate regions but is not well understood in the legume family. Here we examined vernalisation control in the temperate grain legume species, narrow-leafed lupin (Lupinus angustifolius L.), and discovered a candidate gene for an ethylene imine mutation (efl). The efl mutation changes phenology from late to mid-season flowering and additionally causes transformation from obligate to facultative vernalisation requirement. The efl locus was mapped to pseudochromosome NLL-10 in a recombinant inbred line (RIL) mapping population developed by accelerated single seed descent. Candidate genes were identified in the reference genome, and a diverse panel of narrow-leafed lupins was screened to validate mutations specific to accessions with efl. A non-synonymous SNP mutation within an S-adenosyl-L-methionine-dependent methyltransferase protein domain of a Trimethylguanosine Synthase1-like (TGS1) orthologue was identified as the candidate mutation giving rise to efl. This mutation caused substitution of an amino acid within an established motif at a position that is otherwise highly conserved in several plant families and was perfectly correlated with the efl phenotype in F2 and F6 genetic population and a panel of diverse accessions, including the original efl mutant. Expression of the TGS1 homologue did not differ between wild-type and efl genotypes, supporting altered functional activity of the gene product. This is the first time a TGS1 orthologue has been associated with vernalisation response and flowering time control in any plant species.

Novel Salinity Tolerance Loci in Chickpea Identified in Glasshouse and Field Environments.

A better understanding of the genetics of salinity tolerance in chickpea would enable breeding of salt tolerant varieties, offering potential to expand chickpea production to marginal, salinity-affected areas. A Recombinant Inbred Line population was developed using accelerated-Single Seed Descent of progeny from a cross between two chickpea varieties, Rupali (salt-sensitive) and Genesis836 (salt-tolerant). The population was screened for salinity tolerance using high-throughput image-based phenotyping in the glasshouse, in hydroponics, and across 2 years of field trials at Merredin, Western Australia. A genetic map was constructed from 628 unique in-silico DArT and SNP markers, spanning 963.5 cM. Markers linked to two flowering loci identified on linkage groups CaLG03 and CaLG05 were used as cofactors during genetic analysis to remove the confounding effects of flowering on salinity response. Forty-two QTL were linked to growth rate, yield, and yield component traits under both control and saline conditions, and leaf tissue ion accumulation under salt stress. Residuals from regressions fitting best linear unbiased predictions from saline conditions onto best linear unbiased predictions from control conditions provided a measure of salinity tolerance per se, independent of yield potential. Six QTL on CaLG04, CaLG05, and CaLG06 were associated with tolerance per se. In total, 21 QTL mapped to two distinct regions on CaLG04. The first distinct region controlled the number of filled pods, leaf necrosis, seed number, and seed yield specifically under salinity, and co-located with four QTL linked to salt tolerance per se. The second distinct region controlled 100-seed weight and growth-related traits, independent of salinity treatment. Positional cloning of the salinity tolerance-specific loci on CaLG04, CaLG05, and CaLG06 will improve our understanding of the key determinants of salinity tolerance in chickpea.

A QTL on the Ca7 chromosome of chickpea affects resistance to the root-lesion nematode Pratylenchus thornei.

The root-lesion nematode Pratylenchus thornei Sher & Allen, 1953 is a damaging parasite of many crop plants, including the grain legume chickpea (Cicer arietinum L.). Within cultivated chickpea, there are no known sources of strong resistance to P. thornei, but some cultivars have partial resistance. In the research reported here, the genetic basis for differences in P. thornei resistance was analysed using a population derived by accelerated single seed descent from a cross between a partially resistant cultivar, PBA HatTrick, and a very susceptible cultivar, Kyabra. A genetic linkage map was constructed from genotyping-by-sequencing data. Two quantitative trait loci were mapped, one on the Ca4 chromosome and one on the Ca7 chromosome. The Ca7 locus had a greater and more consistent effect than the Ca4 locus. Marker assays designed for single nucleotide polymorphisms on Ca7 were applied to a panel of chickpea accessions. Some of these markers should be useful for marker-assisted selection in chickpea breeding. Haplotype analysis confirmed the Iranian landrace ICC14903 to be the source of the resistance allele in PBA HatTrick and indicated that other Australian cultivars inherited the same allele from other Iranian landraces. A candidate region was defined on the Ca7 pseudomolecule. Within that region, 69 genes have been predicted with high confidence. Among these, two have annotations related to biotic stress response. Three others have previously been reported to be expressed in roots of PBA HatTrick and Kyabra, including one that is more highly expressed in PBA HatTrick than in Kyabra. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01271-8.

Expression Patterns of Key Hormones Related to Pea (Pisum sativum L.) Embryo Physiological Maturity Shift in Response to Accelerated Growth Conditions.

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.

Comparative Reaction of Camelina sativa to Sclerotinia sclerotiorum and Leptosphaeria maculans.

Sclerotinia sclerotiorum and Leptosphaeria maculans are two of the most important pathogens of many cruciferous crops. The reaction of 30 genotypes of Camelina sativa (false flax) was determined against both pathogens. C. sativa genotypes were inoculated at seedling and adult stages with two pathotypes of S. sclerotiorum, highly virulent MBRS-1 and less virulent WW-1. There were significant differences (P < 0.001) among genotypes, between pathotypes, and a significant interaction between genotypes and pathotypes in relation to percent cotyledon disease index (% CDI) and stem lesion length. Genotypes 370 (% CDI 20.5, stem lesion length 1.8 cm) and 253 (% CDI 24.8, stem lesion length 1.4 cm) not only consistently exhibited cotyledon and stem resistance, in contrast to susceptible genotype 2305 (% CDI 37.7, stem lesion length 7.2 cm), but their resistance was independent to S. sclerotiorum pathotype. A F5-recombinant inbred line population was developed from genotypes 370 × 2305 and responses characterized. Low broad-sense heritability indicated a complex pattern of inheritance of resistance to S. sclerotiorum. Six isolates of L. maculans, covering combinations of five different avirulent loci (i.e., five different races), were tested on C. sativa cotyledons across two experiments. There was a high level of resistance, with % CDI < 17, and including development of a hypersensitive reaction. This is the first report of variable reaction of C. sativa to different races of L. maculans and the first demonstrating comparative reactions of C. sativa to S. sclerotiorum and L. maculans. This study not only provides new understanding of these comparative resistances in C. sativa, but highlights their potential as new sources of resistance, both for crucifer disease-resistance breeding in general and to enable broader adoption of C. sativa as a more sustainable oilseed crop in its own right.

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.

Camalexin Production in Camelina sativa is Independent of Cotyledon Resistance to Sclerotinia sclerotiorum.

Camelina sativa (L.) Crantz. has been proposed as a novel source of oilseed resistance to Sclerotinia rot (SR; causal agent Sclerotinia sclerotiorum (Lib.) de Bary). To assess factors likely important in determining the level of resistance to this pathogen, 30 diverse C. sativa genotypes were evaluated using a cotyledon test under controlled environmental conditions. Confirmed cotyledon SR-resistant (CS370) and SR-susceptible (CS2305) genotypes were assessed for camalexin production across time following inoculation at the 1-month vegetative stage of growth. There were significant differences among C. sativa genotypes in response to inoculation with S. sclerotiorum in terms of percent cotyledon disease index (%CDI), with the mean %CDI ranging from 30.9 to 69.4% across germplasm and confirmation screening, respectively. Genotype CS370 consistently showed low %CDI indicating high level of resistance to S. sclerotiorum, whereas CS2305 showed the highest %CDI value. These findings highlight the potential to develop highly SR-resistant cultivars of C. sativa by selection. Furthermore, liquid chromatographic analysis of leaves for both SR-resistant and SR-susceptible genotypes demonstrated that camalexin was produced when inoculated with S. sclerotiorum. However, camalexin production was not linked with disease severity in either genotype, indicating that SR resistance in C. sativa is independent of the level of camalexin production.

Camelina (Camelina sativa (L.) Crantz) as an alternative oilseed: molecular and ecogeographic analyses.

Camelina (Camelina sativa (L.) Crantz) is an oilseed known for its potential as a low-input biofuel feedstock and its high levels of beneficial fatty acids. We investigated the role of geographical origin in genetic variation and fatty acid content, expecting to find significant variability among 53 accessions and a link between ecogeography and both origin and key oil traits. Amplified fragment length polymorphism (AFLP) fingerprinting revealed high levels of diversity within the 53 accessions. Even though sampling was relatively biased towards the Russian-Ukrainian area, this region was identified as a genetic diversity hotspot and possible centre of origin for camelina. The accessions were categorized by principal coordinate analysis using molecular marker data, enabling identification of links between geographical distribution and these categories. The influence of geographic location on four canola oil quality measures in camelina was evaluated using a geographic information system. These measures were (1) more than 30% alpha-linolenic acid, (2) less than 3% erucic acid, (3) less than 10% saturated fatty acids, and (4) a ratio of alpha-linolenic to linoleic acid greater than 1. The results clearly confirm that camelina oil quality characteristics are strongly influenced by environmental factors. The unprecedented high genetic diversity in this group of accessions offers an excellent opportunity to investigate valuable genes for successful adaptation of camelina to specific ecogeographical conditions such as drought.

Doubled-haploid production in chickpea (Cicer arietinum L.): role of stress treatments.

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15-20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4 degrees C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50-400 V with 25 microF capacitance and 25 Omega resistance, (3) anthers were centrifuged at 168-1,509g for 2-15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15-20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1-3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10-30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.