Reverse genetic approaches for breeding nutrient-rich and climate-resilient cereal and food legume crops.

In the last decade, advancements in genomics tools and techniques have led to the discovery of many genes. Most of these genes still need to be characterized for their associated function and therefore, such genes remain underutilized for breeding the next generation of improved crop varieties. The recent developments in different reverse genetic approaches have made it possible to identify the function of genes controlling nutritional, biochemical, and metabolic traits imparting drought, heat, cold, salinity tolerance as well as diseases and insect-pests. This article focuses on reviewing the current status and prospects of using reverse genetic approaches to breed nutrient-rich and climate resilient cereal and food legume crops.

Neurotoxin (N-Oxalyl-L-α,ß-Diamino Propionic Acid) Content in Different Plant Parts of Grass Pea (Lathyrus sativus L.) Spanning Seedling to Maturity Stage: Does It Increase over Time?

ODAP (N-oxalyl-L-2,3-diaminopropionic acid) is present in the seeds of grass pea. In this study, variation of total ODAP accumulation in leaves throughout the crop growth starting from 40 days after sowing to maturity, and the distribution pattern of ODAP in different plant parts including the seeds at the mature stage was analyzed. Five grass pea accessions were evaluated for two subsequent growing seasons in one location of ICARDA, Aleppo (Syria). The results found that the rate of accumulation of total ODAP varied during plant development. Increased rates of synthesis were noticed in young leaves of grass pea. The highest total ODAP content in leaves was noted in the early growth stage (40-50 days after sowing). Mean total ODAP content in leaves ranged from 0.17 to 0.96 percent during 2010-2011 and from 0.19 to 1.28 percent during 2011-2012. During maturity, the total ODAP content was lowest in the seeds than in leaves, stems, pod cover, seed coat, and cotyledons. The ranges of total ODAP content were 0.13 (seed)-0.34 (stem), 0.20 (seed)-1.01 (leaf), 0.22 (seed)-0.62 (leaf), 0.21 (seed)-0.66 (leaf), and 0.21 (seed)-0.78 (leaf) percent in B387, B222, B390, Bio-520, and B587 accessions, respectively, during maturity. The results indicated that the rate of accumulation and synthesis of total ODAP varied during the plant lifespan. The lowest total ODAP content of leaves was observed after 130 days of sowing. The lower total ODAP content after the early vegetative stage of grass pea plants makes them suitable as a feed.

Root-omics for drought tolerance in cool-season grain legumes.

Root traits can be exploited to increase the physiological efficiency of crop water use under drought. Root length, root hairs, root branching, root diameter, and root proliferation rate are genetically defined traits that can help to improve the water productivity potential of crops. Recently, high-throughput phenotyping techniques/platforms have been used to screen the germplasm of major cool-season grain legumes for root traits and their impact on different physiological processes, including nutrient uptake and yield potential. Advances in omics approaches have led to the dissection of genomic, proteomic, and metabolomic structures of these traits. This knowledge facilitates breeders to improve the water productivity and nutrient uptake of cultivars under limited soil moisture conditions in major cool-season grain legumes that usually face terminal drought. This review discusses the advances in root traits and their potential for developing drought-tolerant cultivars in cool-season grain legumes.

Genomics Associated Interventions for Heat Stress Tolerance in Cool Season Adapted Grain Legumes.

Cool season grain legumes occupy an important place among the agricultural crops and essentially provide multiple benefits including food supply, nutrition security, soil fertility improvement and revenue for farmers all over the world. However, owing to climate change, the average temperature is steadily rising, which negatively affects crop performance and limits their yield. Terminal heat stress that mainly occurred during grain development phases severely harms grain quality and weight in legumes adapted to the cool season, such as lentils, faba beans, chickpeas, field peas, etc. Although, traditional breeding approaches with advanced screening procedures have been employed to identify heat tolerant legume cultivars. Unfortunately, traditional breeding pipelines alone are no longer enough to meet global demands. Genomics-assisted interventions including new-generation sequencing technologies and genotyping platforms have facilitated the development of high-resolution molecular maps, QTL/gene discovery and marker-assisted introgression, thereby improving the efficiency in legumes breeding to develop stress-resilient varieties. Based on the current scenario, we attempted to review the intervention of genomics to decipher different components of tolerance to heat stress and future possibilities of using newly developed genomics-based interventions in cool season adapted grain legumes.

Prospects of next generation sequencing in lentil breeding.

Lentil is an important food legume crop that has large and complex genome. During past years, considerable attention has been given on the use of next generation sequencing for enriching the genomic resources including identification of SSR and SNP markers, development of unigenes, transcripts, and identification of candidate genes for biotic and abiotic stresses, analysis of genetic diversity and identification of genes/ QTLs for agronomically important traits. However, in other crops including pulses, next generation sequencing has revolutionized the genomic research and helped in genomic assisted breeding rapidly and cost effectively. The present review discuss current status and future prospects of the use NGS based breeding in lentil.

Multi-location evaluation of mungbean (Vigna radiata L.) in Indian climates: Ecophenological dynamics, yield relation, and characterization of locations.

Crop yield varies considerably within agroecology depending on the genetic potential of crop cultivars and various edaphic and climatic variables. Understanding site-specific changes in crop yield and genotype × environment interaction are crucial and needs exceptional consideration in strategic breeding programs. Further, genotypic response to diverse agro-ecologies offers identification of strategic locations for evaluating traits of interest to strengthen and accelerate the national variety release program. In this study, multi-location field trial data have been used to investigate the impact of environmental conditions on crop phenological dynamics and their influence on the yield of mungbean in different agroecological regions of the Indian subcontinent. The present attempt is also intended to identify the strategic location(s) favoring higher yield and distinctiveness within mungbean genotypes. In the field trial, a total of 34 different mungbean genotypes were grown in 39 locations covering the north hill zone (n = 4), northeastern plain zone (n = 6), northwestern plain zone (n = 7), central zone (n = 11) and south zone (n = 11). The results revealed that the effect of the environment was prominent on both the phenological dynamics and productivity of the mungbean. Noticeable variations (expressed as coefficient of variation) were observed for the parameters of days to 50% flowering (13%), days to maturity (12%), reproductive period (21%), grain yield (33%), and 1000-grain weight (14%) across the environments. The genotype, environment, and genotype × environment accounted for 3.0, 54.2, and 29.7% of the total variation in mungbean yield, respectively (p < 0.001), suggesting an oversized significance of site-specific responses of the genotypes. Results demonstrated that a lower ambient temperature extended both flowering time and the crop period. Linear mixed model results revealed that the changes in phenological events (days to 50 % flowering, days to maturity, and reproductive period) with response to contrasting environments had no direct influence on crop yields (p > 0.05) for all the genotypes except PM 14-11. Results revealed that the south zone environment initiated early flowering and an extended reproductive period, thus sustaining yield with good seed size. While in low rainfall areas viz., Sriganganagar, New Delhi, Durgapura, and Sagar, the yield was comparatively low irrespective of genotypes. Correlation results and PCA indicated that rainfall during the crop season and relative humidity significantly and positively influenced grain yield. Hence, the present study suggests that the yield potential of mungbean is independent of crop phenological dynamics; rather, climatic variables like rainfall and relative humidity have considerable influence on yield. Further, HA-GGE biplot analysis identified Sagar, New Delhi, Sriganganagar, Durgapura, Warangal, Srinagar, Kanpur, and Mohanpur as the ideal testing environments, which demonstrated high efficiency in the selection of new genotypes with wider adaptability.

Morpho-Physiological Traits and Functional Markers Based Molecular Dissection of Heat-Tolerance in Urdbean.

Urdbean (Vigna mungo L. Hepper) is one of the important pulse crops. Its cultivation is not so popular during summer seasons because this crop is unable to withstand excessive heat stress beside lack of humidity in the atmosphere. Therefore, a panel of 97 urdbean diverse genotypes was assessed for yield under stress and non-stress conditions with an aim to identify heat tolerant genotypes. This study identified 8 highly heat tolerant and 35 highly heat sensitive genotypes based on heat susceptibility index. Further, physiological and biochemical traits-based characterization of a group of six highly heat sensitive and seven highly heat tolerant urdbean genotypes showed genotypic variability for leaf nitrogen balance index (NBI), chlorophyll (SPAD), epidermal flavnols, and anthocyanin contents under 42/25°C max/min temperature. Our results showed higher membrane stability index among heat tolerant genotypes compared to sensitive genotypes. Significant differences among genotypes for ETR at different levels of PAR irradiances and PAR × genotypes interactions indicated high photosynthetic ability of a few genotypes under heat stress. Further, the most highly sensitive genotype PKGU-1 showed a decrease in different fluorescence parameters indicating distortion of PS II. Consequently, reduction in the quantum yield of PS II was observed in a sensitive one as compared to a tolerant genotype. Fluorescence kinetics showed the delayed and fast quenching of Fm in highly heat sensitive (PKGU 1) and tolerant (UPU 85-86) genotypes, respectively. Moreover, tolerant genotype (UPU 85-86) had high antioxidant activities explaining their role for scavenging superoxide radicals (ROS) protecting delicate membranes from oxidative damage. Molecular characterization further pinpointed genetic differences between heat tolerant (UPU 85-86) and heat sensitive genotypes (PKGU 1). These findings will contribute to the breeding toward the development of heat tolerant cultivars in urdbean.

Physiological and molecular characterisation for high temperature stress in Lens culinaris.

In the present study, 11 lentil (Lens culinaris Medik) genotypes including heat tolerant and heat sensitive genotypes identified after a screening of 334 accessions of lentil for traits imparting heat tolerance, were characterised based on physiological traits and molecular markers. Results showed a higher reduction in pollen viability among sensitive genotypes (up to 52.3%) compared with tolerant genotypes (up to 32.4%) at 43°C. Higher photosynthetic electron transport rate was observed among heat tolerant genotypes and two heat tolerant lentil genotypes, IG 4258 (0.43) and IG 3330 (0.38) were having highest Fv/Fm values. However, membrane stability was significantly higher in only one heat tolerant genotype, ILL 10712, indicating that different mechanisms are involved to control heat tolerance in lentil. The molecular characterisation of lentil genotypes with 70 polymorphic SSR and genic markers resulted into distinct clusters in accordance with their heat stress tolerance. A functional marker ISM11257 (intron spanning marker) amplifying an allele of 205bp in size was present only among heat tolerant genotypes, and could be further used in a breeding program to identify heat tolerant lentil genotypes. The findings of this study will contribute to the development of heat tolerant lentil cultivars.

Development of Molecular Markers for Iron Metabolism Related Genes in Lentil and Their Expression Analysis under Excess Iron Stress.

Multiple genes and transcription factors are involved in the uptake and translocation of iron in plants from soil. The sequence information about iron uptake and translocation related genes is largely unknown in lentil (Lens culinaris Medik.). This study was designed to develop iron metabolism related molecular markers for Ferritin-1, BHLH-1 (Basic helix loop helix), or FER-like transcription factor protein and IRT-1 (Iron related transporter) genes using genome synteny with barrel medic (Medicago truncatula). The second objective of this study was to analyze differential gene expression under excess iron over time (2 h, 8 h, 24 h). Specific molecular markers were developed for iron metabolism related genes (Ferritin-1, BHLH-1, IRT-1) and validated in lentil. Gene specific markers for Ferritin-1 and IRT-1 were used for quantitative PCR (qPCR) studies based on their amplification efficiency. Significant differential expression of Ferritin-1 and IRT-1 was observed under excess iron conditions through qPCR based gene expression analysis. Regulation of iron uptake and translocation in lentil needs further characterization. Greater emphasis should be given to development of conditions simulating field conditions under external iron supply and considering adult plant physiology.

Lentils (Lens culinaris L.), a rich source of folates.

The potential for genetic biofortification of U.S.-grown lentils ( Lens culinaris L.) with bioavailable folate has not been widely studied. The objectives of this study were (1) to determine the folate concentration of 10 commercial lentil cultivars grown in Minot and McLean counties, North Dakota, USA, in 2010 and 2011, (2) to determine the genotype (G) × environmental (E) interactions for folate concentration in lentil cultivars, and (3) to compare the folate concentration of other pulses [field peas ( Pisum sativum L.) and chickpea ( Cicer arietinum L.)] grown in the United States. Folate concentration in lentil cultivars ranged from 216 to 290 µg/100 g with a mean of 255 µg/100 g. In addition, lentil showed higher folate concentration compared to chickpea (42-125 µg/100 g), yellow field pea (41-55 µg/100 g), and green field pea (50-202 µg/100 g). A 100 g serving of lentils could provide a significant amount of the recommended daily allowance of dietary folates (54-73%) for adults. A significant year × location interaction on lentil folate concentration was observed; this indicates that possible location sourcing may be required for future lentil folate research.