Breeding, genetics, and genomics for tolerance against terminal heat in lentil: Current status and future directions

Heat stress at terminal stage in lentil is one of the major factors causing drastic yield loss. Development of heat tolerant cultivars is one of the ways to tackle this problem. However, heat stress tolerance is a complex trait in nature and many morphological, physiological, biochemical, and metabolic changes are responsible for heat stress tolerance in lentil. Therefore, in the past years, efforts have been made to know genetics and genomics of heat stress tolerance in lentil. In this review, we discussed current progress on breeding, genetics, and genomics including quantitative trait locus (QTL) mapping, transcriptomics, proteomics, and network of metabolic pathways for heat tolerance and future directions for developing heat tolerant cultivars in lentil.


| INTRODUCTION
Lentil (Lens culinaris Medikus) is cultivated globally as a rainfed crop during the winter season. It is mainly cultivated in Indian subcontinent, West-Asia, and Australia where it faces heat stress. Heat stress is a period of hot temperature that causes irreversible damage during growth and development of crop plants (Delahunty, Nuttall, Nicolas, & Brand, 2018). In major growing areas, lentil crop is exposed to temperature >35 C at flowering and pod filling stages during maturity (Delahunty, Nuttall, Nicolas, & Brand, 2015). In rice-lentil crop rotations, the problem is even more serious as late harvesting of the rice crop delays sowing of lentil crop, which may place the reproductive phase of lentil under high temperature stress (Subbarao, Rao, Kumar, Johansen, & Deb, 2001). Therefore, high temperature causes drastic losses of yields due to poor grain filling in lentil. Several days' exposure of the lentil crop to high temperature affects many physiological processes leading to forced grain maturity (Redden et al., 2014). For example, in Australia, a continuous heat wave with a temperature of ≥35 C for 6 days resulted in a yield loss of~70% in lentil (Delahunty et al., 2015). In another study, an 87% yield reduction was observed when lentil plants were grown in the pots under field conditions at temperature of >38/23 C (average day/night temperature) during the reproductive phase (Bhandari et al., 2016). In coming years, higher temperatures due to global warming can be more challenging for the production and productivity of lentil (Kaur, Bains, Bindumadhava, & Nayyar, 2015).
Development of heat tolerant cultivars is one of the ways to tackle the problem of high temperature in lentil under present climatic conditions. However, heat stress tolerance is a complex trait in nature because many morphological, physiological, biochemical, and metabolic changes are responsible as discussed earlier in different crop plants (Abdelrahman, Burritt, Gupta, Tsujimoto, & Tran, 2019;Duan, Liu, Zhang, Li, & Guo, 2019;El Hassouni et al., 2019;Kaur, Sinha, & Bhunia, 2019). Moreover, recent reviews have also discussed different aspects of heat stress tolerance either on food legume crops Liu et al., 2019) or on a specific pulse crop like chickpea (Kaloki, Devasirvatham, & Tan, 2019). However, during the past years, continuous efforts have been made to study the heat stress tolerance in lentil. These studies identified the heat tolerant genotypes and the associated morpho-physio-biochemical traits, characterized the heat tolerance at molecular level, and deciphered the genetics and pathways underlying the heat stress tolerance in lentil Singh et al., 2016;Kumar, Basu, et al., 2018;Singh, Singh, Singh, & Pal, 2017;Delahunty et al., 2018;Singh et al., 2019). Also, knowledge of heat stress in lentil is enriching day by day and is becoming available in the public domain. Therefore, this review is aimed to know the current progress on breeding, genetics, and genomics including quantitative trait locus (QTL) mapping, transcriptomics, proteomics, and network of metabolic pathways for heat tolerance and future strategies of using this knowledge in breeding programs for developing high temperature tolerant lentil cultivars in order to sustain the production and productivity of lentil under changing climatic conditions. The average day and night temperature between 15 C to 25 C and 8 C to 10 C, respectively, during reproductive phase is generally required for normal pods and seeds setting in lentil, and it showed high sensitivity to heat stress when day/night temperatures exceeded from 32/20 C (Ibrahim, 2011). A day/night temperature at or above 35/20 C causes pod abortion, reduction in the number of flowers, pollen viability, pollen germination, stigmatic function, ovular viability, pollen tube elongation, and shortening of flowering time in lentil (Bhandari et al., 2016;. Therefore, for this crop, temperatures >35/25 C is identified as harmful for growth and yield  and >35/25 C can be used as critical temperature for differentiating the heat tolerant and sensitive lentil genotypes (Gaur et al., 2015). However, heat tolerant genotypes can even produce fewer pods up to 40/30 temperature . In other legume crops such as mung bean and common bean, flowering or just prior to flowering stage has been shown to be highly affected by heat stress (Patriyawaty, Rachaputi, & George, 2018), whereas the post-fertilization stages like early pod development has been identified more tolerant to heat stress (Gross & Kigel, 1994). Studies demonstrated that high temperature stress, that is, even a few days exposure to high temperature (30-35 C) affects physiological, metabolic, and molecular function of reproductive organs leading to poor seed set or yield (Gaur et al., 2015;Jiang et al., 2015;Sage et al., 2015).

| Phenomics for identification of heat tolerant genotypes
Heat tolerant genotype can survive/reproduce at/above the critical temperature (>35/25 C; Figure 1). Therefore, screening methods have been developed for identifying heat tolerant genotypes under natural (field or pot) and controlled (hydroponic and pot assay) conditions in lentil Singh et al., 2017). Naturally, lentil genotypes are grown either in earthen pots or fields under late-sown conditions in order to coincide the reproductive stage of plants with high temperature (>35 C), and then, traits like pod setting (seed set) and grain yield are used to identify heat tolerant genotypes (Delahunty et al., 2015;Kumar et al., 2016;Singh et al., 2019;Sita, Sehgal, HanumanthaRao, et al., 2017). However, under field conditions, fluctuating day temperature limits to give uniform temperature during reproductive period across the genotypes when they differ in flowering times . As a result, it is difficult to identify early flowering heat tolerant genotypes due to their maturity before onset of the heat stress. Also, it is a challenging task to measure traits imparting heat tolerance with more accuracy and precision under field conditions. However, use of advance phenomic platforms can help in measuring of several physiological traits with more accuracy and precision directly in the field (Basu et al., 2015). In lentil, high-precision laboratory techniques have been used in measuring the several physiological traits such as photosynthetic rate, pollen viability, and membrane stability taken from field grown samples, and these traits have been used to differentiate heat tolerant and sensitive genotypes .
The controlled conditions give precise monitoring of the traits imparting heat tolerance due to availability of uniform and stable high F I G U R E 1 Sensitivity of lentil plants during reproductive period under the high temperature (>35 C) (a) sensitive, (b) highly sensitive, (c) tolerant, and (d) highly tolerant (Source: Kumar et al., 2016) temperature across the growth and development of crop plants (Basu et al., 2015). In lentil, heat tolerant genotypes have been identified on the basis of growth and development of germinating seeds under high temperature in laboratory or hydroponic conditions (Roy, Tucker, & Tester, 2011;Singh et al., 2016Singh et al., , 2019. However, a heat tolerant genotype identified at seedling stage requires its further validation at reproductive stage as heat stress occurs at terminal stage in lentil. Studies showed that heat tolerant genotypes identified under controlled conditions do not have their reproducibility under field conditions . Therefore, a potential heat tolerant genotype can be identified with more confidence by the use of natural and controlled conditions together. The use of screening methods based on controlled conditions can help to screen the large number of genotypes cost-effectively, which is not possible by using the field screening methods alone. Moreover, controlled conditions are used to provide relevant temperature during targeted stages or for a period of time. In lentil heat stress (35/20 C) applied for 4 h for 7 days from the first day of anthesis under controlled conditions and after that, the normal temperature (27/16 C) was given for pods development . In another study, plants were first grown in pots under natural conditions, and after that, at the flower initiation stage, these pots were moved into the controlled conditions in order to provide relevant temperature during anthesis (Chen et al., 2019;. The focused identification of germplasm strategy (FIGS) is one of the approaches for identifying heat tolerant genotypes in lentil. It works on the concept that environmental conditions strongly help natural selection towards the growing habitat and hence use of lentil accessions from regions exposed to high temperature stress in the screening can have a higher probability of containing traits and genes imparting heat tolerance (Delahunty et al., 2015;Gaur et al., 2015).

| Trait discovery
Phenotypic and physio-biochemical traits have been used to identify the heat tolerant genotypes in lentil (Table 1). As heat stress has the most devastating effects on just before/during flowering in legumes (Patriyawaty et al., 2018), the number of filled pods per plant and seed size under heat stress has been used a key indicator trait to assess heat tolerance among genotypes Singh et al., 2017;Sehgal et al. 2019. In addition to this, different physio-biochemical traits had been examined to differentiate heat tolerant and heat sensitive genotypes that led to identification of key physiological traits in lentil Sita, Sehgal, HanumanthaRao, et al., 2017). These traits listed in Table 1 and also reviewed earlier  are important because they directly help plants for sustaining against heat stress. Therefore, they can be used as indicator traits to identify heat tolerant genotypes in lentil. For example, higher membrane stability and photosynthetic electron transport rate played an important role to tolerate plants against heat stress at vegetative stage in lentil . In lentil, high temperature stress limits the availability of carbohydrates during reproductive development leading to poor seed filling and grain yield (Barghi, Mostafaii, Peighami, & Zakaria, 2012;Bhandari et al., 2016;Sita, Sehgal, HanumanthaRao, et al., 2017). Therefore, activities of sucrose synthase, sucrose phosphate synthase, and acid invertase enzymes can be used as key biochemical traits because activities of these enzymes have been found slow in developing pollen grains leading to poor accumulation of soluble carbohydrates compared with heat tolerant genotypes (Bhandari et al., 2016;Sita, Sehgal, HanumanthaRao, et al., 2017). In other crop plants, heat stress suppressed auxin biosynthesis leading to pollen abnormalities because auxin regulates reproductive process in plants, and hence, its role can be studied in lentil (Ozga, Kaur, Savada, & Reinecke, 2017;Teale, Paponov, & Palme, 2006;Todaka, Nakashima, Shinozaki, & Yamaguchi-Shinozaki, 2012). Therefore, above physiological and biochemical traits can be used for identification of heat tolerant genotypes in breeding program.

| Genetics and exploitation of genetic diversity through conventional breeding
Knowledge of the genetics underlying the traits imparting heat tolerance helps plant breeders for making genetic improvement using either conventional or modern breeding approaches. Considerably large genetic variation has been observed among the lentil germplasm for the traits listed in  Sita, Sehgal, HanumanthaRao, et al., 2017;Bhandari et al., 2016;Chakraborty & Pradhan, 2011;Delahunty et al., 2015;Kumar et al., 2016;Singh et al., , 2019Singh et al., . et al., 2016Singh et al., 2016). However, genetics of few traits responsible for heat stress tolerance has been studied in lentil. In one of the studies, monogenic inheritance has been reported for seedling survival and pod setting under heat stress condition . In this study, a simple inheritance has been reported for a complex trait like pod setting under heat stress condition. However, in snap beans, the pods number per plant under heat stress condition has been controlled by major genes (Rainey & Griffiths, 2005), whereas a quantitative inheritance has been reported for heat stress tolerance in wheat (Barakat, Al-Doss, Elshafei, & Moustafa, 2011;Talukder et al., 2014). Therefore, more studies are required to understand the genetics of many traits imparting heat tolerance in lentil.
Exploiting the genetic diversity in conventional breeding programs is one of the ways to reduce yield losses caused by high-temperature resistance/tolerance to biotic and abiotic stresses (Kumar, Basu, et al., 2018;Kumar, Rajendran, et al., 2015;Sita, Sehgal, HanumanthaRao, et al., 2017). However, limited studies have been conducted for either differentiating the heat sensitive and tolerant genotypes on the basis of genic markers  or mapping a major QTL controlling the seedling survival and pod setting traits under heat stress in lentil . Therefore, in lentil, more efforts are needed to

| Transcriptome analysis for identification of candidate genes responsible for heat tolerance
In lentil, heat and drought stresses occur together during seed filling due to rapid water loss from soil and plant. These two stresses affect different physiological process . For example, activity of RuBisCo and stomatal conductance increases under heat stress but is decreased under drought condition ). In another case, hydrolysis of sucrose has been observed to increase under heat and drought stresses, but it has been inhibited due to combination of stresses ). Effects of heat and drought also observed differently in different parts of lentil plant as starch tends to increase under heat stress in leaves but decreased in seeds.
Whereas it is drastically declined in seeds under drought alone or in combination with heat stress . The effect of heat stress has been observed more on yield traits than drought, whereas drought stress reduced individual seed weights more than heat stress . Therefore, there is a need to identify the candidate genes separately for heat and drought stress. Next generation sequencing (NGS) platforms have opened up the new opportunity for obtaining the genome sequences and transcriptomes. In lentil, NGS based transcriptome analysis provided opportunity to identify candidate genes expressed under biotic and abiotic stress conditions, including heat stress Singh et al., 2019). The transcriptome analysis of heat sensitive and tolerant genotypes led to the identification of candidate genes related to physiological and pollen phenotypes, cell wall, and secondary metabolism in lentil (Singh et al., 2019 conditions has been found to be responsible for producing the phosphoenolpyruvate (PEP). This metabolite is an essential compound of shikimate pathway that is responsible for production of secondary metabolites involved in heat tolerance. These genes are involved in different pathways in cell wall formation and secondary metabolites production that are affected under heat stress (Singh et al., 2019).
These candidate genes can be utilized after validation of their role in breeding programs for developing the heat tolerant genotypes.

| Genes for heat sock proteins
During heat stress, plants generate such type of proteins that stop denaturation and aggregation of functional proteins leading to a proper functioning of various bio-membranes and physiological process such as photosynthesis, assimilate partitioning, water, and nutrient use efficiency in heat tolerant plants (Fragkostefanakis et al., 2016;Ohama et al., 2016). These proteins that act as molecular chaperones are known as heat shock proteins (HSPs), and these are to be 10 to 200 kDa (Schöffl, Prändl, & Reindl, 1999). Therefore, use of the genes encoding synthesis of these HS proteins in breeding programs for developing heat tolerant cultivars can be an important adaptive strategy for tackling the problem of heat stress (Feder & Hofmann, 1999). Therefore, the role of these proteins in the heat stress tolerance has been studied in several legume crops. In soybean, HSPs provided the heat tolerance at seedling stage (Das et al., 2016) and an HSP gene family has been involved to control drought and heat stress at seedling stage (Zhang et al., 2015). In another study, functional markers developed from genes encoding HSP90 of Medicago, HSP70 of Glycine, HSP 17 and HSP 18 of Pisum proteins could not differentiate the heat sensitive and tolerant genotypes of lentil . Therefore, more efforts are required to identify the genes for those HSPs that impart heat tolerance in lentil.

| Network of metabolic pathways responsible for heat tolerance
Heat stress tolerance as a complex trait has a network of different metabolic pathways in crops plants (Bita & Gerats, 2013). Recent reviews have discussed role of different metabolites in controlling heat stress tolerance in food legume crops . In kidney bean, a more accumulation of soluble leaf carbohydrates and higher activity of adenosine-5 0 -diphosphoglucose pyrophosphorylase have been observed under high temperature (Prasad, Boote, Jcv, & Lhjr, 2004). In bermudagrass, upregulation of some important metabolic pathways, including photosynthesis, respiration, amino acid, and GABA shunt led to the synthesis of proteins and metabolites responsible to tolerate heat stress under elevated CO 2 (Yu, Li, Fan, Yang, & Huang, 2017). Several metabolic pathways, including altered energy pathways, lipid super pathway, and increased production of branched-chain amino acids, raffinose family oligosaccharides (RFOs), lipolysis products, and tocopherols help plants to tolerate heat stress (Serrano, Ling, Bahieldin, & Mahfouz, 2019). A crosstalk between carbohydrate and tyrosine metabolism produces metabolites like salidroside that helps plants to recover under heat stress. These metabolic pathways can be considered in plant breeding to maximize crop yields under adverse conditions (Serrano et al., 2019). Further, in cool-season creeping bentgrass, an increased level of the γ-aminobutyric acid (GABA), a nonprotein amino acid, helps to improve heat tolerance because it regulated major metabolic pathways responsible for accumulation of amino acids, organic acids, sugars, and sugar alcohols under heat stress conditions (Li, Yu, Peng, & Huang, 2016 and primary metabolites that were involved in heat tolerance in lentil (Singh et al., 2019). However, metabolomics study can directly exhibit the metabolite changes induced by stress as compared with transcriptomics. Therefore, there is a need to apply metabolomics for exploring the metabolites involved in heat-stress regulation in lentil like other crop plants (De Leonardis et al., 2015;Obata et al., 2015).
Further, a combination of transcriptomics with metabolomics can help to elucidate the gene-to-metabolite pathways as investigated in rice in the response to heat stress (Glaubitz et al., 2017).

| FUTURE WAYS FOR TACKLING THE PROBLEM OF HEAT STRESS
Lentil crop is highly affected by heat stress at terminal stage and in coming years, this problem can be more serious due to global warming. Different ways for tackling the problem of heat stress in lentil has been presented in Figure 2 and discussed below.
• Morphological, physiological, and biochemical traits can be used for developing the heat tolerant cultivars (Kumar, Pratap, & Kumar, 2015). During the past years, in lentil, focus has been made to identify the donors for heat tolerance Singh et al., 2016). However, use of automated phenotyping platforms can accelerate the identification of new genetic resources with more precision and accuracy and their use in future. The significant progress has been made in other crops towards the development of such platforms that are equipped with sensor and image based systems, leading to nondestructive phenotyping of the traits imparting heat tolerance (Kumar, Pratap, & Kumar, 2015). Also, several instruments have been developed for measuring physiological traits like stomatal mechanism and transpiration, osmotic adjustment, leaf water potential, and canopy temperature directly in the field (Basu et al., 2015). These advances are required to be used for heat tolerance studies in lentil and the reverse phenomics can be used to discover the mechanisms and gene(s) for the traits imparting heat tolerance in lentil (Kumar, Pratap, & Kumar, 2015).
• In lentil, limited studies have been conducted on mapping of genes controlling heat tolerance in lentil  that is needed for key physiological traits. As NGS-based transcriptome analysis and comparative genomics have opened the opportunity to identify the expressed sequence tags (ESTs), candidate genes, and metabolic pathways involving in the heat tolerance , more efforts are required to work in this direction. In wheat, new alleles belonging to Hsp26 gene family encoding chloroplast-localized small (s)HSPs have been identified through high throughput Targeting Induced Local Lesions in Genomes (TILLING) approach of reverse genetics. These alleles encode such HSPs that protect the photosynthetic machinery from high temperature stress (Comastri et al., 2018). Therefore, use of this reverse genetic approach can be focused by developing TILLING populations for identification of heat tolerant genes in lentil. Candidate genes or ESTs those are involved in heat tolerance can be used to develop functional markers for breeding . In wheat, a set of Kompetitive Allele Specific PCR (KASP) markers has been developed from genes encoding chloroplast-localized sHSPs in order to identify the heat tolerant genes in breeding populations (Comastri et al., 2018).
• Genome editing is emerging area of genomics for developing the heat tolerant cultivars. The CRISPR/Cas9 based targeted gene editing has great potential to produce high-yielding crops under heat stress (Abdelrahman, Al-Sadi, Pour-Aboughadareh, Burritt, & Tran, 2018). In Arabidopsis thaliana, this approach has been used to modify the abiotic stress response. It also helps to suppress or activate a target gene leading to identification of function of that gene (Osakabe et al., 2016). In cowpea, it has been used to know the function of genes related to nitrogen fixation (Ji et al., 2019 showed an overall high level of genome-wide DNA methylation compared with sensitive lines (Ma et al., 2018). These DNA methylations have occurred in the genes involved in the starch, auxin, and sugar metabolic pathways that are crucial for proper pollen development (Min et al., 2014). Therefore, in future, it needed to handle the heat-methylome variation in breeding programs for developing the heat tolerant cultivars in lentil (Harkess, 2018;Kumar et al., 2019). The breeding strategies of using the epigenetic variation towards the development climate smart pulses have been elaborated in a recent review .
• In crop plants, transcriptional, translational and post-translational mechanisms, and signaling pathways regulate heat tolerance response, and different omics approaches are now available to study the regulation of heat tolerance in crop plants. In lentil, use of these omics approaches is still limited and only few studies used the genomics and transcriptomic approaches for identification of genes responsible for heat tolerance. In crop plants, expressions of several genes are modified towards the tolerance against a particular stress after transcription through non-coding micro RNA (Chinnusamy, Zhu, Zhou, & Zhu, 2007). The role of these micro RNAs for controlling heat tolerance has been studied in different crops under micromics, and heat responsive miRNAs that up-or down-regulate genes responsible for heat tolerance have been F I G U R E 2 Different omics strategies for genetic improvement of heat stress tolerance in lentil. QTL, quantitative trait locus identified (Chinnusamy et al., 2007). Translation of the coding RNA into proteins that work as HSPs (discussed above in details) take part in several biochemical processes leading to the generation of metabolites (metabolome) under heat stress conditions. The role of different metabolites in heat stress tolerance can be studied in lentil as identified in other plant species (Caldana et al., 2011;De Block, Verduyn, De Brouwer, & Cornelissen, 2005;Maruyama et al., 2009;Wienkoop et al., 2008). Thus, advances in different omics approaches can help to integrate the metabolites with transcriptomes for elucidating gene-to-gene and metabolite-to-gene networks in lentil (Hirai et al., 2005). Therefore, a lot of efforts are still required by following different ways of tackling the heat stress problem in lentil.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS
Writing -original draft and review and editing: JK, Writing -review and editing: DSG, All authors read and approved the final manuscript.

FUNDING INFORMATION
Authors work on heat tolerance in lentil is supported by the project "National Innovations in Climate Resilient Agriculture (NICRA)" funded by ICAR (Indian Council of Agricultural Research), New Delhi.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.