**5.4. Molecular markers in breeding for HT**

The efforts by conventional breeding schemes have led to identification of several HT-relevant gene(s) and their inheritance patterns [6, 142]. Recent advances in marker discovery and genotyping assays have led to the precise determination of chromosomal position of the QTLs responsible for HT in different crops [90, 143–145]. The identification of markers linked to QTLs enables breeding of stress-tolerant crops by combining or pyramiding of QTLs governing tolerance to various stresses. An elaborative list of QTLs associated with HT in various crops along with details of mapping populations used, number of QTLs identified, associated markers, chromosomal positions, and phenotypic variation explained (PVE) has been summarized by Jha et al. [146]. Several major or minor QTLs and linked markers for HT have been identified in major food crops such as rice [90, 147–149], wheat [143, 144, 150–152], and maize [153, 154]. QTLs for several HT-related traits have been identified such as cellular membrane stability, pollen germination, and pollen tube growth in maize [153, 154]; stay-green trait, photosynthetic genes, and HSPs in sorghum [155]; and pollen viability in adzuki bean [156].

In wheat, one candidate SNP marker that clearly distinguished heat tolerance and heat-sensitive cultivars was identified [157]. For grain-filling rate (GFR) that governs grain yield in wheat under HS, 12 closely linked SSR markers were identified [158]. Using a SNP marker for a RIL population, five important genomic regions that offered HT in cowpea were identified [159]. Genomewide as well as candidate-gene-based association mapping using SNP and DArT markers in chickpea could establish marker-trait associations for HT [160]. Recently, a major dominant locus *OsHTAS* (*Oryza sativa* heat tolerance at seedling stage) was identified from the genotype HT54, which contributed high-temperature tolerance at 48°C especially during seedling and grain-filling stages [161]. The QTLs identified using molecular markers in different crops provide a way to transfer the causative heat tolerance gene(s)/QTLs to elite cultivars. In parallel, the fine mapping accompanied by cloning of candidate QTL will help the breeders to commence marker-assisted breeding for incorporating HT in various important crops in near future.

Membrane thermostability MTS is considered as a useful component for measuring HT, while assessing genetic variability in different crops [49]. Selection for MTS during anthesis stage under HS led to significant yield improvements in wheat [50]. Various physiological traits and their relative contributions to HT in wheat have been discussed in detail earlier [129]. Screening against HS based on parameters such as electrolyte leakage from cell membrane and chlorophyll fluorescence revealed negative association of membrane injury with specific leaf weight in some legume species including groundnut and soybean [59]. The combination of the two selection parameters, viz. high chlorophyll content and MTS, was implicated to carry out selections in Brassica and wheat [34, 136]. The relative cell injury level (RCIL) under HS could also be taken as a reliable index in determining HS tolerance in

The adaptation of root respiratory carbon metabolism can offer tolerance to soil temperature by managing the ion uptake load as reported in *Agrostis* species [138]. The efficient carbon and protein metabolism is known to confer higher thermotolerance to roots at 45°C in *Agrostis* 

been indicated as a strong indicator of HT [140]. However, stay-green trait is less important in the context of yield on account of disability in translocation of stem reserves to grain under HS [118]. Whereas, the conditions encompassing heat stress alone, as well as heat stress and drought, stay-green trait is measured as normalized difference vegetation index (NDVI) at physiological maturity exhibiting a positive correlation with the yield [141]. Therefore, physiological trait-based breeding remains a promising improvement strategy to develop heat

The efforts by conventional breeding schemes have led to identification of several HT-relevant gene(s) and their inheritance patterns [6, 142]. Recent advances in marker discovery and genotyping assays have led to the precise determination of chromosomal position of the QTLs responsible for HT in different crops [90, 143–145]. The identification of markers linked to QTLs enables breeding of stress-tolerant crops by combining or pyramiding of QTLs governing tolerance to various stresses. An elaborative list of QTLs associated with HT in various crops along with details of mapping populations used, number of QTLs identified, associated markers, chromosomal positions, and phenotypic variation explained (PVE) has been summarized by Jha et al. [146]. Several major or minor QTLs and linked markers for HT have been identified in major food crops such as rice [90, 147–149], wheat [143, 144, 150–152], and maize [153, 154]. QTLs for several HT-related traits have been identified such as cellular membrane stability, pollen germination, and pollen tube growth in maize [153, 154]; stay-green trait, photosynthetic genes, and HSPs in sorghum [155]; and pollen viability in adzuki bean [156]. In wheat, one candidate SNP marker that clearly distinguished heat tolerance and heat-sensitive cultivars was identified [157]. For grain-filling rate (GFR) that governs grain yield in wheat under HS, 12 closely linked SSR markers were identified [158]. Using a SNP marker for a RIL population, five important genomic regions that offered HT in cowpea were identified [159]. Genomewide as well as candidate-gene-based association mapping using SNP and DArT markers in chickpea could establish marker-trait associations for HT [160]. Recently, a major dominant

perennial grass species) [139]. In wheat, stay-green trait associated with CTD has

cotton [137].

56 Next Generation Plant Breeding

*scabra* (a C3

tolerance genotypes without causing yield penalty.

**5.4. Molecular markers in breeding for HT**

For transfer of quantitative traits such as HT, molecular markers would enable the recovery of desirable genotypes in a precise and time-saving fashion [162]. Molecular markers have been useful in identifying heat sensitive advanced generation introgression lines in rice [120, 149]. The near isogenic lines (NILs) created by introducing desirable allele into the heat sensitive cultivar showed considerable reduction in the incidence of heat-induced injuries such as whiteback kernels [163]. Recently in rice, a 1.5-Mb chromosomal region harboring a robust QTL controlling better grain quality under HS has been transferred from "Kokoromachi" to "Tohoku 168" using marker-assisted backcrossing [162]. The resultant NILs had improved grain quality over the susceptible parent.

DNA markers related to various HT/component traits have been identified in different crops such as rice [148, 161, 163–165], wheat [143–145, 150, 152, 166, 167], and cowpea [159, 168]. Once the markers associated with QTLs have been identified, the candidate QTLs can further be introgressed into elite lines through marker-assisted selection (MAS) strategies. One of the difficulties of developing superior genotypes for heat stress is that these traits are generally controlled by small effect QTLs or several epistatic QTLs [3]. To overcome this problem, approaches that can be employed are pyramiding several QTLs in the same genetic background using large populations through marker-assisted recurrent selection (MARS) or genomic selection (GS) [169].

MAS programs for complex traits such as heat tolerance are not effective mainly due to the genotype × environment and gene-gene (i.e., epistasis) interactions, which frequently result in a low breeding efficiency [170]. In contrast to MAS strategies which use markers for which a significant association with a trait has been identified, the GS method predicts breeding values using data derived from a vast number of molecular markers with a high coverage of the genome. Its novelty is that it uses all marker data as predictors of performance and subsequently delivers more accurate predictions [3]. Simulation studies indicated that GS may increase the correlations between predicted and true breeding value over several generations, without the need to re-phenotype. Thus, GS may result in lower analysis costs and increased rates of genetic gain [171, 172].

QTLs often do not translate well across genetic backgrounds and often produce smaller than expected adaptation effects. Thus, improving crop abiotic stress tolerance by exploiting the segregation of natural alleles rather seems challenging for such an adaptive QTL strategy [170]. When quantitative hereditary characteristics such as heat stress tolerance are involved, recurrent selection seems to be one of the most efficient methods in plant breeding. In multiple crosses, the probability is very small of obtaining superior genotypes that reunite all the favorable alleles. However, in this circumstance, a large segregating population is required, aspect that becomes unfeasible in practice [3]. The alternative is to adopt recurrent selection to gradually accumulate, by recombination cycles, the desirable and available alleles in different parents [173]. The main aim of a recurrent selection program is to increase the frequency of favorable alleles for traits of interest, while conserving the genetic variability. The major advantages are: (i) greater genetic variability obtained by intercrossing of multiple parents; (ii) greater opportunity for recombination through successive crossings; (iii) greater efficiency in increasing the favorable gene frequency since it is a repetitive and accumulative process; and (iv) greater facility to incorporate exotic germplasm in the population [174]. In potato breeding program at the Federal University of Lavras, heat stress tolerance genotypes were successfully developed using recurrent selection, which led to gains in tuber production with improved quality [175].

A current approach to the challenge of high-temperature tolerance is "physiological" or "developmental" trait breeding through recurrent selection using crop germplasm from regions with hot growing seasons [127]. In both cases, the targets are loci with high heritability that sustain yield at normal and elevated temperatures. The selection can be on combined heat and drought tolerance, as required by many crops. Recurrent selection has been successfully used to improve heat tolerance in wheat [176, 177] and potato [175]. In wheat, the stable introduction of chromosomes from its wild relative *Leymus racemosus* provided heat tolerance in hot and arid fields [123]. The heat- and drought-tolerant rice variety N22 (*aus* ssp.) has provided QTLs associated with high levels of HSPs in anthers, spikelets, and flag leaves associated with maintenance of yield under high night temperatures [148, 178]. Additional targeted developmental breeding in rice takes advantage of QTLs from *Oryza officinalis* that avoid heat-induced spikelet sterility by promoting dehiscence and fertilization in the cooler early mornings [179]. In time, these approaches may yield loci and knowledge that can accelerate improvement in heat tolerance in combination with drought tolerance.

Field trials under real stress conditions allow for conclusive remarks on stress tolerance and yield performance of a genotype. Development of more precise phenotyping tools that can be applied to field conditions is a prerequisite for enabling the assessment of the complex genetic networks associated with QTLs. The small, yet significant phenotypic changes delivered by introducing single genes into breeding material require precision phenotyping protocols and the resource capacity to carry these out on very large populations. The integrated approach for HT has been illustrated in **Figure 1** with details of whole breeding process.

in model plants such as *Arabidopsis*, tobacco, and rice. Further, it needs to be extended to other

**Figure 1.** Schematic presentation of next-generation (integrated) breeding approach to develop heat tolerant cultivars.

Breeding Cultivars for Heat Stress Tolerance in Staple Food Crops

http://dx.doi.org/10.5772/intechopen.76480

59

To combine stress tolerance with high yield potential while avoiding the negative effects of a stress gene on plant growth under favorable conditions, strategies that spatially and temporally restrict transgene expression via tissue-specific and stress-inducible promoters are used [182]. Engineering promoters will facilitate gene pyramiding through genetic modification, addressing the issue of tolerance to multiple stresses at different stages of plant growth [183]. As an alternative, engineering with specific transcription factors and signaling components could be employed. Ultimately, this leads to the expression of their target transcriptome that consists of several genes involved in the response to stress. Transcriptome engineering emerges thus as a promising avenue for the development of abiotic stress-tolerant crops. Currently, however, plant genetic engineering is hampered by nonbiological constraints mainly related to the commercialization of transgenic crops, particularly in Europe [184]. Thus, the future commercial success of transgenic breeding will depend upon the development of clearly defined and scientifically based regulatory frameworks, and upon public

Enormous sequence information is being generated through new-generation high-throughput DNA sequencing technologies [186]. But, precise, accurate, and high-throughput phenotyping of the traits on a large scale remains strenuous [187]. Field-based phenotyping (FBP)

acceptance of genetically modified plants and their produce [185].

**5.6. Phenomics for precise and high-throughput phenotyping**

agriculturally important field crops.

## **5.5. Transgenic approach**

Tolerant sources if not found in a given species or not giving enough protection, transgenic approach is an alternative option. However, it requires identification of the gene responsible for the desired trait, but poses no barrier for transferring useful genes across different species within the plant kingdom or even from animal systems. Genes of nonplant species could potentially be introgressed as well and generally, several combinations of beneficial genes could be transferred into the same plant [3]. With increasingly refined transformation and regeneration protocols, transgenic techniques are becoming attractive tool for designing both biotic and abiotic stress-tolerant crops via manipulating native genes or introducing gene(s) that lie beyond the crop gene pools [180]. Toward development of transgenics for HT, primarily the focus has been on engineering genes that encode transcription factors (TFs), HSPs, chaperones, organic osmolytes, antioxidants and plant growth regulators. This has been earlier summarized by Grover et al. [181]. Most of the transgenics developed for HT are mostly

parents [173]. The main aim of a recurrent selection program is to increase the frequency of favorable alleles for traits of interest, while conserving the genetic variability. The major advantages are: (i) greater genetic variability obtained by intercrossing of multiple parents; (ii) greater opportunity for recombination through successive crossings; (iii) greater efficiency in increasing the favorable gene frequency since it is a repetitive and accumulative process; and (iv) greater facility to incorporate exotic germplasm in the population [174]. In potato breeding program at the Federal University of Lavras, heat stress tolerance genotypes were successfully developed using recurrent selection, which led to gains in tuber production with

A current approach to the challenge of high-temperature tolerance is "physiological" or "developmental" trait breeding through recurrent selection using crop germplasm from regions with hot growing seasons [127]. In both cases, the targets are loci with high heritability that sustain yield at normal and elevated temperatures. The selection can be on combined heat and drought tolerance, as required by many crops. Recurrent selection has been successfully used to improve heat tolerance in wheat [176, 177] and potato [175]. In wheat, the stable introduction of chromosomes from its wild relative *Leymus racemosus* provided heat tolerance in hot and arid fields [123]. The heat- and drought-tolerant rice variety N22 (*aus* ssp.) has provided QTLs associated with high levels of HSPs in anthers, spikelets, and flag leaves associated with maintenance of yield under high night temperatures [148, 178]. Additional targeted developmental breeding in rice takes advantage of QTLs from *Oryza officinalis* that avoid heat-induced spikelet sterility by promoting dehiscence and fertilization in the cooler early mornings [179]. In time, these approaches may yield loci and knowledge that can accel-

Field trials under real stress conditions allow for conclusive remarks on stress tolerance and yield performance of a genotype. Development of more precise phenotyping tools that can be applied to field conditions is a prerequisite for enabling the assessment of the complex genetic networks associated with QTLs. The small, yet significant phenotypic changes delivered by introducing single genes into breeding material require precision phenotyping protocols and the resource capacity to carry these out on very large populations. The integrated approach

Tolerant sources if not found in a given species or not giving enough protection, transgenic approach is an alternative option. However, it requires identification of the gene responsible for the desired trait, but poses no barrier for transferring useful genes across different species within the plant kingdom or even from animal systems. Genes of nonplant species could potentially be introgressed as well and generally, several combinations of beneficial genes could be transferred into the same plant [3]. With increasingly refined transformation and regeneration protocols, transgenic techniques are becoming attractive tool for designing both biotic and abiotic stress-tolerant crops via manipulating native genes or introducing gene(s) that lie beyond the crop gene pools [180]. Toward development of transgenics for HT, primarily the focus has been on engineering genes that encode transcription factors (TFs), HSPs, chaperones, organic osmolytes, antioxidants and plant growth regulators. This has been earlier summarized by Grover et al. [181]. Most of the transgenics developed for HT are mostly

erate improvement in heat tolerance in combination with drought tolerance.

for HT has been illustrated in **Figure 1** with details of whole breeding process.

improved quality [175].

58 Next Generation Plant Breeding

**5.5. Transgenic approach**

**Figure 1.** Schematic presentation of next-generation (integrated) breeding approach to develop heat tolerant cultivars.

in model plants such as *Arabidopsis*, tobacco, and rice. Further, it needs to be extended to other agriculturally important field crops.

To combine stress tolerance with high yield potential while avoiding the negative effects of a stress gene on plant growth under favorable conditions, strategies that spatially and temporally restrict transgene expression via tissue-specific and stress-inducible promoters are used [182]. Engineering promoters will facilitate gene pyramiding through genetic modification, addressing the issue of tolerance to multiple stresses at different stages of plant growth [183]. As an alternative, engineering with specific transcription factors and signaling components could be employed. Ultimately, this leads to the expression of their target transcriptome that consists of several genes involved in the response to stress. Transcriptome engineering emerges thus as a promising avenue for the development of abiotic stress-tolerant crops. Currently, however, plant genetic engineering is hampered by nonbiological constraints mainly related to the commercialization of transgenic crops, particularly in Europe [184]. Thus, the future commercial success of transgenic breeding will depend upon the development of clearly defined and scientifically based regulatory frameworks, and upon public acceptance of genetically modified plants and their produce [185].
