Advances in Research on Drought Resistance in Rice

Yueming YI Shimei WANG Xinxin CHENG Qian ZHU Yiru LI Fengbin WANG Minghua ZHANG

Abstract Rice is the main grain crop in China. With global warming, drought and water shortage have become one of the important factors limiting rice production. Improving the drought resistance of rice can effectively alleviate the loss of rice yield caused by drought. In this paper, the research progress on rice drought resistance and its evaluation methods, mapping of QTLs for drought resistance-related traits, and mining for drought resistance genes were reviewed, and molecular breeding for drought resistance in rice was prospected.

Key words Rice; Drought resistance; Methods of identification and evaluation; QTL mapping

Received: June 20, 2022  Accepted: August 21, 2022

Supported by Natural Science Foundation of Anhui Province (1908085MC93); Major Science and Technology Project of Anhui Province (2021d06050002); Anhui Province Joint Research on Improved Crop Varieties (Rice).

Yueming YI (1996-), female, P. R. China, devoted to research about rice breeding for drought tolerance.

*Corresponding author.

Rice is one of the important food crops in the world, and it is the main food for more than half of the world’s population. The 2020 World Water Development Report by United Nations in 2020 pointed out that with population growth and economic development, global water consumption is growing steadily at a rate of about 1% per year. Globally, the area of dryland is projected to expand significantly (https:∥www.unwater.org/). The freshwater resources available for agricultural production will continue to decrease, and droughts caused by water shortages will severely limit the normal growth and development of plants. The water consumption for rice production accounts for 70% of the total agricultural water consumption, and the average annual loss of rice yield due to drought is about 143-250 kg/hm2, and it reaches 2 700 kg/hm2 in severe years［1-4］. Therefore, improving drought resistance of rice and cultivating new water-saving and drought-resistant rice varieties are the bottlenecks in alleviating the yield reduction of rice production due to water shortage.

The drought resistance of rice is a complex quantitative trait, and each drought resistance-related trait may involve one or more genes. With the rapid development of molecular marker technology and molecular biotechnology, the analysis methods of classical genetics cannot identify the gene positions that control drought resistance, while molecular biology can help identify quantitative trait loci (QTLs) that have a greater impact on target traits under drought stress to study their impact on yield, which is beneficial to molecular marker-assisted breeding［5-6］. The study of plant drought resistance mechanism and genetic inheritance from the molecular level has brought a new way to analyze the drought resistance of rice［7］.

Drought Resistance in Rice and Its Response Mechanism

Drought resistance in rice

The specific connotation of rice drought resistance includes drought avoidance, drought tolerance, drought recovery and drought escape. Drought avoidance refers to the ability of rice to avoid plant water shortage by reducing water demand during drought periods. Rice maintains a high leaf water potential by absorbing water from the soil through a developed root system, moderately closing the body surface stomata and generating an impermeable waxy layer. Drought tolerance refers to the ability of rice to maintain metabolism and endure drought even under the condition of water shortage in leaves through osmotic regulation of cells, improvement of cell elasticity, and reduction of cell volume. Drought recovery refers to the ability of rice to quickly recover from water stress. After the water stress is over, rice can produce more tillers and obtain sufficient yield. Drought escape means that rice grows while avoiding the period when drought occurs, so as to reduce the adverse effects of drought. Rice is most sensitive to water stress from ear development to flowering. Early-maturing varieties can escape drought stress due to earlier flowering, but late-maturing varieties may be affected by drought. Drought escape is not a drought resistance mechanism in the true sense, but the ability of plants to grow rapidly in a concentrated manner in the rainy season formed by their own selection. Therefore, drought resistance of rice is more a combination of the three mechanisms: drought avoidance, drought tolerance and drought recovery［9-10］. In the early stage of drought stress, drought avoidance plays a major role, and with the aggravation of drought, the role of drought tolerance appears, and drought recovery appears after the relief of drought stress.

Response of rice to drought stress

Drought will affect the morphology, physiology and cells of rice to varying degrees. Morphologically, leaf rolling occurs, and stomata are closed, and transpiration is weakened by reducing leaf area and gas exchange rate［11］. Rice initially senses drought through the roots, and stem cells, cortical cells, and vascular cells subsequently mediate responses to water limitation. Plants constantly adjust root structure, with root tips growing toward areas of higher water content to absorb water from the soil. The plant hormone abscisic acid (ABA) is a major molecule that promotes signal transduction during drought stress response. The dehydration signal stimulates local production of ABA in different organs of plants, and accumulated ABA will activate the downstream signal components, promote the closure of stomata to retain water, regulate the osmotic process in the tissue, and actively maintain physiological water balance［8,12］.

The sensitivity of rice to drought is different at different growth stages. Early water shortage will delay rice germination and have little effect on yield. During the reproductive growth period, rice is most sensitive to drought, and water shortage will affect the development of pollen and reduce the photosynthetic production capacity, resulting in the reduction of above-ground biomass accumulation and severe yield reduction. In the grain-filling stage, the effect of drought stress on yield is relatively small, and the material and energy produced by photosynthesis in the early stage of rice plants is relatively sufficient, and it is difficult for drought to stress the grain-filling stage in the later stage. However, later drought has a greater impact on the grain maturity of late-maturing varieties［13-14］.

Evaluation Methods for Drought Resistance in Rice

The drought resistance of rice is related to the genetic characteristics of rice varieties, soil environment, morphological characters, physiological indexes, and the time and intensity of drought. Due to the differences in growth period and response mechanism of rice when responding to drought stress, the identification method of drought resistance is complex, and the evaluation indexes are different.

Rice drought resistance indexes

The drought resistance of rice is mainly measured by drought resistance identification indexes. There are many kinds of drought resistance identification indexes related to rice, and the views put forward by different scholars are also inconsistent.

During the germination stage of rice, hypertonic fluids such as polyethylene glycol and mannitol are mainly used to simulate drought stress, with germination rate, radicle length, germ length, shoot dry weight, root length, root dry weight, and material transport rate as screening indexes［15］. During the rice seedling stage, plant height, leaf dry weight, leaf fresh weight, leaf wilting degree, leaf rolling degree, root number, root length and other morphological indexes as well as physiological and biochemical indexes such as proline content, relative water content, stomatal resistance, plasma membrane permeability, malondialdehyde, superoxide dismutase and catalase are mainly screened, or rice seedlings are subjected to repeated drought stress treatment to identify their survival rate. Yu et al.［16］ analyzed that root dry weight, root length, leaf dry weight and plant height could be used as indexes for the identification of drought resistance in rice seedlings. The survival rate of rice seedlings under repeated drought is significantly correlated with drought resistance, and the identification is reproducible and simple. The selection of indexes at the rice flowering stage mainly focuses on chlorophyll, relative leaf water content, leaf area, photosynthetic rate, stomatal conductance and some biochemical indexes. The drought resistance index is the ratio of the index value under drought stress and normal water conditions, which is a common index in the identification of rice drought resistance, which can reflect the sensitivity of rice to drought［17］. Different drought resistance identification methods have the same purpose, and yield is the ultimate goal of drought resistance identification. Mau et al.［18］ believed that using yield as an index for drought resistance identification is an effective method to select genotypes that combine drought resistance with high yield potential. However, Cheng et al.［19］ believed that taking the final yield as the drought resistance index has the disadvantage of time-consuming and labor-intensive, and using the neck-panicle node thickness as a single evaluation index can save time and labor.

In the actual drought resistance identification experiments of rice varieties, most of them take seed setting rate as the main index, and drought stress experiments are carried out at the flowering stage of rice to compare and identify the drought resistance grades of varieties. A single index cannot comprehensively evaluate drought resistance, and the drought resistance in a certain period cannot represent the comprehensive drought resistance level of rice. It is necessary to construct a comprehensive index system of drought resistance in terms of morphology, physiology, and biochemistry［20］.

Drought resistance identification method

Drought resistance identification methods mainly include direct identification and indirect identification. Drought resistance identification methods mainly include direct identification and indirect identification. Direct identification is to treat rice under drought stress by regulating the amount of soil moisture in natural fields, dry sheds and pots, and evaluate its drought resistance according to the growth and development and physiological processes in different growth stages of rice and yield results under water stress. Indirect identification is to evaluate the physiological and biochemical indexes of rice in the laboratory［21］. Yang et al.［22］ used the drought resistance identification platform of greenhouse+dry pond to stress rice during the booting stage, and screened out materials with potential drought resistance. Tian et al.［23］ used PEG-6000 hypertonic solution stress instead of soil water drought stress to evaluate the drought resistance of plants during germination, which can be used to screen drought-resistant varieties in large quantities.  Zhang et al.［24］ also believed that the use of PEG treatment in the greenhouse to simulate drought stress is a convenient method, which is easy to maintain the humidity and temperature during the vegetative growth period in greenhouses, and can effectively identify excellent drought-resistant strains.

There are various types of drought resistance evaluation methods. The drought resistance classification method, membership function method, cluster analysis method, principal component analysis method, regression analysis, grey relational analysis and other methods are mainly used for combined evaluation. Liu et al.［25］ used the membership function method to comprehensively evaluate the drought resistance of main rice varieties. The membership function method eliminates the one-sidedness of a single index, making the drought resistance of tested materials comparable, and the evaluation results are more reliable. Wang et al.［26］ screened identification indexes of rice drought resistance using the principal component analysis and stepwise regression analysis, and quantified the drought resistance of rice with the relative value of each index. Li et al.［27］ screened the identification indexes of drought resistance at the full heading stage of rice in cold regions by the logarithmic principal component analysis method according to the value of the grey correlation coefficient and the identification efficiency. The logarithmic principal component evaluation method solved the nonlinear relationship between the indexes and between principal components and the original data, and reduced the deviation of the evaluation results. Sakinah et al.［28］ identified the effectiveness of complex heatmap analysis in screening for drought resistance in rice. Singh et al.［29］ scored and classified the drought resistance of tested rice varieties using the total drought response index (TDRI). In recent years, researchers have begun to use the comprehensive index identification method. The comprehensive drought resistance is the comprehensive score of drought resistance obtained by the principal component evaluation of each test material under water stress, which can comprehensively and systematically evaluate the drought resistance of rice.

Agricultural Biotechnology2022

Research Progress on QTLs Related to Drought Resistance in Rice

QTL mapping method

The selection of the QTL mapping method is mainly based on the type of the target population and the research purpose［30］. The two main strategies for QTL mapping are selective genotyping (SG) and population segregation analysis (BSA) at present. The traditional QTL mapping method is to identify and screen molecular markers, construct a molecular genetic linkage map, and perform correlation analysis between measured phenotype values and genotypes to determine corresponding positions of QTLs on chromosomes. The traditional QTL mapping strategy process is time-consuming and labor-intensive, and cannot adapt to the development of modern biotechnology. Salunkhe et al.［31］ located a QTL related to leaf rolling and leaf dryness using a recombinant inbred line constructed by IR20 and Nootripathu by the BSA method. The RM302-RM8085-RM3825 region is the major QTL for rice drought resistance trait, involving multiple genetic backgrounds, which is helpful for the mining and map-based gene cloning of rice drought resistance genes. With the development of sequencing technology, researchers have explored genome-wide analysis methods such as MutMap and QTL-seq for gene mapping［32］.  Takagi et al.［33］ isolated rice blast resistance gene Pii from the rice plant Hitomebore by the MutMap technology using a mutant strain that lost the function of Pii. The high-throughput genome-assisted QTL-seq strategy, combining methods for BSA and whole-genome sequencing (WGS), has become one of the most popular methods for the rapid discovery and identification of major QTLs in crops［34］. Next-generation sequencing technology has reduced the cost of whole-genome sequencing, making researchers favor the use of sequencing technology to obtain high-density genetic maps. Wang et al.［35］ used whole-genome sequencing technology to sequence a RIL population and constructed a high-density genetic map, which is conducive to further QTL mapping of rice drought resistance. The development of genetic maps based on easily generated, highly codominant and highly specific markers of known linkage groups is an urgent need for crop breeding applications［36］.

QTLs related to drought resistance traits

In recent years, researchers have done a lot of research on rice drought resistance, and located many QTL loci related to drought resistance traits. As of February 2, 2022, the Gramene database (https://gramene.org/) has published more than 800 QTLs for quantitative traits related to drought resistance in rice.

Mapping of QTLs for yield traits

Drought will directly lead to the reduction of rice yield, and yield traits under water stress conditions are one of the important indexes for evaluating drought resistance［38］. Bernier et al.［39］ used the F3 population constructed by drought-sensitive parent Way Rarem and drought-resistant parent Vandana to locate several QTLs for yield traits. Among them, a locus qDT12.1, which has a greater impact on grain yield under drought conditions, was located on chromosome 12. The study of Asmuni et al.［40］ proved that this QTL is a locus with large effect and continuous influence.  Shailesh et al.［41］ locate QTLs related to grain yield, days from sowing to heading, and plant height using Swarna and Dular as parents. Yuan et al.［42］ detected QTLs related to plant height, heading date and effective panicle number using a recombinant inbred line population constructed by Zhong 156 and Gumei 2. The results showed that the heading stage had an inhibitory effect on the expression of QTLs related to plant height and effective panicle number. Venuprasad et al.［43］ found a major QTL related to grain yield under stress on chromosome 1 in five different rice populations: qDTY1.1, which explained 58% of the genetic variation of this trait, and is a potential candidate locus for molecular breeding for drought tolerance in rice. Nitika et al.［44］ identified QTLs with large high-yielding effect and strong consistency under drought conditions: qDTY1.2, qDTY2.2 and qDTY1.3, qDTY2.3, and concluded that these QTLs can contribute to the study of improved varieties with high yield potential. Some major QTLs affecting rice yield can be located in different populations and years, indicating that these loci are important prerequisites for breeding high-yielding and drought-tolerant varieties through molecular marker selection, as shown in Table 1.

QTLs for root traits

Rice roots are the organ that is in direct contact with the soil and directly senses the soil water status. Therefore, many studies on rice drought resistance are mostly focused on the root system. Under drought conditions, rice roots absorb water and nutrients from deep soil for osmotic regulation by optimizing root traits and improving root activity, thereby resisting water stress and improving rice yield. The effects of root traits on yield are more important under drought conditions than under wet conditions, and drought-tolerant rice varieties usually have well-developed root systems［45-47］.

Ray et al.［48］ used restriction fragment length polymorphism (RFLP) molecular markers to identify a recombinant inbred line population, and located QTLs related to the total root number, root penetration index, and tiller number. It is the first study using the RFLP quantitative trait analysis to analyze root-related QTLs. Uga et al.［49］ located and cloned a QTL related to the ratio of deep roots in the 608.4 Kb region of chromosome 9: Dro1, using a recombinant inbred line population constructed by rice variety IR64 with a low ratio of deep roots and upland rice variety Kinandang Patong with a high ratio of deep roots. DRO1 is negatively regulated by auxin and involved in rice root tip cell elongation, and the effects of drought can be avoided by using DRO1 to increase root depth. Sabar et al.［50］ used the F2 population of IR55419-04 and Super Basmati as the mapping population, and detected one QTL related to root dry weight, three QTLs related to deep root length, two QTLs related to the surface area of deep roots, one QTL related to deep root volume, two QTLs related to the diameter of deep root. These QTLs can be used for molecular marker-assisted breeding of new rice varieties and can be used for fine mapping.  Mu et al.［51］ studied the root system of upland rice, and detected QTLs related to the number of roots, the thickness of the root base, and the maximum root length. Wang et al.［52］ located multiple QTLs related to the root system using the population of upland rice introgression lines.   Patil et al.［53］ used a combination of QTL mapping and next-generation RNA sequencing to fine-map target QTLs. rdw8.1, a major QTL controlling root dry weight and root length on chromosome 8, was identified as a wound-inducible protein, which is involved in rice drought resistance and has potential value in rice drought resistance breeding. rdw8.1, a major QTL controlling root dry weight and root length on chromosome 8, was identified as a wound-inducible protein, which is involved in rice drought resistance and has potential value in rice drought resistance breeding. The mapping results of QTLs for root traits are shown in Table 2.

QTLs for physiological traits related to drought resistance

Plants will produce a series of physiological and biochemical reactions under water stress, such as stomatal conductance, chlorophyll content, transpiration rate, photosynthesis rate and relative water content, which all decrease. Optimizing these physiological and biochemical processes can improve the drought resistance of rice［54］.

Price et al.［55］ used the F2 population of drought-resistant cultivar Azucena and Bala to locate QTLs related to leaf rolling degree, stomatal conductance, and stomatal closure rate. Meanwhile, differences in leaf rolling and stomatal behavior of rice were analyzed, revealing gene regions of value or potential value in rice drought resistance breeding. Ranjan et al.［56］ constructed 190 recombinant F7 inbred lines and identified QTLs that control the relative content of chlorophyll, chlorophyll A and proline content, qRCC1.1, qCHLa1.1 and qPRO3.1, which will provide reference for improving rice drought resistance by molecular marker-assisted breeding.  Yang et al.［57］ obtained 135 DH lines using typical indica rice IR64 and japonica rice Azucena as parents, and detected a QTL related to chlorophyll content in leaves (qChl3), a QTL related to stomatal resistance in leaves (qRs3), and QTLs (qT1, qT8, qT9, qT11) related to leaf temperature. Wang et al.［58］ locate two loci related to the relative water content of leaves using an F2 population. Pan et al.［59］ located five loci related to leaf water potential in rice tillering stage. The QTLs for physiological traits related to drought resistance are shown in Table 3.

Research Progress on Cloning and Genetic Expression of Drought Resistance Genes

The premise of gene cloning is to locate drought-resistance-related QTLs and inducible genes, understand the signal transduction and functional expression of drought-response genes, and conduct transgenic research after gene cloning［60］. To date, many transgenic rice plants have shown strong drought resistance. Transgenic drought-resistant rice typically utilizes protein-coding genes that control drought regulatory networks to modulate the effects of drought stress. These proteins include transcription factors, protein kinases, receptor-like kinases, enzymes involved in the synthesis of osmoprotectants or plant hormones, and other regulatory or functional proteins［61］. DRO1 is the first drought resistance gene cloned based on map-based cloning technology under drought stress. DRO1 is negatively regulated by auxin and participates in the elongation of root tip cells to improve the drought resistance of rice［49］. Qin et al.［62］ showed that rice drought-responsive gene OsPUB67 encodes a U-boxE3 ubiquitin ligase. Overexpression of OsPUB67 could enhance the scavenging ability of reactive oxygen species and stomatal closure, and played a role under drought stress. Cai et al.［63］ found that the OsABA8ox3 gene not only responded to drought stress, but also responded to seed germination and post-germination growth. Reducing the expression of OsABA8ox3 could enhance the drought resistance of rice. Joo et al.［64］ transformed OsSCE1 and OsSCE3 into rice, and concluded that overexpression of OsSCE3 gene could improve drought resistance. OsSCE1 is mainly involved in carbohydrate metabolism, while OsSCE3 is mainly involved in drought stress response. Kaur et al.［65］ cloned the OsMIZ1 gene from five rice varieties, and their studies showed that it was involved in the drought stress mechanism and was gene-specific. Liu et al.［66］ constructed a TAC library of wild medicinal rice and transferred favorable genes combining specific cloning screening and genetic transformation methods, which is beneficial to the breeding of drought-tolerant rice. Some drought response-related genes OsDSR-1, OsERF65, OsAHL1, and OsUGT55, have also been discovered and cloned one after another, but the genes that are actually used in rice production are still very few.

Discussion and Prospect

In recent years, although some progress has been made in rice drought resistance research at home and abroad, the research progress of using QTL mapping for variety breeding is slow.

One is that mapping of QTLs related to drought resistance is difficult to apply in actual breeding work. The same population has different numbers and effects of QTLs detected in different environments, and there may be epistatic effects or interaction effects between loci of various traits and between loci and environments［67］. There are many QTLs mapped for rice drought resistance-related traits, and some QTLs have been verified and cloned, but their molecular genetic mechanism has not been clearly explored. The functional studies of drought response and tolerance-related genes are lagging behind, and the currently known genes do not provide practical solutions to drought problems［68］. Molecular marker technology and genomics methods have brought new approaches to rice breeding. It is necessary to strengthen the positioning of favorable genes for drought resistance, high yield and high quality, and to dig deep into the genetic mechanism of the genes, so as to enrich the resources of rice drought resistance genes and promote the in-depth transgenic research of rice drought resistance［69］. Using CRISPR/Cas9 technology for editing and cloning drought-tolerant genes in rice will be an ideal choice for breeding drought-tolerant rice varieties.

The second is that most of breeders’ studies take high yield as the main goal. Rice drought resistance breeding is mainly based on yield indexes for field selection, and lack comprehensive consideration of yield, quality, resistance and other factors［11］. QTL combinations should be optimized under different genetic backgrounds to realize QTL recombination and breed varieties with excellent comprehensive traits under drought conditions［70］.

The third is that the drought resistance evaluation system and the QTLs data system are not perfect. The duration and intensity of drought stress is dynamic and highly unpredictable. The genetic basis of drought resistance is complex, and the genetic variation of yield and its related traits under drought conditions is very small, and the drought resistance mechanism also differs among varieties, regions and stages. Many scholars have proposed different identification indexes of drought resistance, which complicates the evaluation of drought resistance. Therefore, it is necessary to establish a systematic identification standard for drought resistance, and to construct a comprehensive evaluation system for screening in terms of yield, morphology, physiology and biochemistry［71］. A simple reproducible drought resistance screening technology capable of predicting target environments should be developed to classify the types of drought and use irrigation systems and remote sensing technology to monitor field soil moisture status［14］. Meanwhile, a QTL data system can be established to integrate the information of various high-quality drought resistance genes and molecular markers, so as to establish a foundation for the application of molecular marker-assisted breeding.

Breeders have bred a variety of new high-yielding and high-quality rice varieties through hybrid breeding, mutation breeding, haploid breeding, polyploid breeding, and cell engineering breeding. However, there are limitations due to regional climate conditions and other conditions.  The ultimate goal of rice breeding for drought resistance is to increase the drought resistance of rice on the basis of maintaining the original comprehensive traits such as high yield and disease resistance. In recent years, the development of functional genomics has made it possible to perform high-throughput crop genome analysis［73］. Combining traditional breeding methods with molecular and genomic approaches can unearth favorable drought resistance genes, enabling breeders to achieve high-precision crop trait improvement in a short period of time. Rice gene sequencing and gene cloning will help to better understand the genetic basis of drought resistance, improve the use efficiency of drought resistance genes in drought resistance genetics and breeding, and promote the application and development of molecular marker-assisted breeding technology and transgenic technology in crop drought resistance breeding［74］.

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