Assessment of molecular markers and marker-assisted selection for drought tolerance in barley(Hordeum vulgare L.)

This review updates the present status of the field of molecular markers and marker-assisted selection(MAS),using the example of drought tolerance in barley.The accuracy of selected quantitative trait loci(QTLs),candidate genes and suggested markers was assessed in the barley genome cv.Morex.Six common strategies are described for molecular marker development,candidate gene identification and verification,and their possible applications in MAS to improve the grain yield and yield components in barley under drought stress.These strategies are based on the following five principles:(1)Molecular markers are designated as genomic‘tags’,and their‘prediction’is strongly dependent on their distance from a candidate gene on genetic or physical maps;(2)plants react differently under favourable and stressful conditions or depending on their stage of development;(3)each candidate gene must be verified by confirming its expression in the relevant conditions,e.g.,drought;(4)the molecular marker identified must be validated for MAS for tolerance to drought stress and improved grain yield;and(5)the small number of molecular markers realized for MAS in breeding,from among the many studies targeting candidate genes,can be explained by the complex nature of drought stress,and multiple stress-responsive genes in each barley genotype that are expressed differentially depending on many other factors.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax(Linum usitatissimum L.)is a versatile crop and its seeds are a major source of unsaturated fatty acids.Stearoyl-acyl carrier protein desaturase(SAD)is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses.However,the function of SAD orthologs from L.usitatissimum has not been assessed.Here,we found that two LuSAD genes,LuSAD1 and LuSAD2,are present in the genome of L.usitatissimum cultivar‘Longya 10’.Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds.Interestingly,ectopic expression of LuSAD2 in A.thaliana caused altered plant architecture.Similarly,the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds.Furthermore,we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels,as well as reducing membrane damage.These findings not only broaden our knowledge of the LuSAD functions in plants,but also offer promising targets for improving the quantity and quality of oil,and the abiotic stress tolerance of oil-producing crops,through molecular manipulation.

Overexpression of the peroxidase gene ZmPRX1 increases maize seedling drought tolerance by promoting root development and lignification

Drought is a main abiotic stress factor hindering plant growth,development,and crop productivity.Therefore,it is crucial to understand the mechanisms by which plants cope with drought stress.Here,the function of the maize peroxidase gene ZmPRX1 in drought stress tolerance was investigated by measurement of its expression in response to drought treatment both in a ZmPRX1 overexpression line and a mutant line.The higher root lignin accumulation and seedling survival rate of the overexpression line than that of the wild type or mutant support a role for ZmPRX1 in maize drought tolerance by regulating root development and lignification.Additionally,yeast one-hybrid,Dule luciferase and ChIP-qPCR assays showed that ZmPRX1 is negatively regulated by a nuclear-localized ZmWRKY86 transcription factor.The gene could potentially be used for breeding of drought-tolerant cultivars.

Morphological and Molecular Characterizations of Country Bean (Lablab purpureus L.) Genotypes for Drought Tolerance

Country bean, Lablab purpureus (L.) is considered one of the most important leguminous crops, but their cultivation under drought stress condition encounters challenges. In this study, an experiment has been conducted among 30 genotypes under drought condition to explore morphological diversity of qualitative and quantitative, biochemical, molecular analysis. The study identified significant variations in eight traits among the genotypes examined, with phenotypic variance exceeding genotypic variance, indicating both genetic and environmental influences. High heritability and genetic advance were observed in primary, secondary, and tertiary branch lengths, suggesting these traits are likely controlled by additive gene effects, making them effective targets for selection. Principal component analysis identified three components that made a substantial contribution, accounting for approximately 73.06% of the overall quantitative variations. Among the quantitative traits, the highest coefficient of variation (CV%) has been found in number of flowers (55.05%). While number of primary branches, primary branch length, number of secondary branches, secondary branch length, number of tertiary branches, tertiary branch length has individually more than 20% of CV%. The genotypes have been grouped into three clusters based on quantitative traits. Analysis of protein reveals that the genotypes of DS28 and DS29 have higher protein content than other genotypes. Dehydrogenase responsive genotypes have been found on DS28 and DS29 from the molecular analysis. The results suggest that the genotypes DS28 and DS29 could contribute as genetic resource of high protein content and DREB responsive, and the eight quantitative traits of 30 genotypes could be used for further breeding programme.

Evaluation of Chlorophyll Content and Fluorescence Parameters as Indicators of Drought Tolerance in Barley

Drought is a major abiotic stress that severely affects food production worldwide. Agronomic and physiological traits associated with drought tolerance are suitable indicators for selection of drought tolerance genotypes to reduce the impact of water deficit on crop yield in breeding program. The objective of this study was to identify indicators related to drought tolerance through analysis of photosynthetic traits in barley （Hordeum vulgare L.）. These traits included chlorophyll content, initial fluorescence （Fo）, maximum primary yield of photochemistry of photosystem Ⅱ （Fv /Fo） and maximum quantum yield of photosystem Ⅱ （Fv/Fm）. Four genotypes （Tadmor, Arta, Morocco9-75 and WI2291） variable in drought tolerance were used to investigate the correlation between these traits and drought tolerance. The results reflected that all of these traits were affected negatively in the four genotypes at different levels of post-anthesis drought stress, but the decrease in drought tolerant genotypes was much less than that of drought sensitive genotypes. The results further revealed that the components of the photosynthetic apparatus could be damaged significantly in drought sensitive genotypes, while drought tolerant genotypes were relatively less affected. On the other hand, the values of chlorophyll content, Fo, Fv/Fo and Fv/Fm in drought tolerance genotypes were significantly higher than those in drought sensitive genotypes under drought stress. It was concluded that chlorophyll content, Fo, Fv/Fo and Fv/Fm could be considered as reliable indicators in screening barley germplasm for drought tolerance.

Study on Change Laws of Cold Tolerance in Wheat under Drought Stress

In order to study the change laws of cold tolerance in wheat under drought stress, 11 wheat varieties planted in Huang-Huai wheat area and one variety representative in middle-lower Yangtze River wheat area were selected, and the their change laws of cold tolerance under normal moisture condition and drought stress condition. The results showed that under drought condition, the cold tolerance of wheat in various growth stages was remarkably lower than that under nondrought condition, and the decreasing amplitudes were the largest in overwintering stage with an average of 4.91 ℃, the smallest in regreening stage, and in the middle in the jointing stage. Under non-drought condition, the cold tolerance of semi- winter varieties in various growth stages was better than that of spring varieties, there were significant differences between different varieties, and the cold tolerance of all varieties in overwintering stage was improved compared with before winter, and rapidly decreased after overwintering. Semiwinter varieties showed cold tolerance remarkably enhanced in overwintering stage, and the cold tolerance of spring varieties was also improved to a certain degree.

Evaluation of Cold Tolerance of Sugarcane under Drought Frost Condition

[Objective]The aim was to understand the change characteristics of sugarcane traits and evaluate the cold tolerance of sugarcane varieties under the drought and frost conditions.[Method] The experiment was carried out in Ziyuan County,Guangxi Province where the frost occurred often with 21 domestic and abroad sugarcane varieties（elites）.[Result] There were significant changes in the brix,green leaf number and photosynthetic rate of sugarcane before and after light frost and decreased more in cold-sensitive varieties.However,the correlation was only significant between the damage rate of stem length and internode,percentage of green leaves after heavy frost in relation to sugarcane brix and brix changes after light frost.Further analysis showed that the evaluation for cold tolerance of sugarcane would be more simple and reliable with traits of the damage rate of stem length and percentage of upper green leaves.The evaluation for varieties indicated that if it was not carried out for cold-tolerance identification in breeding program,the percentage of sugarcane varieties with good cold-tolerance will be lower than30%,and higher than 60% with poor cold-tolerance in subtropical and tropical regions.[Conclusion] This study had provided theoretical basis for the cold-resistant evaluation of sugar cane and the breeding of the varieties of cold-resistant.

Association Mapping for Drought Tolerance of Rice (Oryza sativa L.) at Vegetative Stage

[Objective] This study aimed to identify possible quantitative trait loci (QTL) for vegetative drought tolerance traits of rice (Oryza sativa L.). [Method] A total of 184 rice genotypes were field screened and 156 SSR markers randomly distributed at each 3 Mb bin were used through genome-wide scanning method and association analysis to detect QTLs for vegetative drought tolerance traits leaf rolling (LER), leaf drying (LED) and drought recovery rate (DRR). [Result] The experimental rice genotypes showed varied response under vegetative drought, LER, LED and DRR were highly correlated. Population structure was detected at K=3 and K=7, certain extent of admixture existed in the experimental rice genotypes, relative kinship of the rice genotypes ranged from 0 to 0.924 5. Significant linkage disequilibrium among SSR markers was detected. Sixteen SSR markers have been detected to be associated with vegetative drought tolerance traits, four for LER, eight for LED and four for DDR. Most of the markers were associated with more than one trait, indicating a single mechanism might involve in expression of several related traits, i.e. osmotic adjustment. RM107 (Chr.9) was associated with all the three traits and fell exactly within or closely nearby to previous reported regions, was a major QTL for vegetative drought tolerance, RM477 (Chr.8) was significantly associated with DRR and extremely significantly associated with LER was probably another major QTL for vegetative drought tolerance. [Conclusion] Association mapping is a very effective method for describing complex traits like drought tolerance.

Identification of Drought Tolerance in Peanut (Arachis hypogaea) Germplasm at Seedling Stage

[Objective] This study was conducted to identify the drought tolerance of peanut（Arachis hypogaea） seedlings. [Method] The plant height, fresh weight and dry weight of 80 peanut varieties or lines were determined under the drought stress simulated using 15% PEG6000 in laboratory. Relative values of the three indices were calculated to measure the drought tolerance of the 80 peanut varieties or lines. [Result] The growth of peanut seedlings was significantly inhibited after 10 d of drought treatment. Cluster analysis revealed that at Euclidean distance D=15, the80 peanut varieties or lines were divided into four categories A, B, C and D. Among them, category B had better drought tolerance while category C was sensitive to drought. In all the four categories, the approved peanut varieties or lines showed better drought tolerance than the unapproved ones. [Conclusion] The drought tolerant and the drought sensitive peanut varieties or lines screened in this study can be used for further basic research and germplasm improvement.

Grey Correlation Analysis of Physiological Traits Related to Drought Tolerance in Pennisetum sp.

The drought resistance of Pennisetum sp. was analyzed under different drought stress conditions simulated by various concentrations of PEG-6000. Changesofa series of indexes such as chlorophyll content, water content, relative electrical conductivity of osmotic solution, MDA content, proline content, soluble polysaccharide content and soluble protein content in the leaves were measured.The correlation of these indexes with the drought resistance of Pennisetum sp. was an- alyzed by the method of grey correlation analysis. The results showed that the chlorophyll, water content, relative electrical conductivity and soluble protein con- tentexhibited obvious negative correlation with the stress concentration, which de- creasedfollowing the increase of PEG-6000 concentration. The soluble polysaccha- ride contentshowed an increasing trend,the MDA content showed a rise at first fol- lowed by a declinetrend, while the prolinecontent was just opposite to MDA. The changes of the 3 indexes showed obvious positive correlation with thestress con- centration. The correlation of these indexes with the drought resistance of Pennise- tum sp. were ranked as follows： leaf water content （0.980 8）, chlorophyll content （0.967 9）, MDA content （0.876 0）, soluble polysaccharide content （0.839 5）, soluble protein content（0.827 5）, relative electrical conductivity of osmotic solution（0.670 8）, and proline content（0.571 3）.

Comprehensive Evaluation on Drought Tolerance of Widely-used Rice Varieties with Subordinate Function Method

Using drought-tolerant cultivar IAPAR 9 （upland rice from Brazil） and drought-sensitive varieties IR64 as the controls, the drought tolerances of total 26 rice varieties widely used in production were evaluated with subordinate function method based on morphological, physiological and yield traits. The results showed that the trait values of grain yield per plant （GYP）, effective panicles per plant （EPP）, seed-setting rate （SSR）, panicle length （PL）, leaf relative water content （RWC） and chlorophyll content （SPAD） in all tested varieties decreased significantly under the conditions of drought stress. The subordinate function values of these traits of all tested rice varieties were thus calculated varieties. Based on the aver- age subordinate function values of all these traits, total 7 （i.e. Y Liangyou 1） and 3 （i.e. Liangyou Peijiu） rice varieties were identified as drought-tolerant and drought- sensitive varieties, respectively.

Physiological Mechanism for Anthocyanins to Strengthen the Drought Tolerance of Plants

This paper summarized the possible physiological mechanism by which anthocyanins strengthen the tolerance of plants to drought. Drought stress can in-duce plant cel s to synthesize and accumulate anthocyanins. The photochemical properties, subcel ular accumulation sites and spatial distributions in plant organs and tissues of anthocyanins determine their function of strengthening plant tolerance, which is realized by three possible physiological mechanisms： （1） anthocyanins and their chelated metal ions can optimize the osmoregulation ability of the plant cel s by directly acting as the osmoregulation substances of the cel s, （2） anthocyanins with suitable spatial locations can reduce the photoinhibition of the plants under drought stresses, （3） anthocyanins can effectively maintain and improve the active oxygen-scavenging capacity of the plant cel s under drought conditions. Therein, that the anthocyanins enhance the antioxidant capacity of the plant cel s under drought stresses is probably the main reason for the anthocyanins to strengthen the drought tolerance of plants. This review could provide a reference for the mechanism re-search of the drought resistance and the breeding of the drought-resistant cultivars for the plants holding the ability to synthesize and accumulate anthocyanins.

The ZOS7-MYB60 module confers drought-stress tolerance in rice

Shanlan upland rice is an important landrace resource with high drought stress(DS)tolerance.Despite its importance,genes responsible for yield in Shanlan upland rice have yet to be discovered.Our previous study identified a drought-responsive zinc finger protein,ZOS7,as highly expressed in Shanlandao upland rice.However,the function of this gene in controlling drought tolerance remains largely unexplored.In this study,we found that overexpressing ZOS7,a drought-responsive zinc finger protein,in rice increased biomass and yield under drought stress.Co-overexpressing ZOS7 and MYB60,encoding a protein with which ZOS7 interacted,intensified the yield increase.ZOS7 and MYB60 appear to form a module that confers drought tolerance by regulating stomatal density and wax biosynthesis.The ZOS7-MYB60module could be used in molecular breeding for drought tolerance in rice.

Analysis on Drought Tolerance of TaPIMP1 Transgenic Wheat

[Objective] This study aimed to evaluate the application potential of wheat MYB protein-encoding gene TaPIMP1 in the breeding of drought-tolerant wheat germplasm. [Method] The homozygous transgenic lines with TaPIMP1 gene which is driven by ubiquitin promoter and their recipient Yangmai 158 were cultivated under simulated drought condition. Then, seed germination, seedling growth, and some biochemical parameters associated with abiotic stress of the three cultivars were an- alyzed. [Result] In the medium containing 20% PEG6000, the expression level of TaPIMP1 gene in all the three wheat cultivars changed greatly within the first 24 h; the seed germination rate, the lengths of coleoptile and radical of two transgenic lines B64 and B208 were significantly higher than those of control Yangmai 158. In the medium containing 10%-20% PEG6000, the leaf relative water content and sol- uble sugar contents of two TaPIMP1 transgenic lines were significantly higher than those in control. In the medium containing 15%-20% PEG6000, the MDA content of two transgenic lines was significantly lower than that of the control. [Conclusion] The drought tolerance of the two transgenic lines has been improved obviously com- pared with Yangmai 158, so the TaPIMP1 gene can be introduced to develop new drought-tolerant wheat cultivars.

Analysis of Combining Ability of Drought Tolerance in Maize

By using complete-diallel cross design（Griffing method I）, the influence of the combination of different high-yield genotypes of maize on the drought tolerance of their offspring, the general combining ability, the specific combining ability and the back cross effect of drought tolerance between parents and the main genetic parameters for drought tolerance were analyzed. The result indicated that there were significant differences in general combining ability effects（GCA） of maize; there were highly significant differences in special combining ability effects（SCA）; there was no significant difference in reciprocal effects（R）. There were apparent differences in drought tolerance among six parents; to be specific, Zheng 58 had the highest drought tolerance, while PH4CV had the lowest. Improving drought-tolerant parents with Zheng 58, Ji 853 and Xinzi 8717 had gain superiority effects on the increase of drought tolerance in offspring. The influence of the genetic additive effect on the drought tolerance of offsprings varied with different parents and combinations. Therefore, the expression of drought tolerance inheritance genes was determined only by the additive and non-additive genetic effects but had little relationship with reciprocal effects. The selection of drought tolerance of maize should be conducted at higher generations.

Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance

The morphological development of rice(Oryza sativa L.)leaves is closely related to plant architecture,physiological activities,and resistance.However,it is unclear whether there is a co-regulatory relationship between the morphological development of leaves and adaptation to drought environment.In this study,a drought-sensitive,roll-enhanced,and narrow-leaf mutant(renl1)was induced from a semi-rolled leaf mutant(srl1)by ethyl methane sulfonate(EMS),which was obtained from Nipponbare(NPB)through EMS.Map-based cloning and functional validation showed that RENL1 encodes a cellulose synthase,allelic to NRL1/OsCLSD4.The RENL1 mutation resulted in reduced vascular bundles,vesicular cells,cellulose,and hemicellulose contents in cell walls,diminishing the water-holding capacity of leaves.In addition,the root system of the renl1 mutant was poorly developed and its ability to scavenge reactive oxygen species(ROS)was decreased,leading to an increase in ROS after drought stress.Meanwhile,genetic results showed that RENL1 and SRL1 synergistically regulated cell wall components.Our results revealed a theoretical basis for further elucidating the molecular regulation mechanism of cellulose on rice drought tolerance,and provided a new genetic resource for enhancing the synergistic regulation network of plant type and stress resistance,thereby realizing simultaneous improvement of multiple traits in rice.

Piriformospora indica confers drought tolerance on Zea mays L.through enhancement of antioxidant activity and expression of drought-related genes

Drought stress is one of the most severe environmental constraints to plant growth and crop productivity. Plant growth is greatly affected by drought stress, and plants, to survive,adapt to this stress by invoking different pathways. Piriformospora indica, a root-colonizing endophytic fungus of Sebacinales, promotes plant growth and confers resistance to biotic and abiotic stresses, including drought stress, by affecting the physiological properties of the host plant. The fungus strongly colonizes the roots of maize(Zea mays L.) and promotes shoot and root growth under both normal growth conditions and drought stress. We used polyethylene glycol(PEG-6000) to mimic drought stress and found that root fresh and dry weight, leaf area, SPAD value, and leaf number were increased in P. indica-colonized plants.The antioxidative activities of catalases and superoxide dismutases were upregulated within 24h in the leaves of P. indica-colonized plants. Drought-related genes DREB2A, CBL1,ANAC072, and RD29A were upregulated in drought-stressed leaves of P. indica-colonized plants. Furthermore, after drought treatment, proline content increased, whereas accumulation of malondialdehyde(MDA), an indicator of membrane damage, decreased in P. indica-colonized maize. We conclude that P. indica-mediated plant protection against the detrimental effects of drought may result from enhanced antioxidant enzyme activity,proline accumulation, and expression of drought-related genes and lower membrane damage in maize plants.

QTL Mapping for Drought Tolerance at Stages of Germination and Seedling in Wheat (Triticum aestivum L.) Using a DH Population

Drought is a major constraint in many wheat( Triticum aestivum L.) production regions. Quantitative trait loci (QTLs) conditioning drought tolerance at stages of germination and seedling in wheat were identified in a double haploid (DH) population derived from the cross, Hanxuan10×Lumai14, using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Interval mapping analysis revealed that QTLs for drought tolerance at germination stage were located on chromosomes 1B, 2B, 5A, 6B, 7A and 7B, respectively, and the most effective QTL was mapped on chromosome 2B, explaining 27.2% of phenotypic variance. The QTLs for drought tolerance at seedling stage were located on 1B, 3B and 7B, respectively, and the most effective QTL was mapped on chromosome 3B, explaining 21.6% of phenotypic variance. Their positions were different from those of QTLs conferring drought tolerance at germination stage, indicating that drought tolerance at germination stage and seedling stage was controlled by different loci. Most of the identified QTLs explained 18% or more of phenotypic variance for drought tolerance at germination and seedling stage, and would be useful in future for marker assisted selection programs and cultivar improvement.

Drought stress tolerance analysis of Populus ussuriensis clones with different ploidies

The selection of drought-tolerant plants is an important aspect of plant breeding.We studied physiological and biochemical mechanisms of different ploidies of Populus ussuriensis Kom.that relate to drought stress tolerance.We used a 5%(v/v)polyethylene glycol(PEG-6000)solution to simulate drought stress.We recorded leaf phenotypes including color,dry area and curl degree.We evaluated sequential variations in some drought stress tolerance-related physiological and biochemical indices and compared these among diploid clones(CK),triploid clones(T12)and tetraploid clones(F20).T12 leaves exhibited slightly more drought stress damage than CK and F20 leaves.CK leaves suffered the most severe drought stress damage.The physiological and biochemical indices of the different ploidies differed significantly 12 days after drought stress treatment.The activities of superoxide dismutase,peroxidase,catalase and proline in the triploid(T12)leaves were the highest.The relative electric conductivity and malondialdehyde content of T12 leaves were the lowest.The index values of F20 were between those ofthe diploid and triploid.In consideration of these results,the drought resistance of the three different ploidies of P.ussuriensis can be ranked as T12>F20>CK.We speculate that the gene expression patterns of polyploid clones of poplar will change after genome doubling and that some of the drought stress tolerance-related physiological and biochemical indices will be improved,resulting in greater drought tolerance of polyploid clones.

A new method for evaluating the drought tolerance of upland rice cultivars

Worldwide, approximately 27 million ha of rice are grown in upland rather than paddy fields, and is subject to drought stress. To counter this stress, it is desirable to breed new rice cultivars with improved drought tolerance. For breeding purposes, especially for breeding upland rice, it is desirable to develop a simple and accurate method to evaluate rice drought tolerance. We describe a new method that can be used to evaluate efficiently the drought tolerance degree(DTD) of upland rice cultivars, and call it the DTD method.DTD is defined as the mean of the ratios of green leaf length to total leaf length of the top three leaves in every rice seedling after drought treatment, and thus takes values from zero to one. To test whether the DTD method works effectively to evaluate drought tolerance of upland rice cultivars, we determined the DTD values of 13 upland rice cultivars showing varying degrees of drought tolerance in drought-tolerance trials. The idrl-1 mutant, which displayed the strongest drought tolerance of the 13 cultivars as identified by drought-tolerance trials under severe drought stress, had the highest DTD value and297-28, displaying the weakest drought tolerance, had the lowest DTD value. Further analyses of water potential, survival rate, panicles per plant, spikelets per panicle, seed setting rate, yield per plant, and contents of proline, chlorophyll, and malondialdehyde(MDA) indicated that DTD values are in general correlated with the values of these traits,making this new method useful for assessing the drought tolerance of upland rice cultivars.These results show that the DTD method is a simple, direct and relatively accurate evaluation method for drought-tolerance breeding of upland rice.