Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (Paeonia lactiflora Pall.) in Response to Drought Stress

Herbaceous peony(Paeonia lactiflora Pall.),as a high-end cut flower in the international market,has high ornamental and medicinal values.But in Northern China,drought is a major environmental factor influencing the growth and development of P.lactiflora.Quantitative real-time polymerase chain reaction(qRT-PCR)can evaluate gene expression levels under different stress conditions,and stable internal reference is the key for qRT-PCR.At present,there is no systematic screening of internal reference for correcting gene expressions of P.lactiflora in response to drought stress.In this study,10 candidate genes[ubiquitin(UBQ2),UBQ1,elongation factor 1-α(EF-1α),Histidine(His),eukaryotic initiation factor(eIF),tubulin(TUB),actin(ACT),UBQ3,ACT2,RNA polymerase II(RNA Pol II)]were chosen,and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress.Due to the difference of operation methods,the results of different analysis were distinct,and the final comprehensive analysis indicated that EF-1αwas a relatively stable internal reference gene for P.lactiflora under drought stress.Also,UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis.This study will lay a foundation for screening the key genes of P.lactiflora in response to drought stress.

OsMas1,a novel maspardin protein gene,confers tolerance to salt and drought stresses by regulating ABA signaling in rice

Drought and salt stresses,the major environmental abiotic stresses in agriculture worldwide,affect plant growth,crop productivity,and quality.Therefore,developing crops with higher drought and salt tolerance is highly desirable.This study reported the isolation,biological function,and molecular characterization of a novel maspardin gene,OsMas1,from rice.The OsMas1 protein was localized to the cytoplasm.The expression levels of OsMas1 were up-regulated under mannitol,PEG6000,NaCl,and abscisic acid(ABA) treatments in rice.The OsMas1 gene was introduced into the rice cultivar Zhonghua 11(wild type,WT).OsMas1-overexpression(OsMas1-OE) plants exhibited significantly enhanced salt and drought tolerance;in contrast,OsMas1-interference(OsMas1-RNAi) plants exhibited decreased tolerance to salt and drought stresses,compared with WT.OsMas1-OE plants exhibited enhanced hypersensitivity,while OsMas1-RNAi plants showed less sensitivity to exogenous ABA treatment at both germination and post-germination stages.ABA,proline and K+ contents and superoxide dismutase(SOD),catalase(CAT),peroxidase(POD),and photosynthesis activities were significantly increased.In contrast,malonaldehyde(MDA),hydrogen peroxide(H2O2),superoxide anion radical(O2-··),and Na+ contents were significantly decreased in OsMas1-OE plants compared with OsMas1-RNAi and WT plants.Overexpression of OsMas1 up-regulated the genes involved in ABA signaling,proline biosynthesis,reactive oxygen species(ROS)-scavenging system,photosynthesis,and ion transport under salt and drought stresses.Our results indicate that the OsMas1 gene improves salt and drought tolerance in rice,which may serve as a candidate gene for enhancing crop resistance to abiotic stresses.

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.

Biotic and abiotic stress-responsive genes are stimulated to resist drought stress in purple wheat

Triticum aestivum L. cv. Guizi 1(GZ1) is a drought-tolerant local purple wheat cultivar. It is not clear how purple wheat resists drought stress, but it could be related to anthocyanin biosynthesis. In this study, transcriptome data from droughttreated samples and controls were compared. Drought slightly reduced the anthocyanin, protein and starch contents of GZ1 grains and significantly reduced the grain weight. Under drought stress, 16 682 transcripts were reduced, 27 766 differentially expressed genes(DEGs) were identified, and 379 DEGs, including DREBs, were related to defense response. The defense-response genes included response to water deprivation, reactive oxygen, bacteria, fungi, etc. Most of the structural and regulatory genes in anthocyanin biosynthesis were downregulated, with only Ta DFR, Ta OMT, Ta5,3GT, and Ta MYB-4 B1 being upregulated. Ta CHS, Ta F3H, TaCHI, Ta4CL, and TaF3’H are involved in responses to UV, hormones, and stimulus. Ta CHS-2D1, Ta DFR-2D2, Ta DFR-7D, TaOMT-5A, Ta5,3 GT-1B1, Ta5,3GT-3A, and Ta5,3GT-7B1 connect anthocyanin biosynthesis with other pathways, and their interacting proteins are involved in primary metabolism, genetic regulation, growth and development, and defense responses. There is further speculation about the defense-responsive network in purple wheat. The results indicated that biotic and abiotic stress-responsive genes were stimulated to resist drought stress in purple wheat GZ1, and anthocyanin biosynthesis also participated in the drought defense response through several structural genes.

Identification of genes for drought resistance and prediction of gene candidates in soybean seedlings based on linkage and association mapping

Drought is one of the primary abiotic stress factors affecting the yield,growth,and development of soybeans.In extreme cases,drought can reduce yield by more than 50%.The seedling stage is an important determinant of soybean growth:the number and vigor of seedlings will affect growth and yield at harvest.Therefore,it is important to study the drought resistance of soybean seedlings.In this study,a recombinant inbred line(RIL)population comprising 234 F_(6:10)lines(derived from Zhonghuang35×Jindou 21)and a panel of 259 soybean accessions was subjected to drought conditions to identify the effects on phenotypic traits under these conditions.Using a genetic map constructed by single nucleotide polymorphism(SNPs)markers,18 quantitative trait loci(QTL)on 7 soybean chromosomes were identified in two environments.This included 9 QTL clusters identified in the RIL population.Fifty-three QTL were identified in 19 soybean chromosomes by genome-wide association analysis(GWAS)in the panel of accessions,including 69 significant SNPs(-log_(10)(P)≥3.97).A combination of the two populations revealed that two SNPs(-log_(10)(P)≥3.0)fell within two of the QTL(qPH7-4 and qPH7-6)confidence intervals.We not only re-located several previously reported drought-resistance genes in soybean and other crops but also identified several non-synonymous stress-related mutation site differences between the two parents,involving Glyma.07 g093000,Glyma.07 g093200,Glyma.07 g094100 and Glyma.07 g094200.One previously unreported new gene related to drought stress,Glyma.07 g094200,was found by regional association analysis.The significant SNP CHR7-17619(G/T)was within an exon of the Glyma.07 g094200 gene.In the RIL population,the DSP value of the"T"allele of CHR7-17619 was significantly(P<0.05)larger than the"G"allele in different environments.The results of our study lay the groundwork for cloning and molecular marker-assisted selection of droughtresistance genes in soybeans at the seedling stage.

Poaceae Orthologs of Rice OsSGL,DUF1645 Domain-Containing Genes,Positively Regulate Drought Tolerance,Grain Length and Weight in Rice

Grain yield is a polygenic trait that can be influenced by environmental factors and genetic compositions at all plant growth stages.Currently,the molecular mechanisms behind the coordination of the interaction between grain yield-related traits remain unknown.In this study,we characterized the function of four STRESS_tolerance and GRAIN_LENGTH(Os SGL)Poaceae ortholog genes that are transcribed into DUF1645 domain-containing proteins in relation to the grain length,grain weight,and drought stress-tolerance of rice.The transgenic plants with overexpressing or heterologous high levels of Poaceae OsSGL ortholog genes exhibited longer grain size than the wild type plants.Larger cells were seen in panicles of the four transgenic lines with paraffin sectioning and scanning electron microscopy analyses.In addition,four Poaceae OsSGL ortholog genes positively affected the drought tolerance of rice.Four transgenic plants displayed higher resistance to drought stress at the seedling and vegetative stages.RNA-sequencing and qRT-PCR results indicated that over-or heterologous-expression of four Poaceae OsSGL ortholog genes also affected the transcriptome of rice plants.These genes may play a role in auxin and cytokinin biosynthesis and their transduction pathways.Taken together,these results suggested that the four OsSGL orthologs have a conserved function in the regulation of stress-tolerance and cell growth by modulating hormonal biosynthesis and signaling.

Evaluation of Inherited Resistance Genes of Bacterial Leaf Blight, Blast and Drought Tolerance in Improved Rice Lines

Improved rice lines were developed frome three parents with the resistance or tolerance to bacterial leaf blight,blast and drought stress,respectively,using single-,double-and three-way crosses.The improved lines were assessed for agro-morphological and yield traits under non-drought stress(NS)and reproductive-stage drought stress(RS)treatments.The mean comparison of traits measured between parent plants and progenies(improved lines)were similar,and there were significant and non-significant differences among the parents and improved lines(genotypes)under NS and RS.Smilarly,there was significant and non-significant differences in the interaction among both parent varieties and improved lines for NS and RS.Cluster and 3D-model of principal component analysis did not generate categorical clusters according to crossing methods,and there were no exclusive crossing method inclined variations under the treatments.The improved lines were high-yielding,disease resistant,and drought-tolerant compared with their parents.All the crossing methods were good for this crop improvement program without preference to any,despite the number of genes introgressed.

Improvement of Upland Rice Variety by Pyramiding Drought Tolerance QTL with Two Major Blast Resistance Genes for Sustainable Rice Production

Varalu is an early maturing rice variety widely grown in the rainfed ecosystem preferred for its grain type and cooking quality.However,the yield of Varalu is substantially low since it is being affected by reproductive drought stress along with the blast disease.The genetic improvement of Varalu was done by introgressing a major yield QTL,qDTY_(12.1),along with two major blast resistance genes i.e.Pi54 and Pi1 through marker-assisted backcross breeding.Both traits were transferred till BC_(2) generation and intercrossing was followed to pyramid the two traits.Stringent foreground selection was carried out using linked markers as well as peak markers(RM28099,RM28130,RM511 and RM28163)for the targeted QTL(qDTY_(12.1)),RM206 for Pi54 and RM224 for Pi1.Extensive background selection was done using genome-wide SSR markers.Six best lines(MSM-36,MSM-49,MSM-53,MSM-57,MSM-60 and MSM-63)having qDTY_(12.1) and two blast resistance genes in homozygous condition with recurrent parent genome of 95.0%-96.5% having minimal linkage drag of about 0.1 to 0.7 Mb were identified.These lines showed yield advantage under drought stress as well as irrigated conditions.MSM-36 showed better performance in the national coordinated trials conducted across India,which indicated that improved lines of Varalu expected to replace Varalu and may have an important role in sustaining rice production.The present study demonstrated the successful marker-assisted pyramiding strategy for introgression of genes/QTLs conferring biotic stress resistance and yield under abiotic stress in rice.

Identification of QTLs and candidate genes for physiological traits associated with drought tolerance in cotton

Background:Cotton is mainly grown for its natural fiber and edible oil.The fiber obtained from cotton is the indispensable raw material for the textile industries.The ever changing climatic condition,threatens cotton production due to a lack of sufficient water for its cultivation.Effects of drought stress are estimated to affect more than 50%of the cotton growing regions.To elucidate the drought tolerance phenomenon in cotton,a backcross population was developed from G.tomentosum,a drought tolerant donor parent and G.hirsutum which is highly susceptible to drought stress.Results:A genetic map of 10888 SNP markers was developed from 200 BC_2F_2 populations.The map spanned 4191.3 centi-Morgan(c M),with an average distance of 0.1047 c M,covering 51%and 49%of At and Dt sub genomes,respectively.Thirty stable Quantitative trait loci(QTLs)were detected,in which more than a half were detected in the At subgenome.Eighty-nine candidate genes were mined within the QTL regions for three traits:cell membrane stability(CMS),saturated leaf weight(SLW)and chlorophyll content.The genes had varied physiochemical properties.A majority of the genes were interrupted by introns,and only 15 genes were intronless,accounting for 17%of the mined genes.The genes were found to be involved molecular function(MF),cellular component(CC)and biological process(BP),which are the main gene ontological(GO)functions.A number of mi RNAs were detected,such as mi R164,which is associated with NAC and MYB genes,with a profound role in enhancing drought tolerance in plants.Through RT-q PCR analysis,5 genes were found to be the key genes involved in enhancing drought tolerance in cotton.Wild cotton harbors a number of favorable alleles,which can be exploited to aid in improving the narrow genetic base of the elite cotton cultivars.The detection of 30 stable QTLs and 89 candidate genes found to be contributed by the donor parent,G.tomentosum,showed the significant genes harbored by the wild progenitors which can be exploited in developing more robust cotton genotypes with diverse tolerance levels to various environmental stresses.Conclusion:This was the first study involving genome wide association mapping for drought tolerance traits in semi wild cotton genotypes.It offers an opportunity for future exploration of these genes in developing highly tolerant cotton cultivars to boost cotton production.

Identification of candidate genes for drought stress tolerance in rice by the integration of a genetic (QTL) map with the rice genome physical map

Genetic improvement for drought stress tolerance in rice involves the quantitative nature of the trait, which reflects the additive effects of several genetic loci throughout the genome. Yield components and related traits under stressed and well-water conditions were assayed in mapping populations derived from crosses of Azucena×IR64 and Azucena×Bala. To find the candidate rice genes underlying Quantitative Trait Loci (QTL) in these populations, we conducted in silico analysis of a candidate region flanked by the genetic markers RM212 and RM319 on chromosome 1, proximal to the semi-dwarf (sd1) locus. A total of 175 annotated genes were identified from this region. These included 48 genes annotated by functional homology to known genes, 23 pseudogenes, 24 ab initio predicted genes supported by an alignment match to an EST (Expressed sequence tag) of unknown function, and 80 hypothetical genes predicted solely by ab initio means. Among these, 16 candidate genes could potentially be involved in drought stress response.

Genome-wide identification and comparative analysis of drought related genes in roots of two maize inbred lines with contrasting drought tolerance by RNA sequencing

Drought is one of the most important abiotic stresses affecting maize growth and development and therefore resulting in yield loss.Thus it is essential to understand molecular mechanisms of drought stress responses in maize for drought tolerance improvement.The root plays a critical role in plants sensing water deficit.In the present study,two maize inbred lines,H082183,a drought-tolerant line,and Lv28,a drought-sensitive line,were grown in the field and treated with different water conditions(moderate drought,severe drought,and well-watered conditions)during vegetative stage.The transcriptomes of their roots were investigated by RNA sequencing.There were 1428 and 512 drought-responsive genes(DRGs)in Lv28,688 and 3363 DRGs in H082183 under moderate drought and severe drought,respectively.A total of 31 Gene Ontology(GO)terms were significantly over-represented in the two lines,13 of which were enriched only in the DRGs of H082183.Based on results of Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis,"plant hormone signal transduction"and"starch and sucrose metabolism"were enriched in both of the two lines,while"phenylpropanoid biosynthesis"was only enriched in H082183.Further analysis revealed the different expression patterns of genes related to abscisic acid(ABA)signal pathway,trehalose biosynthesis,reactive oxygen scavenging,and transcription factors might contribute to drought tolerance in maize.Our results contribute to illustrating drought-responsive molecular mechanisms and providing gene resources for maize drought improvement.

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship(R^2=0.7763, P<0.05). This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

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.

Drought-responsive genes expressed predominantly in root tissues are enriched with homotypic cis-regulatory clusters in promoters of major cereal crops

The root appears to be the most relevant organ for breeding drought stress tolerance.However, our knowledge about temporal and spatial regulation of drought-associated genes in the root remains fragmented, especially in crop plants. We performed a meta-analysis of expression divergence of essential drought-inducible genes and analyzed their association with cis-elements in model crops and major cereal crops. Our analysis of42 selected drought-inducible genes revealed that these are expressed primarily in roots,followed by shoot, leaf, and inflorescence tissues, especially in wheat. Quantitative real-time RT-PCR analysis confirmed higher expression of TaDREB2 and TaAQP7 in roots,correlated with extensive rooting and drought-stress tolerance in wheat. A promoter scan up to 2 kb upstream of the translation start site using phylogenetic footprinting revealed708 transcription factor binding sites, including drought response elements(DREs), auxin response elements(Aux REs), MYCREs/MYBREs, ABAREs, and ERD1 in 19 selected genes.Interestingly, these elements were organized into clusters of overlapping transcription factor binding sites known as homotypic clusters(HCTs), which modulate drought physiology in plants. Taken together, these results revealed the expression preeminence of major drought-inducible genes in the root, suggesting its crucial role in drought adaptation. The occurrence of HCTs in drought-inducible genes highlights the putative evolutionary modifications of crop plants in developing drought adaptation. We propose that these DNA motifs can be used as molecular markers for breeding drought-resilient cultivars, particularly in the cereal crops.

Selection and Verification of Reference Genes for qRT-PCR Analysis in Iris domestica under Drought

Iris domestica is a plant of the Iridaceae family and is drought-tolerant,but its drought-resistance mechanism is not yet clear.Analysing the gene expression changes of I.domestica by qRT-PCR is an important mean to understand its drought resistance characteristics.Nevertheless,a lack of reference genes greatly hinders investigation and research on the adaptation of I.domestica to drought at the molecular and genetic levels.In this study,we assessed the expression stability of 11 candidate gene in I.domestica under drought stress conditions and different tissues using geNorm,NormFinder,BestKeeper and RefFinder tools.The results showed that EF1βwas the most stable reference genes under drought stress and in different tissues.To validate further the stability of the identified reference genes,the expression patterns of VP gene in I.domestica was analysed.These results will be conducive to more accurate quantification of gene expression levels in I.domestica.

Genome wide identification and characterization of MAPK genes reveals their potential in enhancing drought and salt stress tolerance in Gossypium hirsutum

Background:The cotton crop is universally considered as protein and edible oil source besides the major contributor of natural fiber and is grown in tropical and subtropical regions around the world Unpredicted environmental stresses are becoming significant threats to sustainable cotton production,ultimately leading to a substantial irreversible economic loss.Mitogen-activated protein kinase(MAPK)is generally considered essential for recognizing environmental stresses through phosphorylating downstream signal pathways and plays a vital role in numerous biological processes.Results:We have identified 74 MAPK genes across cotton,41 from G.hirsutum,19 from G.raimondii,whereas 14 have been identified from G.arboreum.The MAPK gene-proteins have been further studied to determine their physicochemical characteristics and other essential features.In this perspective,characterization,phylogenetic relationship,chromosomal mapping,gene motif,cis-regulatory element,and subcellular localization were carried out.Based on phylogenetic analysis,the MAPK family in cotton is usually categorized as A,B,C,D,and E clade.According to the results of the phylogenic relationship,cotton has more MAPKS genes in Clade A than Clade B.The cis-elements identified were classified into five groups(hormone responsiveness,light responsiveness,stress responsiveness,cellular development,and binding site).The prevalence of such elements across the promoter region of these genes signifies their role in the growth and development of plants.Seven GHMAPK genes(GH_A07G1527,GH_D02G1138,GH_D03G0121,GH_D03G1517,GH_D05G1003,GH_D11G0040,and GH_D12G2528)were selected,and specific tissue expression and profiling were performed across drought and salt stresses.Results expressed that six genes were upregulated under drought treatment except for GH_D11G0040 which is downregulated.Whereas all the seven genes have been upregulated at various hours of salt stress treatment.Conclusions:RNA sequence and qPCR results showed that genes as differentially expressed across both vegetative and reproductive plant parts.Similarly,the qPCR analysis showed that six genes had been upregulated substantially through drought treatment while all the seven genes were upregulated across salt treatments.The results of this study showed that cotton GHMPK3 genes play an important role in improving cotton resistance to drought and salt stresses.MAPKs are thought to play a significant regulatory function in plants’responses to abiotic stresses according to various studies.MAPKs’involvement in abiotic stress signaling and innovation is a key goal for crop species research,especially in crop breeding.

Characters and structures of the nucleobase-ascorbate transporters(NAT)family genes in Gossypium hirsutum and their roles in responding to salt and drought stresses

Background: Nucleobase-ascorbate transporters(NAT), synonymously called nucleobase-cation symporter 2(NCS2) proteins, were earlier reported to be involved in plant growth, development and resistance to stress. Previous studies concluded that s a polymorphic SNP associated with NAT12 was significant di erent between salt-tolerant and salt-sensitive materials of upland cotton. In current study, a comprehensive analysis of NAT family genes was conducted for the first time in cotton.Results: In this study, we discovered 32, 32, 18, and 16 NAT genes in Gossypium hirsutum, G. barbadense, G. raimondii and G. arboreum, respectively, which were classified into four groups(groups I–IV) based on the multiple sequence analysis. These GhNAT genes were unevenly distributed on At and Dt sub-genome in G. hirsutum. Most GhNAT members in the same group had similar gene structure characteristics and motif composition. The collinearity analysis revealed segmental duplication as well as tandem duplication contributing to the expansion of the GhNATs. The analysis of cis-acting regulatory elements of GhNATs showed that the function of GhNAT genes in cotton might be related to plant hormone and stress response. Under di erent conditions, the expression levels further suggested the GhNAT family genes were associated with plant response to various abiotic stresses. GhNAT12 was detected in the plasma membrane. And it was validated that the GhNAT12 gene played an important role in regulating cotton resistance to salt and drought stress through the virus-induced gene silencing(VIGS) analysis.Conclusions: A comprehensive analysis of NAT gene family was performed in cotton, including phylogenetic analysis, chromosomal location, collinearity analysis, motifs, gene structure and so on. Our results will further broaden the insight into the evolution and potential functions of NAT genes in cotton. Current findings could make significant contribution towards screening more candidate genes related to biotic and abiotic resistance for the improvement in cotton.

Genome wide identification and characterization of light-harvesting Chloro a/b binding(LHC)genes reveals their potential role in enhancing drought tolerance in Gossypium hirsutum

Background: Cotton is an important commercial crop for being a valuable source of natural fiber.Its production has undergone a sharp decline because of abiotic stresses,etc.Drought is one of the major abiotic stress causing significant yield losses in cotton.However,plants have evolved self-defense mechanisms to cope abiotic factors like drought,salt,cold,etc.The evolution of stress responsive transcription factors such as the trihelix,a nodule-inception-like protein(NLP),and the late embryogenesis abundant proteins have shown positive response in the resistance improvement to several abiotic stresses.Results: Genome wide identification and characterization of the effects of Light-Harvesting Chloro a/b binding(LHC)genes were carried out in cotton under drought stress conditions.A hundred and nine proteins encoded by the LHC genes were found in the cotton genome,with 55,27,and 27 genes found to be distributed in Gossypium hirsutum,G.arboreum,and G.raimondii,respectively.The proteins encoded by the genes were unevenly distributed on various chromosomes.The Ka/Ks(Non-synonymous substitution rate/Synonymous substitution rate)values were less than one,an indication of negative selection of the gene family.Differential expressions of genes showed that majority of the genes are being highly upregulated in the roots as compared with leaves and stem tissues.Most genes were found to be highly expressed in MR-85,a relative drought tolerant germplasm.Conclusion: The results provide proofs of the possible role of the LHC genes in improving drought stress tolerance,and can be explored by cotton breeders in releasing a more drought tolerant cotton varieties.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Paeonia ostii T.Hong et J.X.Zhang Leaves Exposed to Different Drought Stress Conditions

The definition of relatively stable expressed internal reference genes is essential in both traditional blotting quantification and as a modern data quantitative strategy.Appropriate internal reference genes can accurately standardize the expression abundance of target genes to avoid serious experimental errors.In this study,the expression profiles of ten candidate genes,ACT1,ACT2,GAPDH,eIF1,eIF2,α-TUB,β-TUB,TBP,RNA Pol II and RP II,were calculated for a suitable reference gene selection in Paeonia ostii T.Hong et J.X.Zhang leaves under various drought stress conditions.Data were processed by the four regularly used evaluation software.A comprehensive analysis revealed that RNA Pol II was the most stable gene and eIF2 was the least stable one.In addition,the geNorm program provided the optimal choice of two reference gene combination,RNA Pol II andβ-TUB,for qRT-PCR normalization in P.ostii subjected to different drought stress levels.Our research provided convenience for gene expression analysis in P.ostii under drought stress and promoted research of effective methods to alleviate P.ostii drought stress in the future.

Overexpression of the Suaeda salsa SsNHX1 gene confers enhanced salt and drought tolerance to transgenic Zea mays

Maize is one of the most important crops worldwide, but it suffers from salt stress when grown in saline-alkaline soil. There is therefore an urgent need to improve maize salt tolerance and crop yield. In this study, the SsNHX1 gene of Suaeda salsa, which encodes a vacuolar membrane Na^+/H^+ antiporter, was transformed into the maize inbred line 18-599 by Agrobacterium-mediated transformation. Transgenic maize plants overexpressing the SsNHX1 gene showed less growth retardation when treated with an increasing NaCl gradient of up to 1%, indicating enhanced salt tolerance. The improved salt tolerance of transgenic plants was also demonstrated by a significantly elevated seed germination rate(79%) and a reduction in seminal root length inhibition. Moreover, transgenic plants under salt stress exhibited less physiological damage. SsNHX1-overexpressing transgenic maize accumulated more Na^+ and K^+ than wild-type(WT) plants particularly in the leaves, resulting in a higher ratio of K^+/Na^+ in the leaves under salt stress. This result revealed that the improved salt tolerance of SsNHX1-overexpressing transgenic maize plants was likely attributed to SsNHX1-mediated localization of Na^+ to vacuoles and subsequent maintenance of the cytosolic ionic balance. In addition, SsNHX1 overexpression also improved the drought tolerance of the transgenic maize plants, as rehydrated transgenic plants were restored to normal growth while WT plants did not grow normally after dehydration treatment. Therefore, based on our engineering approach, SsNHX1 represents a promising candidate gene for improving the salt and drought tolerance of maize and other crops.