Cotton (Gossypium spp.) is one of the major fibre crops of the world. Although it is classified as salt tolerant crop, cotton growth and productivity are adversely affected by high salinity, especially at germination and seedling stages. Identification of genes and miRNAs responsible for salt tolerance in upland cotton (Gossypium hirsutum L.) would help reveal the molecular mechanisms of salt tolerance. We performed physiological experiments and transcriptome sequencing (mRNA-seq and small RNA-seq) of cotton leaves under salt stress using Illumina sequencing technology. We investigated two distinct salt stress phases—dehydration (4 h) and ionic stress (osmotic restoration; 24 h)—that were identified by physiological changes of 14-day-old seedlings of two cotton genotypes, one salt tolerant and the other salt sensitive, during a 72-h NaCl exposure. A comparative transcriptomics was used to monitor gene and miRNA differential expression at two time points (4 and 24 h) in leaves of the two cotton genotypes under salinity conditions. The expression patterns of differentially co-expressed unigenes were divided into six groups using short time-servies expression miner software. During a 24-h salt exposure, 819 transcription factor unigenes were differentially expressed in both genotypes, with 129 unigenes specifically expressed in the salt-tolerant genotype. Under salt stress, 108 conserved miRNAs from known families were differentially expressed at two time points in the salt-tolerant genotype. We further analyzed the predicted target genes of these miRNAs along with the transcriptome for each time point. Important expressed genes encoding membrane receptors, transporters, and pathways involved in biosynthesis and signal transduction of calcium-dependent protein kinase, mitogen-activated protein kinase, and hormones (abscisic acid and ethylene) were up-regulated. We also analyzed the salt stress response of some key miRNAs and their target genes and found that the expressions of five of nine target genes exhibited significant inverse correlations with their corresponding miRNAs. On the basis of these results, we constructed molecular regulatory pathways and a potential regulatory network for these salt-responsive miRNAs. Our comprehensive transcriptome analysis has provided new insights into salt-stress response of upland cotton. The results should contribute to the development of genetically modified cotton with salt tolerance.

Simple sequence repeats (SSRs) have been widely used in maize genetics and breeding, because they are co-dominant, easy to score, and highly abundant. In this study, we used whole-genome sequences from 16 maize inbreds and 1 wild relative to determine SSR abundance and to develop a set of high-density polymorphic SSR markers. A total of 264 658 SSRs were identified across the 17 genomes, with an average of 135 693 SSRs per genome. Marker density was one SSR every of 15.48 kb. (C/G)n, (AT)n, (CAG/CTG)n, and (AAAT/ATTT)n were the most frequent motifs for mono, di-, tri-, and tetra-nucleotide SSRs, respectively. SSRs were most abundant in intergenic region and least frequent in untranslated regions, as revealed by comparing SSR distributions of three representative resequenced genomes. Comparing SSR sequences and e-polymerase chain reaction analysis among the 17 tested genomes created a new database, including 111 887 SSRs, that could be develop as polymorphic markers in silico. Among these markers, 58.00, 26.09, 7.20, 3.00, 3.93, and 1.78% of them had mono, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs, respectively. Polymorphic information content for 35 573 polymorphic SSRs out of 111 887 loci varied from 0.05 to 0.83, with an average of 0.31 in the 17 tested genomes. Experimental validation of polymorphic SSR markers showed that over 70% of the primer pairs could generate the target bands with length polymorphism, and these markers would be very powerful when they are used for genetic populations derived from various types of maize germplasms that were sampled for this study.

The capacity for ion compartmentalization among different tissues and cells is the key mechanism regulating salt tolerance in plants. In this study, we investigated the ion compartmentalization capacity of two upland cotton genotypes with different salt tolerances under salt shock at the tissue, cell and molecular levels. We found that the leaf glandular trichome could secrete more salt ions in the salt-tolerant genotype than in the sensitive genotype, demonstrating the excretion of ions from tissue may be a new mechanism to respond to short-term salt shock. Furthermore, an investigation of the ion distribution demonstrated that the ion content was significantly lower in critical tissues and cells of the salt-tolerant genotype, indicating the salt-tolerant genotype had a greater capacity for ion compartmentalization in the shoot. By comparing the membrane H⁺-ATPase activity and the expression of ion transportation-related genes, we found that the H⁺-ATPase activity and Na⁺/H⁺antiporter are the key factors determining the capacity for ion compartmentalization in leaves, which might further determine the salt tolerance of cotton. The novel function of the glandular trichome and the comparison of Na⁺compartmentalization between two cotton genotypes with contrasting salt tolerances provide a new understanding of the salt tolerance mechanism in cotton.

Drought stress is one of the major limiting factors for maize production. With the availability of maize B73 reference genome and whole-genome resequencing of 15 maize inbreds, common variants (CV) and clustering analyses were applied to identify non-synonymous SNPs (nsSNPs) and corresponding candidate genes for drought tolerance. A total of 524 nsSNPs that were associated with 271 candidate genes involved in plant hormone regulation, carbohydrate and sugar metabolism, signaling molecules regulation, redox reaction and acclimation of photosynthesis to environment were detected by CV and cluster analyses. Most of the nsSNPs identified were clustered in bin 1.07 region that harbored six previously reported QTL with relatively high phenotypic variation explained for drought tolerance. Genes Ontology (GO) analysis of candidate genes revealed that there were 35 GO terms related to biotic stimulus and membrane-bounded organelle, showing significant differences between the candidate genes and the reference B73 background. Changes of expression level in these candidate genes for drought tolerance were detected using RNA sequencing for fertilized ovary, basal leaf meristem tissue and roots collected under drought stressed and well-watered conditions. The results indicated that 70% of candidate genes showed significantly expression changes under two water treatments and our strategies for mining candidate genes are feasible and relatively efficient. Our results successfully revealed candidate nsSNPs and associated genes for drought tolerance by comparative sequence analysis of 16 maize inbred lines. Both methods we applied were proved to be efficient for identifying candidate genes for complex traits through the next-generation sequencing technologies (NGS). These selected genes will not only facilitate understanding of genetic basis of drought stress response, but also accelerate genetic improvement through marker-assisted selection in maize.

Summary: Root architecture remodelling is critical for forage moisture in water‐limited soil. DEEPER ROOTING 1 (DRO1) in Oryza, Arabidopsis, and Prunus has been reported to improve drought avoidance by promoting roots to grow downward and acquire water from deeper soil. In the present study, we found that ZmDRO1 responded more strongly to abscisic acid (ABA)/drought induction in Zea mays ssp. mexicana, an ancestral species of cultivated maize, than in B73. It was proposed that this is one of the reasons why Zea mays ssp. mexicana has a more noticeable change in the downward direction angle of the root and fewer biomass penalties under water‐deficient conditions. Thus, a robust, synthetic ABA/drought‐inducible promoter was used to control the expression of ZmDRO1^B73 in Arabidopsis and cultivated maize for drought‐resistant breeding. Interestingly, ABA‐inducible ZmDRO1 promoted a larger downward root angle and improved grain yield by more than 40% under water‐limited conditions. Collectively, these results revealed that different responses to ABA/drought induction of ZmDRO1 confer different drought avoidance abilities, and we demonstrated the application of ZmDRO1 via an ABA‐inducible strategy to alter the root architecture of modern maize to improve drought adaptation in the field.

Although the root system is indispensable for absorption of nutrients and water, it is poorly studied in maize owing to the difficulties of direct measurement of roots. Here, 103 maize lines were used to compare root architectures under well-watered and water-stressed conditions. Significant genetic variation, with medium to high heritability and significant correlations, was observed for root traits. Total root length (TRL) and total root surface area (TSA) had high phenotypical diversity, and TRL was positively correlated with TSA, root volume, and root forks. The first two principal components explained 94.01% and 91.15% of total root variation in well-watered and water-stressed conditions, respectively. Thus, TRL and TSA, major contributors to root variation, can be used as favorable selection criteria at the seedling stage. We found that stiff stalk and non-stiff stalk groups (temperate backgrounds) showed relatively higher mean values for root morphological diversity than the TST group (tropical/subtropical background). Of the tested lines, 7, 42, 45, and 9 were classified as drought sensitive, moderately sensitive, moderately drought tolerant, and highly drought tolerant, respectively. Seven of the 9 extremely drought tolerant lines were from the TST group, suggesting that TST germplasms harbor valuable genetic resources for drought tolerance that could be used in breeding to improve abiotic stress tolerance in maize.