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Abstract: Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are an important gene regulator, controlling almost all biological and metabolic processes, in both plants and animals. In this study, we investigated the effect of drought and salinity stress on the expression of miRNAs and their targets in cotton (Gossypium hirsutum L.). Our results show that the expression change of miRNAs and their targets were dose-dependent and tissue-dependent under salinity and drought conditions. The expression of miRNAs in leaf was down-regulated under higher salinity stress while shows variable patterns in other conditions. The highest fold-changes of miRNAs were miR398 in roots with 28.9 fold down-regulation under 0.25% NaCl treatment and miR395 in leaves with 7.6 fold down-regulation under 1% PEG treatment. The highest up-regulation of miRNA targets was AST in roots with 4.7 fold-change under 2.5% PEG and the gene with highest down-regulation was CUC1 in leaves with 25.6 fold-change under 0.25% NaCl treatment. Among seven miRNA-target pairs we studied, five pairs, miR156–SPL2, miR162–DCL1, miR159–TCP3, miR395–APS1 and miR396–GRF1, show significant regulation relationship in roots and leaves under salinity stress concentration. [Copyright &y& Elsevier]

Abstract: No study has been performed on identifying microRNAs (miRNAs) and their targets in cotton although cotton is one of the most important fiber and economic crops around the world. In this study, we found 30 potential cotton miRNAs using a comparative genomic approach based on genomic survey sequence analysis and miRNA secondary structure. These cotton miRNAs belong to 22 miRNA families. Expressed sequence tag (EST) analysis indicated that the predicted miRNAs were expressed in cotton plants. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 139 potential miRNA targets were identified in cotton genome. A majority of these targets belong to transcriptional factors which regulate cotton growth and development, including leaf, root, stem, flower, and even fiber development. Those miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and secondary wall synthesis and deposition. Some of the newly identified miRNA targets may be unique to cotton species. In this study, we found that at least 3 miRNA families (miR 396, 414, and 782) target callous synthase, fiber protein Fb23, and fiber quinone-oxidoreductase, suggesting that miRNAs play an important role in cotton fiber differentiation and development. [Copyright &y& Elsevier]

Cotton is one of the most important economic and fiber crops in the world. KNOX is one class of universal transcription factors, which plays important roles in plant growth and development as well as response to different stresses. Although there are many researches on KNOXs in other plant species, there are few reports on cotton. In this study, we systematically and comprehensively identified all KNOX genes in upland cotton and its two ancestral species; we also studied their functions by employing RNA-seq analysis and virus-induced gene silence (VIGS). A total of 89 KNOX genes were identified from three cotton species. Among them, 44 were from upland cotton, 22 and 23 were found in its ancestral species G. raimondii and G. arboreum , respectively. Plant polyploidization and domestication play a selective force driving KNOX gene evolution. Phylogenetic analysis displayed that KNOX genes were evolved into three Classes. The intron length and exon number differed in each Class. Transcriptome data showed that KNOX genes of Class II were widely expressed in multiple tissues, including fiber. The majority of KNOX genes were induced by different abiotic stresses. Additionally, we found multiple cis -elements related to stress in the promoter region of KNOX genes. VIGS silence of GhKNOX4-A and GhKNOX22-D genes showed significant growth and development effect in cotton seedlings under salt and drought treatments. Both GhKNOX4-A and GhKNOX22-D regulated plant tolerance; silencing both genes induced oxidative stresses, evidenced by reduced SOD activity and induced leave cell death, and also enhanced stomatal open and water loss. Thus, GhKNOX4-A and GhKNOX22-D may contribute to drought response by regulating stomata opening and oxidative stresses. • A total of 89 KNOX genes were identified from upland cotton and its two ancestral species • Plant polyploidization and domestication play a selective force driving KNOX gene evolution • VIGS silence of GhKNOX4-A Gh and GhKNOX22-D genes showed significant growth and development effect in cotton seedlings under salt and drought treatment • Silencing GhKNOX4-A and GhKNOX22-D induced oxidative and abiotic stresses • GhKNOX4-A and GhKNOX22-D may contribute to drought response by regulating stomata opening and oxidative stresses [ABSTRACT FROM AUTHOR]

Grafting is a classical technique to increase plant stress resistance and yield in horticultural crops. This review takes cotton (Gossypium spp.) as an example to summarize the application of grafting in sustainable development for the following subjects: (1) cotton production, such as improving stress tolerance, increasing nutrient and water use efficiency, and cultivating cotton perennially for high yield; (2) cotton breeding, such as conserving and propagating special germplasms, utilizing heterosis, breeding new varieties, improving the survival rate of transplanted plantlets, and accelerating virus-induced gene silencing (VIGS); and (3) cotton physiology and molecular biology research, such as the mechanism underlying root-shoot communication. In particular, cotton is a unique non-horticultural field crop, which can grow annually or perennially, depending on winter temperature. Therefore, cotton grafting has some unique aspects, e.g., annual cotton cultivars can be used in perennial cultivation when grafted on cold-resistant rootstocks to improve the over-winter survival abilities, and graft-propagated male sterile plants can be used for multi-year heterosis utilization in near tropics, which could be used to build a sustainable agricultural system. Moreover, future promises associated with grafting have been presented and discussed, such as whether cotton grafting could promote the exchange of genetic material (especially DNA) between rootstock and scion, resulting in stable inheritable variations that remains to be verified by molecular genetics. • Cotton grafting has been widely used to study root-shoot communication mechanism. • Grafting may become a new technology for cotton germplasm innovation. • Grafting can be applied to sustainable production and perennial cultivation. • Grafting is important for reproduction of sterile cotton. [ABSTRACT FROM AUTHOR]

• Around 340 circRNAs were identified from leaf, fiber and ovule in upland cotton. • Single parent expressed circRNAs played an essential role in heterosis. • Thirty-two key circRNAs related to formation of heterosis were identified. • A cotton fiber development related potential ceRNA-PPI network were established. Circular RNA (CircRNA) is a type of single-stranded RNA that plays a vital role in gene regulation during and after transcription. However, the functional role of circRNA in polyploid crop hybridization has not yet been reported. In this study, a high-yield elite upland cotton (Gossypium hirsutum) inbred line, 4133Bt, crossed with a high-quality elite inbred line, J02–247, produced a high -yield and high-quality F 1 hybrid. An RNA-sequence library was constructed from three biological replicates of the parents and F 1 hybrids in the vegetative and reproductive growth periods. A total of 340 high-confidence circRNAs were identified. Our results showed that the circRNAs were more active in ovules than in fiber and leaves and that the host genes of ovule-specific circRNAs were involved in nucleoside metabolic processes. In addition, single parent expressed (SPE) circRNAs and extremely nonadditively expressed (ENAE) circRNAs had different expression patterns in the F 1 hybrid. Moreover, 32 out of the 340 circRNAs were found to be dominant (expression level dominance, ELD) or overdominant (transgressive) at the expression level. These circRNAs might regulate the expression of their host genes through competitive inhibition. Eight of these 32 circRNAs might regulate gene expression through a putative ceRNA network. At 0 days post-anthesis (0 DPA) ovule, five circRNAs were found to interact with six miRNAs that are associated with the regulation of cytoskeleton- and plant cell wall-related genes during the initiation stage of cotton fiber development. Two circRNAs that interacted with two miRNAs to regulate vesicle transport and signal transduction pathways during the fiber elongation stage were identified at 10 DPA (fiber). These results indicate that circRNA may regulate the initiation and elongation stages of cotton fiber via a sponge model during the hybridization process. This study provides a deeper understanding of the molecular mechanism of hybrid breeding from a new perspective. [ABSTRACT FROM AUTHOR]