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Significant differences exist in aroma and taste of different grades of large-leaf black tea. In this study, sensory histology combined with metabolomics were used to investigate the sensory characteristics and phytochemical profiles of different grades of Huangpu black tea (HPBT). Sensory evaluation showed that high grade HPBT had high intensity of pekoe, fresh aroma and umami, with aroma and taste scores declining with decreasing grades. 173 non-volatiles were identified, of which 23 marker metabolites could be used as discrimination of different grades HPBT taste. In addition, 154 volatile compounds were identified in the different grades of HPBT, with 15 compounds as key odorants for distinguishing the aroma of different grades of HPBT. Furthermore, correlation analysis revealed that linalool, geraniol and nonanal contributed to the aroma quality score of HPBT. This study will provide a more comprehensive understanding for processing, quality evaluation and grade evaluation system of large-leaf black tea.

Genetic maps highlight the genome organization and structure but also provide the chance of tagging superior traits for crop improvement through marker-assisted selection. Amino acids are building blocks of proteins and perform crucial function in regulating the signaling of molecules involved in the development and growth of plants. Plant architecture also have an impact on crop productivity. In order to select elite cultivars for breeding and identification of favorable alleles and their functional properties, a deep understanding of genetic architecture and development of genetic map is essential. In present investigation, an interspecific cross of Gossypium barbadense XH-18 × G. darwinii 5-7 was made to develop a genetic map utilizing single sequence repeat markers for the dissection of amino acids involved in genetic architecture of G. barbadense and G. darwinii. We measured chromosomal distribution of 20 amino acids across the whole genome of both species. The map consists of 613 markers spread across all 26 chromosomes, covering 2371.4 cM of cotton genome with an average inter-marker distance of 9.35 cM. The marker number anchored on the chromosomes varied from 5 to 76 with an average of 23.57 on each chromosome. The Dt sub-genome had more markers (83.03%) than the At sub-genome (15.66%). Moreover, the longest chromosome was 143.387 cM, the shortest was 58.430 cM, and the average length was 91.207 cM. The Dt subgenome spans a greater genomic distance than the At subgenome. A sum of 21,035 genes were discovered, covering the complete genome of G. barbadense; G. darwinii and have been found to be involved in tRNA 3'-trailer cleavage, macromolecule modification, peptide deformylase activity, response to biotic stimulus and defense response. The minimum Glutamic acid (Glu), Histidine (His), and Lysine (Lys) were found on Chr.13 (0.00-17.74), Chr.02 (0.00-8.01), and Chr.06 (0.00-17.97), respectively found through chromosomal amino acid dissection. The genome-wide SSR interspecific genetic map of G. barbadense and G. darwinii is first of its kind, and studying chromosomal distribution of amino acids will set a landmark step to dissect the genome structure of G. darwinii.

Abstract Background The diagnostic criteria for abdominal obesity are usually waist circumference or waist-to-hip ratio. The magnitude of the risks for cancers of the digestive system and abdominal obesity is unknown. To assess whether abdominal obesity increases the risk of digestive cancer, we conducted a systematic review and meta-analysis of prospective cohort studies in a database. Methods PubMed, Embase, and Web of Science databases were searched from their inception to December 2022. The 9-star Newcastle Ottawa Scale was used to assess study quality. Pooled relative risks and 95% confidence intervals were calculated using fixed or random effect models respectively. The stability of the results was explored by one-by-one exclusion. Subgroup analysis was conducted to explore sources of heterogeneity. Publication bias was evaluated by Begg’s and Egger’s tests. Results A total of 43 cohort studies were included. There were 42 and 31 studies in the meta-analysis of waist circumference and waist-to-hip ratio on digestive system cancer, respectively. The results of the meta-analysis revealed that the greater waist circumference and waist-to-hip ratio were correlated with increased incidence of digestive system cancers: waist circumference: RR 1.48, 95% CI 1.38-1.59, p < 0.001; waist-to-hip ratio: RR 1.33, 95% CI 1.28-1.38, p = 0.001. Subgroup analysis by cancer type showed that higher WC and WHR would increase the prevalence of LC, PC, GC, EC, and CRC. The sensitivity analysis was conducted by a one-by-one elimination method, and the results of the meta-analysis remained stable. It is proved that the results were robust by the trim-and-fill method. Conclusions There was evidence to suggest that abdominal obesity increased the incidence of digestive cancer, it is necessary to take appropriate measures to reduce abdominal obesity. Waist circumference and waist-to-hip ratio may be better predictors of digestive system cancers. However, the association between waist circumference and digestive system cancer was greater, so more attention should be paid to measuring abdominal obesity with waist circumference.

Yellow tea is a lightly fermented tea with unique sensory qualities and health benefits. However, chemical composition and sensory quality of yellow tea products have rarely been studied. 12 representative yellow teas, which were basically covered the main products of yellow tea, were chosen in this study. Combined analysis of non-targeted/targeted metabolomics and electronic sensor technologies (E-eye, E-nose, E-tongue) revealed the chemical and sensor variation. The results showed that yellow big tea differed greatly from yellow bud teas and yellow little teas, but yellow bud teas could not be effectively distinguished from yellow little teas based on chemical constituents and electronic sensory characteristics. Sensor variation of yellow teas might be attributed to some compounds related to bitterness and aftertaste-bitterness (4′-dehydroxylated gallocatechin-3-O-gallate, dehydrotheasinensin C, myricitin 3-O-galactoside, phloroglucinol), aftertaste-astringency (methyl gallate, 1,5-digalloylglucose, 2,6-digalloylglucose), and sweetness (maltotriose). This study provided a comprehensive understanding of yellow tea on chemical composition and sensory quality.

Abstract Background The cyclic nucleotide-gated channel (CNGC) gene family plays a significant role in the uptake of both essential and toxic cations, and has a role in enhancing tolerance to various forms of abiotic stresses as well as the modulation of the heavy metal toxicity to plant through the absorption of heavy metals. Results A complete genome-wide identification and functional characterization of the cotton CNGC genes was carried out, in which 55, 28, and 29 CNGC genes were identified in Gossypium hirsutum, G. raimondii, and G. arboreum, respectively. The protein encoded by the CNGC genes exhibited GRAVY value below zero, indicating their hydrophilic property. CNGC genes were unevenly distributed in 19 out of 26 chromosomes, in which the highest density were observed on Ah05, with 8 genes. High gene coverage was observed among the diploid cotton species, with CNGC genes mapped on all A chromosomes and on 11 out of 13 of D chromosomes. The majority of CNGC proteins were localized in the endoplasmic reticulum, nucleus, and plasma membrane. Gene expression analysis revealed the up-regulation of Gh_A01G0520 (CNGC4) and Gh_D13G1974 (CNGC5) across various forms of abiotic stresses. Moreover, down-regulation of Gh_A01G0520 (CNGC4) and Gh_D13G1974 (CNGC5) in CNGC s silenced plants caused the significantly reduced ability to tolerate drought and salt stresses. All CNGCs silenced plants were recorded to have significantly low content of antioxidants but relatively higher content of oxidant, including MDA and H2O2. Furthermore, SPAD, CMS (cell membrane stability), ELWL (excised leaf water loss), SDW (shoot dry matter weight), and RDW (root dry matter weight) were all lower in CNGCs silenced plants compared with the wild type plants. Conclusion Significant reduction in antioxidant content and negative effects of physiological and morphological characters in CNGCs silenced plants has revealed the novel role of CNGC genes in enhancing cell integrity under abiotic stress conditions. These results provide vital information that will expand our understanding of the CNGC gene family in cotton and other plants, thus promoting the integration of these genes in the development of the environmental resilient plants.

Abstract Background The development of cotton fiber is regulated by the orchestrated binding of regulatory proteins to cis-regulatory elements associated with developmental genes. The cis–trans regulatory dynamics occurred throughout the course of cotton fiber development are elusive. Here we generated genome-wide high-resolution DNase I hypersensitive sites (DHSs) maps to understand the regulatory mechanisms of cotton ovule and fiber development. Results We generated DNase I hypersensitive site (DHS) profiles from cotton ovules at 0 and 3 days post anthesis (DPA) and fibers at 8, 12, 15, and 18 DPA. We obtained a total of 1185 million reads and identified a total of 199,351 DHSs through ~ 30% unique mapping reads. It should be noted that more than half of DNase-seq reads mapped multiple genome locations and were not analyzed in order to achieve a high specificity of peak profile and to avoid bias from repetitive genomic regions. Distinct chromatin accessibilities were observed in the ovules (0 and 3 DPA) compared to the fiber elongation stages (8, 12, 15, and 18 DPA). Besides, the chromatin accessibility during ovules was particularly elevated in genomic regions enriched with transposable elements (TEs) and genes in TE-enriched regions were involved in ovule cell division. We analyzed cis-regulatory modules and revealed the influence of hormones on fiber development from the regulatory divergence of transcription factor (TF) motifs. Finally, we constructed a reliable regulatory network of TFs related to ovule and fiber development based on chromatin accessibility and gene co-expression network. From this network, we discovered a novel TF, WRKY46, which may shape fiber development by regulating the lignin content. Conclusions Our results not only reveal the contribution of TEs in fiber development, but also predict and validate the TFs related to fiber development, which will benefit the research of cotton fiber molecular breeding.

Abstract Objective: To determine the appropriateness of three widely used formulas estimating 24-h urinary Na (24hUNa) from spot urine samples in the Chinese population. Design: Systematic review and meta-analysis. Setting: Literature review was conducted to identify studies for estimating 24hUNa using the Kawasaki, Tanaka and INTERSALT formulas simultaneously in PubMed, Embase and the Cochrane library databases. The mean difference (MD) and correlation coefficients (r) between measures and estimates from different formulas were assessed. Participants: Information extraction and quality assessment were performed in thirteen studies involving 8369 subjects. Results: Two studies which affected the overall robustness were excluded in the ‘leave-one-out’ sensitivity analyses. Within the final meta-analysis included eleven studies and 7197 participants, 36·07 mmol/d (95 %CI 16·89, 55·25) of MD was observed in the Kawasaki formula, and –19·62 mmol/d (95 %CI –37·37, –1·87) in the Tanaka formula and –35·78 mmol/d (95 %CI –50·76, –20·80) in the INTERSALT formula; a pooled r-Fisher’s Z of 0·39 (95 %CI 0·32, 0·45) in the Kawasaki formula, 0·43 (95 %CI 0·37, 0·49) in the Tanaka formula and 0·36 (95 %CI 0·31, 0·42) in the INTERSALT formula. Subgroup analyses were conducted to explore the possible factors affecting the accuracy of the formula estimation from three mainly aspects: population types, Na intake levels and urine specimen types. Conclusions: The meta-analysis suggested that the Tanaka formula performed a more accurate estimate in Chinese population. Time of collecting spot urine specimens and Na intake level of the sample population might be the main factors affecting the accuracy of the formula estimation.

Abstract Background Plant pollen has diverse morphological characteristics that can be consistently passed down from generation to generation. Information on pollen morphology is thus immensely important for plant classification and identification. In the genus Gossypium, however, in-depth research on pollen morphology is lacking, with only few reports on limited cotton species. To evaluate the diversity of pollen in Gossypium, we therefore conducted a comprehensive analysis of the pollen morphology of 33 cotton species and varieties using scanning electron microscopy. Results The 33 analyzed cotton samples exhibited common pollen morphological features, including spherical shapes, radial symmetry, echination, panporation, and operculation, while the pollen size, spine shape, spine density and length showed distinctive features. Pollen size varied significantly among species, with diameters ranging from 62.43 μm in G. harknessii to 103.41 μm in G. barbadense. The exine had an echinate sculptural texture, and spines were mostly conical or sharply conical but occasionally rod-like. Spine density varied from 173 in G. incanum to 54 in G. gossypioides, while spine length ranged from 3.53 μm in G. herbaceum to 9.47 μm in G. barbadense. In addition, the 33 cotton species and varieties were grouped at a genetic distance of 3.83 into three clusters. Cluster I comprised five allotetraploid AD-genome cotton species, four D-genome species, and one K-genome species. Cluster II included 13 diploid species from A, B, D, E, and G genomes, whereas Cluster III only consisted one E-genome species G. incanum. Conclusions Although pollen characteristics alone are not enough to resolve taxonomic and systematic relationships within the genus Gossypium, our results add to knowledge on palynomorphology and contribute to phenological information on these taxa. Our findings should aid future systematic and phylogenetic studies of the Gossypium genus.

Salinity is a major threat to the yield and productivity of cotton seedlings. In the present study, we developed a BC2F2 population of cotton plants from Gossypium darwinii (5–7) and Gossypium hirsutum (CCRI 12–4) salt-susceptible parents to identify salt-resistant candidate genes. The Illumina HiSeq™ strategy was used with bulked segregant analysis. Salt-resistant and salt-susceptible DNA bulks were pooled by using 30 plants from a BC2F2 population. Next-generation sequencing (NGS) technology was used for the sequencing of parents and both bulks. Four significant genomic regions were identified: the first genomic region was located on chromosome 18 (1.86 Mb), the second and third genomic regions were on chromosome 25 (1.06 Mb and 1.94 Mb, respectively), and the fourth was on chromosome 8 (1.41 Mb). The reads of bulk1 and bulk2 were aligned to the G. darwinii and G. hirsutum genomes, respectively, leading to the identification of 20,664,007 single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels). After the screening, 6,573 polymorphic markers were obtained after filtration of the candidate regions. The SNP indices in resistant and susceptible bulks and Δ(SNP-index) values of resistant and susceptible bulks were measured. Based on the higher Δ(SNP-index) value, six effective polymorphic SNPs were selected in a different chromosome. Six effective SNPs were linked to five candidate genes in four genomic regions. Further validation of these five candidate genes was carried out using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), resulting in an expression profile that showed two highly upregulated genes in the salt-tolerant species G. darwinii, i.e., Gohir.D05G367800 and Gohir.D12G239100; however, the opposite was shown in G. hirsutum, for which all genes, except one, showed partial expression. The results indicated that Gohir.D05G367800 and Gohir.D12G239100 may be salt-tolerant genes. We are confident that this study could be helpful for the cloning, transformation, and development of salt-resistant cotton varieties.

Purple tea, renowned for its anthocyanin content and distinctive purple hue, has gained prominence. The anthocyanin content in purple tea can exceed three times that of traditional green-leaf tea. Purple tea harbors various anthocyanins, implicating intricate pathways of biosynthesis and transcriptional regulation. Concurrently, owing to its distinctive chemical composition, the processing of purple tea may be constrained, potentially influencing the sensory attributes and flavor profile of the tea. The richness of anthocyanins in purple tea has yielded potential health benefits, including antioxidative and anti-cancer properties, rendering purple tea a sought-after commodity in the tea market. However, current research on purple tea remains incomplete, including indistinct networks of anthocyanin biosynthesis and regulatory mechanisms, incomplete chemical characterization, and a need for comprehensive investigations into its biological activities. The limited research foundation has greatly reduced the popularity and consumption of purple tea. This paper aims to provide an overview of recent advancements in the biosynthesis and regulation of anthocyanins, as well as the chemical compositions, processing, and health benefits of purple tea. This review will provide the groundwork for future efforts in the selection and innovation of purple tea germplasm, purple tea processing, and the expansion of the market for purple tea consumption.

Eurotium is the teleomorph genus associated with the section Aspergillus. Eurotium comprises approximately 20 species, which are widely distributed in nature and human environments. Eurotium is usually the key microorganism for the fermentation of traditional food, such as Fuzhuan brick tea, Liupao tea, Meju, and Karebushi; thus, Eurotium is an important fungus in the food industry. Eurotium has been extensively studied because it contains a series of interesting, structurally diverse, and biologically important secondary metabolites, including anthraquinones, benzaldehyde derivatives, and indol diketopiperazine alkaloids. These secondary metabolites have shown multiple biological activities, including antioxidative, antimicrobial, cytotoxic, antitumor, insecticidal, antimalarial, and anti-inflammatory activities. This study presents an up-to-date review of the phytochemistry and biological activities of all Eurotium species. This review will provide recent advances on the secondary metabolites and their bioactivities in the genus Eurotium for the first time and serve as a database for future research and drug development from the genus Eurotium.

ObjectiveWe conducted a meta-analysis to assess the efficacy of prophylactic human papillomavirus (HPV) vaccines against cervical cancer precursors and HPV persistent infection among Asian populations.MethodsRandomized controlled clinical trials conducted in Asian countries were identified from three electronic databases (PubMed, EMBASE and the Cochrane Library). Publication retrieval was performed on September 1, 2022 and only those written in English were included. The data were analyzed with Cochrane Review Manager (version 5.3) and Stata/SE (15.1). Effect sizes were presented as risk ratios (RRs) and 95% confidence intervals (CIs).ResultsTen articles were considered in the meta-analysis, without significant heterogeneity among them. The fixed-effect RRs and 95% CIs for cervical intraepithelial neoplasia grade 1 (CIN1+) and CIN2+ were 0.10 (0.05–0.21) and 0.11 (0.04–0.27), respectively. Positive effect of HPV vaccination on 6- and 12-month persistent infection were observed, with the respective pooled RRs of 0.05 (95% CI: 0.03–0.09) and 0.09 (95% CI: 0.05–0.15). HPV vaccination has a positive effect on the incidence of cytological abnormalities associated with HPV 16/18 (RR, 0.13; 95% CI (0.09–0.20)). Positive effects of HPV vaccination were also observed for HPV 16- and 18-specific immunogenicity (RR, 235.02; 95% CI (82.77–667.31) and RR, 98.24; 95% CI (50.36–191.67), respectively). Females receiving an initial vaccination showed significant decreased incidences of cervical intraepithelial neoplasia, HPV persistent infection and cytological abnormalities and a significantly higher antibody positive conversion rate compared with non-vaccination counterparts.ConclusionProphylactic HPV vaccines are highly efficacious in preventing cervical cancer in Asian females. The government should accelerate the processes of vaccine introduction and vaccination implementation by prioritizing them in public health policies, which should be helpful to enhance Asian females’ awareness of receiving HPV vaccination volitionally.

Abstract Background Cotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally. The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions. However, plants have evolved a number of survival strategies, among them is the induction of various stress-responsive genes such as the ribosomal protein large (RPL) gene. The RPL gene families encode critical proteins, which alleviate the effects of drought and salt stress in plants. In this study, comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses. Results Based on the genome-wide evaluation, 26, 8, and 5 proteins containing the RPL14B domain were identified in Gossypium hirsutum, G. raimondii, and G. arboreum, respectively. Furthermore, through bioinformatics analysis, key cis-regulatory elements related to RPL14B genes were discovered. The Myb binding sites (MBS), abscisic acid-responsive element (ABRE), CAAT-box, TATA box, TGACG-motif, and CGTCA-motif responsive to methyl jasmonate, as well as the TCA-motif responsive to salicylic acid, were identified. Expression analysis revealed a key gene, Gh_D01G0234 (RPL14B), with significantly higher induction levels was further evaluated through a reverse genetic approach. The knockdown of Gh_D01G0234 (RPL14B) significantly affected the performance of cotton seedlings under drought/salt stress conditions, as evidenced by a substantial reduction in various morphological and physiological traits. Moreover, the level of the antioxidant enzyme was significantly reduced in VIGS-plants, while oxidant enzyme levels increased significantly, as demonstrated by the higher malondialdehyde concentration level. Conclusion The results revealed the potential role of the RPL14B gene in promoting the induction of antioxidant enzymes, which are key in oxidizing the various oxidants. The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.

Endophytes are critical for plant growth and health. Tea is an economically important crop in China. However, little is known about the distribution pattern and potential functions of endophytic communities in tea trees. In this study, two genotypes (BXZ and MF) cultivated under the same conditions were selected, and endophytic bacteria and fungi were analyzed through 16S rRNA and ITS high-throughput sequencing technologies, respectively. For endophytic bacteria, root tissues harbored the most diverse endophytes, followed by stems and old leaves, and new leaves possessed the lowest diversity. In contrast, old leave tissues harbored more diverse endophytic fungi than did root and stem tissues. Most of the dominant endophytes showed obvious cultivar and tissue preferences. Tissue type played a more important role in shaping community structure than did cultivar. Nevertheless, some endophytic bacterial groups, which mainly affiliated to Chryseobacterium, Sphingomonas, Rhizobium, Morganella, Methylobacterium and Comamonadaceae, could parasitize different tissues, and the average relative abundance of endophytic bacteria was as high as 72.57%. Some endophytic fungal populations, such as Colletotrichum, Uwebraunia, Cladosporium, and Devriesia, could also parasitize tea, and the relative abundance accounted for approximately 25.70–97.26%. The cooperative relationship between endophytic bacteria and fungi in the new leaves was stronger than that in the old leaves, which can better participate in the metabolism of tea material.

Lint percentage is one of the most essential yield components and an important economic index for cotton planting. Improving lint percentage is an effective way to achieve high-yield in cotton breeding worldwide, especially upland cotton (Gossypium hirsutum L.). However, the genetic basis controlling lint percentage has not yet been systematically understood. Here, we performed a genome-wide association mapping for lint percentage using a natural population consisting of 189 G. hirsutum accessions (188 accessions of G. hirsutum races and one cultivar TM-1). The results showed that 274 single-nucleotide polymorphisms (SNPs) significantly associated with lint percentage were detected, and they were distributed on 24 chromosomes. Forty-five SNPs were detected at least by two models or at least in two environments, and their 5 Mb up- and downstream regions included 584 makers related to lint percentage identified in previous studies. In total, 11 out of 45 SNPs were detected at least in two environments, and their 550 Kb up- and downstream region contained 335 genes. Through RNA sequencing, gene annotation, qRT-PCR, protein–protein interaction analysis, the cis-elements of the promotor region, and related miRNA prediction, Gh_D12G0934 and Gh_A08G0526 were selected as key candidate genes for fiber initiation and elongation, respectively. These excavated SNPs and candidate genes could supplement marker and gene information for deciphering the genetic basis of lint percentage and facilitate high-yield breeding programs of G. hirsutum ultimately.

Abstract 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.

Drought seriously threats the growth and development of Gossypium hirsutum L. To dissect the genetic basis for drought tolerance in the G. hirsutum L. germplasm, a population, consisting of 188 accessions of G. hirsutum races and a cultivar (TM-1), was genotyped using the Cotton80KSNP biochip, and 51,268 high-quality single-nucleotide polymorphisms (SNPs) were obtained. Based on the phenotypic data of eight drought relative traits from four environments, we carried out association mapping with five models using GAPIT software. In total, thirty-six SNPs were detected significantly associated at least in two environments or two models. Among these SNPs, 8 and 28 (including 24 SNPs in 5 peak regions) were distributed in the A and D subgenome, respectively; eight SNPs were found to be distributed within separate genes. An SNP, TM73079, located on chromosome D10, was simultaneously associated with leaf fresh weight, leaf wilted weight, and leaf dry weight. Another nine SNPs, TM47696, TM33865, TM40383, TM10267, TM59672, TM59675, TM59677, TM72359, and TM72361, on chromosomes A13, A10, A12, A5, D6, and D9, were localized within or near previously reported quantitative trait loci for drought tolerance. Moreover, 520 genes located 200 kb up- and down-stream of 36 SNPs were obtained and analyzed based on gene annotation and transcriptome sequencing. The results showed that three candidate genes, Gh_D08G2462, Gh_A03G0043, and Gh_A12G0369, may play important roles in drought tolerance. The current GWAS represents the first investigation into mapping QTL for drought tolerance in G. hirsutum races and provides important information for improving cotton cultivars.

Abstract Background Segregation distortion (SD) is a common phenomenon among stable or segregating populations, and the principle behind it still puzzles many researchers. The F2:3 progenies developed from the wild cotton species of the D genomes were used to investigate the possible plant transcription factors within the segregation distortion regions (SDRs). A consensus map was developed between two maps from the four D genomes, map A derived from F2:3 progenies of Gossypium klotzschianum and G. davidsonii while Map B from G. thurberi and G. trilobum F2:3 generations. In each map, 188 individual plants were used. Results The consensus linkage map had 1 492 markers across the 13 linkage groups with a map size of 1 467.445 cM and an average marker distance of 1.037 0 cM. Chromosome D502 had the highest percentage of SD with 58.6%, followed by Chromosome D507 with 47.9%. Six thousand and thirty-eight genes were mined within the SDRs on chromosome D502 and D507 of the consensus map. Within chromosome D502 and D507, 2 308 and 3 730 genes were mined, respectively, and were found to belong to 1 117 gourp out of which 622 groups were common across the two chromosomes. Moreover, genes within the top 9 groups related to plant resistance genes (R genes), whereas 188 genes encoding protein kinase domain (PF00069) comprised the largest group. Further analysis of the dominant gene group revealed that 287 miRNAs were found to target various genes, such as the gra-miR398, gra-miR5207, miR164a, miR164b, miR164c among others, which have been found to target top-ranked stress-responsive transcription factors such as NAC genes. Moreover, some of the stress- responsive cis-regulatory elements were also detected. Furthermore, RNA profiling of the genes from the dominant family showed that higher numbers of genes were highly upregulated under salt and osmotic stress conditions, and also they were highly expressed at different stages of fiber development. Conclusion The results indicated the critical role of the SDRs in the evolution of the key regulatory genes in plants.

Abstract Background Wild species of cotton are excellent resistance to abiotic stress. Diploid D-genome cotton showed abundant phenotypic diversity and was the putative donor species of allotetraploid cotton which produce the largest textile natural fiber. Results A total of 41,053 genes were expressed in all samples by mapping RNA-seq Illumina reads of G. thurberi (D1), G. klotzschianum (D3-k), G. raimondii (D5) and G. trilobum (D8) to reference genome. The numbers of differently expressed genes (DEGs) were significantly higher under cold stress than salt stress. However, 34.1% DEGs under salt stress were overlapped with cold stress in four species. Notably, a potential shared network (cold and salt response, including 16 genes) was mined out by gene co-expression analysis. A total of 47,180–55,548 unique genes were identified in four diploid species by De novo assembly. Furthermore, 163, 344, 330, and 161 positively selected genes (PSGs) were detected in thurberi, G. klotzschianum, G. raimondii and G. trilobum by evolutionary analysis, respectively, and 9.5–17% PSGs of four species were DEGs in corresponding species under cold or salt stress. What’s more, most of PSGs were enriched GO term related to response to stimulation. G. klotzschianum showed the best tolerance under both cold and salt stress. Interestingly, we found that a RALF-like protein coding gene not only is PSGs of G. klotzschianum, but also belongs to the potential shared network. Conclusion Our study provided new evidence that gene expression variations of evolution by natural selection were essential drivers of the morphological variations related to environmental adaptation during evolution. Additionally, there exist shared regulated networks under cold and salt stress, such as Ca2+ signal transduction and oxidation-reduction mechanisms. Our work establishes a transcriptomic selection mechanism for altering gene expression of the four diploid D-genome cotton and provides available gene resource underlying multi-abiotic resistant cotton breeding strategy.

Abstract 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 10 888 SNP markers was developed from 200 BC2F2 populations. The map spanned 4 191.3 centi-Morgan (cM), with an average distance of 0.104 7 cM, 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 miRNAs were detected, such as miR164, which is associated with NAC and MYB genes, with a profound role in enhancing drought tolerance in plants. Through RT-qPCR 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.

Abstract Background Cotton grows in altering environments that are often unfavorable or stressful for its growth and development. Consequently, the plant must cope with abiotic stresses such as soil salinity, drought, and excessive temperatures. Alkali-salt stress response remains a cumbersome biological process and is regulated via a multifaceted transcriptional regulatory network in cotton. Results To discover the molecular mechanisms of alkali-salt stress response in cotton, a comprehensive transcriptome analysis was carried out after alkali-salt stress treatment in three accessions of Gossypium hirsutum with contrasting phenotype. Expression level analysis proved that alkali-salt stress response presented significant stage-specific and tissue-specific. GO enrichment analysis typically suggested that signal transduction process involved in salt-alkali stress response at SS3 and SS12 stages in leaf; carbohydrate metabolic process and oxidation-reduction process involved in SS48 stages in leaf; the oxidation-reduction process involved at all three phases in the root. The Co-expression analysis suggested a potential GhSOS3/GhCBL10-SOS2 network was involved in salt-alkali stress response. Furthermore, Salt-alkali sensitivity was increased in GhSOS3 and GhCBL10 Virus-induced Gene Silencing (VIGS) plants. Conclusion The findings may facilitate to elucidate the underlying mechanisms of alkali-salt stress response and provide an available resource to scrutinize the role of candidate genes and signaling pathway governing alkali-salt stress response.

Epigallocatechin gallate (EGCG) is an important contributor to bitterness and astringency in summer tea leaves; however, the transcriptional regulatory mechanisms of EGCG biosynthesis remain unclear. In this study, EGCG content was significantly decreased after foliar spraying with nano-Se fertilizers in tea leaves. A WRKY transcription factor (TF), CsWRKY70, was found to be positively related to EGCG content. The open reading frame of CsWRKY70 was 891 bp encoding 296 amino acids. CsWRKY70 is localized to the nucleus and has transcriptional activation activity. The electrophoretic mobility shift assay indicated that CsWRKY70 can directly bind to the promoters of CsLAR and CsUGT84A containing W-box (5′-C/TTGACT/C-3′) sequences. Dual-luciferase reporter experiment verified that CsWRKY70 activated CsLAR and CsUGT84A expressions in tobacco leaves. In summary, these results demonstrated that CsWRKY70 may reduce EGCG biosynthesis by inhibiting the CsLAR and CsUGT84A expressions under nano-Se treatment. Our findings provide new insight into the regulatory mechanism of WRKY TFs involved in catechin biosynthesis and offer a theoretical basis for breeding low or high EGCG content tea cultivars.

Low temperature is a common biological abiotic stress in major cotton-growing areas. Cold stress significantly affects the growth, yield, and yield quality of cotton. Therefore, it is important to develop more robust and cold stress-resilient cotton germplasms. In response to climate change and erratic weather conditions, plants have evolved various survival mechanisms, one of which involves the induction of various stress responsive transcript factors, of which the C-repeat-binding factors (CBFs) have a positive effect in enhancing plants response to cold stress. In this study, genomewide identification and functional characterization of the cotton CBFs were carried out. A total of 29, 28, 25, 21, 30, 26, and 15 proteins encoded by the CBF genes were identified in seven Gossypium species. A phylogenetic evaluation revealed seven clades, with Clades 1 and 6 being the largest. Moreover, the majority of the proteins encoded by the genes were predicted to be located within the nucleus, while some were distributed in other parts of the cell. Based on the transcriptome and RT-qPCR analysis, Gthu17439 (GthCBF4) was highly upregulated and was further validated through forward genetics. The Gthu17439 (GthCBF4) overexpressed plants exhibited significantly higher tolerance to cold stress, as evidenced by the higher germination rate, increased root growth, and high-induction levels of stress-responsive genes. Furthermore, the overexpressed plants under cold stress had significantly reduced oxidative damage due to a reduction in hydrogen peroxide (H2O2) production. Moreover, the overexpressed plants under cold stress had minimal cell damage compared to the wild types, as evidenced by the Trypan and 3,3′-Diaminobenzidine (DAB) staining effect. The results showed that the Gthu17439 (GthCBF4) could be playing a significant role in enhancing cold stress tolerance in cotton and can be further exploited in developing cotton germplasm with improved cold-stress tolerance.

Cotton is an excellent model for studying crop polyploidization and domestication. Chromatin profiling helps to reveal how histone modifications are involved in controlling differential gene expression between A and D subgenomes in allotetraploid cotton. However, the detailed profiling and functional characterization of broad H3K4me3 and H3K27me3 are still understudied in cotton. In this study, we conducted H3K4me3- and H3K27me3-related ChIP-seq followed by comprehensively characterizing their roles in regulating gene transcription in cotton. We found that H3K4me3 and H3K27me3 exhibited active and repressive roles in regulating the expression of genes between A and D subgenomes, respectively. More importantly, H3K4me3 exhibited enrichment level-, position-, and distance-related impacts on expression levels of related genes. Distinct GO term enrichment occurred between A/D-specific and homeologous genes with broad H3K4me3 enrichment in promoters and gene bodies, suggesting that broad H3K4me3-marked genes might have some unique biological functions between A and D subgenome. An anticorrelation between H3K27me3 enrichment and expression levels of homeologous genes was more pronounced in the A subgenome relative to the D subgenome, reflecting distinct enrichment of H3K27me3 in homeologous genes between A and D subgenome. In addition, H3K4me3 and H3K27me3 marks can indirectly influence gene expression through regulatory networks with TF mediation. Thus, our study provides detailed insights into functions of H3K4me3 and H3K27me3 in regulating differential gene expression and subfunctionalization of homeologous genes, therefore serving as a driving force for polyploidization and domestication in cotton.

Drought and low-temperature stresses are the most prominent abiotic stresses affecting cotton. Wild cotton being exposed to harsh environments has more potential to cope with both biotic and abiotic stresses. Exploiting wild cotton material to induce resistant germplasm would be of greater interest. The candidate gene was identified in the BC2F2 population among Gossypium tomentosum and Gossypium hirsutum as wild male donor parent noted for its drought tolerance and the recurrent parent and a high yielding but drought susceptible species by genotyping by sequencing (GBS) mapping. Golden2-like (GLK) gene, which belongs to the GARP family, is a kind of plant-specific transcription factor (TF) that was silenced by virus-induced gene silencing (VIGS). Silencing of GhGLK1 in cotton results in more damage to plants under drought and cold stress as compared with wild type (WT). The overexpression of GhGLK1 in Arabidopsis thaliana showed that the overexpressing plants showed more adaptability than the WT after drought and cold treatments. The results of trypan blue and 3,3′-diaminobenzidine (DAB) staining showed that after drought and cold treatment, the leaf damage in GhGLK1 overexpressed plants was less as compared with the WT, and the ion permeability was also lower. This study suggested that the GhGLK1 gene may be involved in the regulation of drought and cold stress response in cotton. Our current research findings add significantly to the existing knowledge of cold and drought stress tolerance in cotton.

Polyphenol oxidase (PPO) plays a role in stress response, secondary metabolism, and other physiological processes during plant growth and development, and is also a critical enzyme in black tea production. However, the regulatory mechanisms of PPO genes and their activity in tea plants are still unclear. In this study, we measured PPO activity in two different tea cultivars, Taoyuandaye (TYDY) and Bixiangzao (BXZ), which are commonly used to produce black tea and green tea, respectively. The expression pattern of CsPPO1 was assessed and validated via transcriptomics and quantitative polymerase chain reaction in both tea varieties. In addition, we isolated and identified an R2R3-MYB transcription factor CsMYB59 that may regulate CsPPO1 expression. CsMYB59 was found to be a nuclear protein, and its expression in tea leaves was positively correlated with CsPPO1 expression and PPO activity. Transcriptional activity analysis showed that CsMYB59 was a transcriptional activator, and the dual-luciferase assay indicated that CsMYB59 could activate the expression of CsPPO1 in tobacco leaves. In summary, our study demonstrates that CsMYB59 represents a transcriptional activator in tea plants and may mediate the regulation of PPO activity by activating CsPPO1 expression. These findings provide novel insights into the regulatory mechanism of PPO gene in Camellia sinensis, which might help to breed tea cultivars with high PPO activity.

Cotton is one of the most important fiber crops globally. Despite this, various abiotic stresses, including drought, cause yield losses. We used transcriptome profiles to investigate the co-expression patterns of gene networks associated with drought stress tolerance. We identified three gene modules containing 3,567 genes highly associated with drought stress tolerance. Within these modules, we identified 13 hub genes based on intramodular significance, for further validation. The yellow module has five hub genes (Gh_A07G0563, Gh_D05G0221, Gh_A05G3716, Gh_D12G1438, and Gh_D05G0697), the brown module contains three hub genes belonging to the aldehyde dehydrogenase (ALDH) gene family (Gh_A06G1257, Gh_A06G1256, and Gh_D06G1578), and the pink module has five hub genes (Gh_A02G1616, Gh_D12G2599, Gh_D07G2232, Gh_A02G0527, and Gh_D07G0629). Based on RT-qPCR results, the Gh_A06G1257 gene has the highest expression under drought stress in different plant tissues and it might be the true candidate gene linked to drought stress tolerance in cotton. Silencing of Gh_A06G1257 in cotton leaves conferred significant sensitivity in response to drought stress treatments. Overexpression of Gh_A06G1257 in Arabidopsis also confirms its role in drought stress tolerance. L-valine, Glutaric acid, L-proline, L-Glutamic acid, and L-Tryptophan were found to be the most significant metabolites playing roles in drought stress tolerance. These findings add significantly to existing knowledge of drought stress tolerance mechanisms in cotton.

The acyl-coenzyme A oxidase 3 (ACX3) gene involved in the β-oxidation pathway plays a critical role in plant growth and development as well as stress response. Earlier on, studies focused primarily on the role of β-oxidation limited to fatty acid breakdown. However, ACX3 peroxisomal β-oxidation pathways result in a downstream cascade of events that act as a transduction of biochemical and physiological responses to stress. A role that is yet to be studied extensively. In this study, we identified 20, 18, 22, 23, 20, 11, and 9 proteins in Gossypium hirsutum, G. barbadense, G. tomentosum, G. mustelinum, G. darwinii, G. arboretum, and G. raimondii genomes, respectively. The tetraploid cotton genome had protein ranging between 18 and 22, while diploids had between 9 and 11. After analyzing the gene family evolution or selection pressure, we found that this gene family undergoes purely segmental duplication both in diploids and tetraploids. W-Box (WRKY-binding site), ABRE, CAAT–Box, TATA-box, MYB, MBS, LTR, TGACG, and CGTCA-motif are abiotic stress cis-regulatory elements identified in this gene family. All these are the binding sites for abiotic stress transcription factors, indicating that this gene is essential. Genes found in G. hirsutum showed a clear response to drought and salinity stress, with higher expression under drought and salt stress, particularly in the leaf and root, according to expression analysis. We selected Gh_DO1GO186, one of the highly expressed genes, for functional characterization. We functionally characterized the GhACX3 gene through overexpression and virus-induced gene silencing (VIGS). Overexpression of this gene enhanced tolerance under stress, which was exhibited by the germination assay. The overexpressed seed growth rate was faster relative to control under drought and salt stress conditions. The survival rate was also higher in overexpressed plants relative to control plants under stress. In contrast, the silencing of the GhACX3 gene in cotton plants resulted in plants showing the stress susceptibility phenotype and reduced root length compared to control. Biochemical analysis also demonstrated that GhACX3-silenced plants experienced oxidative stress while the overexpressed plants did not. This study has revealed the importance of the ACX3 family during stress tolerance and can breed stress-resilient cultivar.

Abstract Background The efficient detection and initiation of appropriate response to abiotic stresses are important to plants survival. The plant G-protein coupled receptors (GPCRs) are diverse membranous proteins that are responsible for signal transduction. Results In this research work, we identified a novel gene of the GPCR domain, transformed and carried out the functional analysis in Arabidopsis under drought and cold stresses. The transgenic lines exposed to drought and cold stress conditions showed higher germination rate, increased root length and higher fresh biomass accumulation. Besides, the levels of antioxidant enzymes, glutathione (GSH) and ascorbate peroxidase (APX) exhibited continuously increasing trends, with approximately threefold higher than the control, implying that these ROS-scavenging enzymes were responsible for the detoxification of ROS induced by drought and cold stresses. Similarly, the transgenic lines exhibited stable cell membrane stability (CMS), reduced water loss rate in the detached leaves and significant values for the saturated leaves compared to the wild types. Highly stress-responsive miRNAs were found to be targeted by the novel gene and based on GO analysis; the protein encoded by the gene was responsible for maintaining an integral component of membrane. In cotton, the virus-induced gene silencing (VIGS) plants exhibited a higher susceptibility to drought and cold stresses compared to the wild types. Conclusion The novel GPCR gene enhanced drought and cold stress tolerance in transgenic Arabidopsis plants by promoting root growth and induction of ROS scavenging enzymes. The outcome showed that the gene had a role in enhancing drought and cold stress tolerance, and can be further exploited in breeding for more stress-resilient and tolerant crops.

Abstract Background Auxins play an important role in plant growth and development; the auxins responsive gene; auxin/indole-3-acetic acid (Aux/IAA), small auxin-up RNAs (SAUR) and Gretchen Hagen3 (GH3) control their mechanisms. The GH3 genes function in homeostasis by the catalytic activities in auxin conjugation and bounding free indole-3-acetic acid (IAA) to amino acids. Results In our study, we identified the GH3 genes in three cotton species; Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii, analyzed their chromosomal distribution, phylogenetic relationships, cis-regulatory element function and performed virus induced gene silencing of the novel Gh_A08G1120 (GH3.5) gene. The phylogenetic tree showed four clusters of genes with clade 1, 3 and 4 having mainly members of the GH3 of the cotton species while clade 2 was mainly members belonging to Arabidopsis. There were no paralogous genes, and few orthologous genes were observed between Gossypium and other species. All the GO terms were detected, but only 14 genes were found to have described GO terms in upland cotton, more biological functions were detected, as compared to the other functions. The GH3.17 subfamily harbored the highest number of the cis-regulatory elements, most having promoters towards dehydration-responsiveness. The RNA expression analysis revealed that 10 and 8 genes in drought and salinity stress conditions respectively were upregulated in G. hirsutum. All the genes that were upregulated in plants under salt stress conditions were also upregulated in drought stress; moreover, Gh_A08G1120 (GH3.5) exhibited a significant upregulation across the two stress factors. Functional characterization of Gh_A08G1120 (GH3.5) through virus-induced gene silencing (VIGS) revealed that the VIGS plants ability to tolerate drought and salt stresses was significantly reduced compared to the wild types. The chlorophyll content, relative leaf water content (RLWC), and superoxide dismutase (SOD) concentration level were reduced significantly while malondialdehyde concentration and ion leakage as a measure of cell membrane stability (CMS) increased in VIGS plants under drought and salt stress conditions. Conclusion This study revealed the significance of the GH3 genes in enabling the plant’s adaptation to drought and salt stress conditions as evidenced by the VIGS results and RT-qPCR analysis.

Abstract Background Cotton (Gossypium hirsutum) provides the largest natural fiber for the textile manufacturing industries, but its production is on the decline due to the effects of salinity. Soil salt-alkalization leads to damage in cotton growth and a decrease in yields. Hyperosmolality-gated calcium-permeable channels (OSCA) have been found to be involved in the detection of extracellular changes which trigger an increase in cytosolic free calcium concentration. Hyperosmolality-induced calcium ion increases have been widely speculated to be playing a role in osmosensing in plants. However, the molecular nature of the corresponding calcium ion channels remains unclearly. In this research work, we describe the OSCA genes and their putative function in osmosensing in plants by carrying out genome-wide identification, characterization and functional analysis of the significantly up-regulated OSCA gene, GhOSCA1.1 through reverse genetics. Result A total of 35, 21 and 22 OSCA genes were identified in G. hirsutum, G. arboreum, and G. raimondii genomes, respectively, and were classified into four different clades according to their gene structure and phylogenetic relationship. Gene and protein structure analysis indicated that 35 GhOSCA genes contained a conserved RSN1_7TM (PF02714) domain. Moreover, the cis-regulatory element analysis indicated that the OSCA genes were involved in response to abiotic stress. Furthermore, the knockdown of one of the highly up-regulated genes, Gh_OSCA1.1 showed that the virus-induced gene silenced (VIGS) plants were highly sensitive to dehydration and salinity stresses compared with the none VIGS plants as evident with higher concentration levels of oxidant enzymes compared with the antioxidant enzymes on the leaves of the stressed plants. Conclusion This study provides the first systematic analysis of the OSCA gene family and will be important for understanding the putative functions of the proteins encoded by the OSCA genes in cotton. These results provide a new insight of defense responses in general and lay the foundation for further investigation of the molecular role played by the OSCA genes, thereby providing suitable approaches to improve crop performance under salinity and drought stress conditions.

Electronic-cigarette smoke (eCS) has been shown to cause a degree of oxidative stress and inflammatory damage in lung tissue. The aim of this study was to evaluate the repair mechanism of Eurotium cristatum fermented loose dark tea (ECT) and Eurotium cristatum particle metabolites (ECP) sifted from ECT after eCS-induced injury in mice. Sixty C57BL/6 mice were randomly divided into a blank control group, an eCS model group, an eCS + 600 mg/kg ECP treatment group, an eCS + 600 mg/kg ECT treatment group, an eCS + 600 mg/kg ECP prevention group, and an eCS + 600 mg/kg ECT prevention group. The results show that ECP and ECT significantly reduced the eCS-induced oxidative stress and inflammation and improved histopathological changes in the lungs in mice with eCS-induced liver injury. Western blot analysis further revealed that ECP and ECT significantly inhibited the eCS-induced upregulation of the phosphorylation levels of the extracellular Regulated protein Kinases (ERK), c-Jun N-terminal kinase (JNK) and p38mitogen-activated protein kinases (p38MAPK) proteins, and significantly increased the eCS-induced downregulation of the expression levels of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins. Conclusively, these findings show that ECP and ECT have a significant repairing effect on the damage caused by eCS exposure through the MAPK and PXR/AhR signaling pathways; ECT has a better effect on preventing eCS-induced injury and is suitable as a daily healthcare drink; ECP has a better therapeutic effect after eCS-induced injury, and might be a potential therapeutic candidate for the treatment of eCS-induced injury.

Abstract Background Fluorescence in situ hybridization (FISH) is an efficient cytogenetic technology to study chromosome structure. Transposable element (TE) is an important component in eukaryotic genomes and can provide insights in the structure and evolution of eukaryotic genomes. Results A FISH probe derived from bacterial artificial chromosome (BAC) clone 299N22 generated striking signals on all 26 chromosomes of the cotton diploid A genome (AA, 2x=26) but very few on the diploid D genome (DD, 2x=26). All 26 chromosomes of the A sub genome (At) of tetraploid cotton (AADD, 2n=4x=52) also gave positive signals with this FISH probe, whereas very few signals were observed on the D sub genome (Dt). Sequencing and annotation of BAC clone 299N22, revealed a novel Ty3/gypsy transposon family, which was named as ‘CICR’. This family is a significant contributor to size expansion in the A (sub) genome but not in the D (sub) genome. Further FISH analysis with the LTR of CICR as a probe revealed that CICR is lineage-specific, since massive repeats were found in A and B genomic groups, but not in C–G genomic groups within the Gossypium genus. Molecular evolutionary analysis of CICR suggested that tetraploid cottons evolved after silence of the transposon family 1–1.5 million years ago (Mya). Furthermore, A genomes are more homologous with B genomes, and the C, E, F, and G genomes likely diverged from a common ancestor prior to 3.5–4 Mya, the time when CICR appeared. The genomic variation caused by the insertion of CICR in the A (sub) genome may have played an important role in the speciation of organisms with A genomes. Conclusions The CICR family is highly repetitive in A and B genomes of Gossypium, but not amplified in the C–G genomes. The differential amount of CICR family in At and Dt will aid in partitioning sub genome sequences for chromosome assemblies during tetraploid genome sequencing and will act as a method for assessing the accuracy of tetraploid genomes by looking at the proportion of CICR elements in resulting pseudochromosome sequences. The timeline of the expansion of CICR family provides a new reference for cotton evolutionary analysis, while the impact on gene function caused by the insertion of CICR elements will be a target for further analysis of investigating phenotypic differences between A genome and D genome species.

Segregation distortion (SD) is a genetic mechanism commonly found in segregating or stable populations. The principle behind this puzzles many researchers. The F2 generation developed from wild Gossypium darwinii and G. hirsutum CCRI12 species was used to investigate the possible transcription factors within the segregation distortion regions (SDRs). The 384 out of 2763 markers were distorted in 29 SDRs on 18 chromosomes. Good collinearity was observed among genetic and physical maps of G. hirsutum and G. barbadense syntenic blocks. Total 568 genes were identified from SDRs of 18 chromosomes. Out of these genes, 128 belonged to three top-ranked salt-tolerant gene families. The DUF597 contained 8 uncharacterized genes linked to Pkinase (PF00069) gene family in the phylogenetic tree, while 15 uncharacterized genes clustered with the zinc finger gene family. Two hundred thirty four miRNAs targeted numerous genes, including ghr-miR156, ghr-miR399 and ghr-miR482, while others targeted top-ranked stress-responsive transcription factors. Moreover, these genes were involved in the regulation of numerous stress-responsive cis-regulatory elements. The RNA sequence data of fifteen upregulated genes were verified through the RT-qPCR. The expression profiles of two highly upregulated genes (Gh_D01G2015 and Gh_A01G1773) in salt-tolerant G. darwinii showed antagonistic expression in G. hirsutum. The results indicated that salt-tolerant genes have been possibly transferred from the wild G. darwinii species. A detailed functional analysis of these genes can be carried out which might be helpful in the future for gene cloning, transformation, gene editing and the development of salt-resistant cotton varieties.

Background: It is well known that obesity induced by high-fat diet (HFD) poses a serious threat to people’s health. Fuzhuan brick tea, one of the most popular beverages, is reported to possess a significant effect on regulating lipid metabolism, attributed to its many bioactive ingredients. However, the efficacy and mechanism of compound Fuzhuan brick tea (CFBT) made from Fuzhuan brick tea and other six Chinese herbal medicines are still not well defined. Methods: Sixty mice were divided into six groups: normal control group (CK), high-fat model group (NK), positive control group with anti-hyperlipidemic drug (YK), CFBT at low-(FL), medium-(FM) and high-(FH) dosage. Intervening for 30 days, conventional indexes analysis combined with metabolomics were performed to evaluate the changes in biochemical indexes and liver metabolic profiles in mice submitted to HFD. Results: CFBT treatment was able to ameliorate obesity, serum biochemical parameters, antioxidant activity and hepatic steatosis. In addition, significant alterations in the liver tissue metabolic profiles were observed, with most of these associated with inflammation, glucose and lipid metabolism. Conclusions: This study provides evidence that consumption of CFBT is capable of preventing dyslipidemia, reducing weight gain, restoring liver injury, as well as improving metabolic disorders.

The activity of genome-specific repetitive sequences is the main cause of genome variation between Gossypium A and D genomes. Through comparative analysis of the two genomes, we retrieved a repetitive element termed ICRd motif, which appears frequently in the diploid Gossypium raimondii (D5) genome but rarely in the diploid Gossypium arboreum (A2) genome. We further explored the existence of the ICRd motif in chromosomes of G. raimondii, G. arboreum, and two tetraploid (AADD) cotton species, Gossypium hirsutum and Gossypium barbadense, by fluorescence in situ hybridization (FISH), and observed that the ICRd motif exists in the D5 and D-subgenomes but not in the A2 and A-subgenomes. The ICRd motif comprises two components, a variable tandem repeat (TR) region and a conservative sequence (CS). The two constituents each have hundreds of repeats that evenly distribute across 13 chromosomes of the D5genome. The ICRd motif (and its repeats) was revealed as the common conservative region harbored by ancient Long Terminal Repeat Retrotransposons. Identification and investigation of the ICRd motif promotes the study of A and D genome differences, facilitates research on Gossypium genome evolution, and provides assistance to subgenome identification and genome assembling.

The extrusion of toxins and substances at a cellular level is a vital life process in plants under abiotic stress. The multidrug and toxic compound extrusion (MATE) gene family plays a large role in the exportation of toxins and other substrates. We carried out a genome-wide analysis of MATE gene families in Gossypium raimondii and Gossypium arboreum and assessed their expression levels under salt, cadmium and drought stresses. We identified 70 and 68 MATE genes in G. raimondii and G. arboreum, respectively. The majority of the genes were predicted to be localized within the plasma membrane, with some distributed in other cell parts. Based on phylogenetic analysis, the genes were subdivided into three subfamilies, designated as M1, M2 and M3. Closely related members shared similar gene structures, and thus were highly conserved in nature and have mainly evolved through purifying selection. The genes were distributed in all chromosomes. Twenty-nine gene duplication events were detected, with segmental being the dominant type. GO annotation revealed a link to salt, drought and cadmium stresses. The genes exhibited differential expression, with GrMATE18, GrMATE34, GaMATE41 and GaMATE51 significantly upregulated under drought, salt and cadmium stress, and these could possibly be the candidate genes. Our results provide the first data on the genome-wide and functional characterization of MATE genes in diploid cotton, and are important for breeders of more stress-tolerant cotton genotypes.

Abstract Background Late embryogenesis abundant (LEA) proteins are large groups of hydrophilic proteins with major role in drought and other abiotic stresses tolerance in plants. In-depth study and characterization of LEA protein families have been carried out in other plants, but not in upland cotton. The main aim of this research work was to characterize the late embryogenesis abundant (LEA) protein families and to carry out gene expression analysis to determine their potential role in drought stress tolerance in upland cotton. Increased cotton production in the face of declining precipitation and availability of fresh water for agriculture use is the focus for breeders, cotton being the backbone of textile industries and a cash crop for many countries globally. Results In this work, a total of 242, 136 and 142 LEA genes were identified in G. hirsutum, G. arboreum and G. raimondii respectively. The identified genes were classified into eight groups based on their conserved domain and phylogenetic tree analysis. LEA 2 were the most abundant, this could be attributed to their hydrophobic character. Upland cotton LEA genes have fewer introns and are distributed in all chromosomes. Majority of the duplicated LEA genes were segmental. Syntenic analysis showed that greater percentages of LEA genes are conserved. Segmental gene duplication played a key role in the expansion of LEA genes. Sixty three miRNAs were found to target 89 genes, such as miR164, ghr-miR394 among others. Gene ontology analysis revealed that LEA genes are involved in desiccation and defense responses. Almost all the LEA genes in their promoters contained ABRE, MBS, W-Box and TAC-elements, functionally known to be involved in drought stress and other stress responses. Majority of the LEA genes were involved in secretory pathways. Expression profile analysis indicated that most of the LEA genes were highly expressed in drought tolerant cultivars Gossypium tomentosum as opposed to drought susceptible, G. hirsutum. The tolerant genotypes have a greater ability to modulate genes under drought stress than the more susceptible upland cotton cultivars. Conclusion The finding provides comprehensive information on LEA genes in upland cotton, G. hirsutum and possible function in plants under drought stress.

The phytochrome-interacting factors (PIFs) proteins belong to the subfamily of basic helix–loop–helix (bHLH) transcription factors and play important roles in chloroplast development and chlorophyll biosynthesis. Currently, knowledge about the PIF gene family in Camellia sinensis remains very limited. In this study, seven PIF members were identified in the C. sinensis genome and named based on homology with AtPIF genes in Arabidopsis thaliana. All C. sinensis PIF (CsPIF) proteins have both the conserved active PHYB binding (APB) and bHLH domains. Phylogenetic analysis revealed that CsPIFs were clustered into four groups—PIF1, PIF3, PIF7, and PIF8—and most CsPIFs were clustered in pairs with their corresponding orthologs in Populus tremula. CsPIF members in the same group tended to display uniform or similar exon–intron distribution patterns and motif compositions. CsPIF genes were differentially expressed in C. sinensis with various leaf colors and strongly correlated with the expression of genes involved in the chlorophyll metabolism pathway. Promoter analysis of structural genes related to chlorophyll metabolism found DNA-binding sites of PIFs were abundant in the promoter regions. Protein–protein interaction networks of CsPIFs demonstrated a close association with phytochrome, PIF4, HY5, TOC1, COP1, and PTAC12 proteins. Additionally, subcellular localization and transcriptional activity analysis suggested that CsPIF3b was nuclear localized protein and possessed transcriptional activity. We also found that CsPIF3b could activate the transcription of CsHEMA and CsPOR in Nicotiana benthamiana leaves. This work provides comprehensive research of CsPIFs and would be helpful to further promote the regulation mechanism of PIF on chlorophyll metabolism in C. sinensis.

Abstract Fu brick tea is a unique post-fermented tea product which is fermented with microorganism during the manufacturing process. Metabolic analysis showed that most metabolites content were decreased during the manufacturing process of Fu brick tea, except GA (gallic acid). Illumina MiSeq sequencing of ITS gene amplicons was applied to analyze the fungal community succession. The genera Aspergillus, Cyberlindnera and Candida were predominant at the early stage of manufacturing process (from “primary dark tea” to “fermentation for 3 days”), but after the stage of “fermentation for 3 days” only Aspergillus was still dominated, and maintain a relatively constant until to the end of manufacturing process. The effects of metabolites on the structure of the fungal community were analyzed by redundancy analysis (RDA) and variation partitioning analysis (VPA). The results indicated that GCG (gallocatechin gallate), EGCG (epigallocatechin gallate) and GA as well as the interactions among them were the most probably ones to influence, or be influenced by the fungal communities during the fermentation process of Fu brick tea. This study revealed fungal succession, metabolite changes and their relationships, provided new insights into the mechanisms for manufacturing process of Fu brick tea.

Cytogenetic maps of Gossypium hirsutum (Linnaeus, 1753) homoeologous chromosomes Ah01 and Dh01 were constructed by fluorescence in situ hybridization (FISH), using eleven homoeologous-chromosomes-shared bacterial artificial chromosomes (BACs) clones and one chromosome-specific BAC clone respectively. We compared the cytogenetic maps with the genetic linkage and draft genome assembly maps based on a standardized map unit, relative map position (RMP), which allowed a global view of the relationship of genetic and physical distances along each chromosome, and assembly quality of the draft genome assembly map. By integration of cytogenetic maps with sequence maps of the two chromosomes (Ah01 and Dh01), we inferred the locations of two scaffolds and speculated that some homologous sequences belonging to homoeologous chromosomes were removed as repetitiveness during the sequence assembly. The result offers molecular tools for cotton genomics research and also provides valuable information for the improvement of the draft genome assembly.

Abstract Background Chromosome microdissection is one of the most important techniques in molecular cytogenetic research. Cotton (Gossypium Linnaeus, 1753) is the main natural fiber crop in the world. The resistance gene analog (RGA) cloning after its single chromosome microdissection can greatly promote cotton genome research and breeding. Results Using the linker adaptor PCR (LA-PCR) with the primers of rice disease-resistance homologues, three nucleotide sequences PS016 (KU051681), PS054 (KU051682), and PS157 (KU051680) were obtained from the chromosome Ah01 of upland cotton (cv. TM-1). The Blast results showed that the three sequences are the nucleotide binding site-leucine rich repeat (NBS-LRR) type RGAs. Clustering results indicated that they are homologous to these published RGAs. Thus, the three RGAs can definitely be confirmed as NBS-LRR class of RGAs in upland cotton. Conclusions Using single chromosome microdissection technique, DNA libraries containing cotton RGAs were obtained. This technique can promote cotton gene cloning, marker development and even the improvement of cotton genome research and breeding.

We found 33, 17, and 20 Alba genes in Gossypium hirsutum, Gossypium arboretum, and Gossypium raimondii, respectively. The Alba protein lengths ranged from 62 to 312 aa, the molecular weight (MW) from 7.003 to 34.55 kDa, grand average hydropathy values of −1.012 to 0.609 and isoelectric (pI) values of −3 to 11. Moreover, miRNAs such as gra-miR8770 targeted four genes, gra-miR8752 and gra-miR8666 targeted three genes, and each and gra-miR8657 a, b, c, d, e targeted 10 genes each, while the rests targeted 1 to 2 genes each. Similarly, various cis-regulatory elements were detected with significant roles in enhancing abiotic stress tolerance, such as CBFHV (RYCGAC) with a role in cold stress acclimation among others. Two genes, Gh_D01G0884 and Gh_D01G0922, were found to be highly induced under water deficit and salt stress conditions. Through virus-induced gene silencing (VIGS), the VIGS cotton plants were found to be highly susceptible to both water deficit and salt stresses; the VIGS plants exhibited a significant reduction in root growth, low cell membrane stability (CMS), saturated leaf weight (SLW), chlorophyll content levels, and higher excised leaf water loss (ELWL). Furthermore, the stress-responsive genes and ROS scavenging enzymes were significantly reduced in the VIGS plants compared to either the wild type (WT) and or the positively controlled plants. The VIGS plants registered higher concentration levels of hydrogen peroxide and malondialdehyde, with significantly lower levels of the various antioxidants evaluated an indication that the VIGS plants were highly affected by salt and drought stresses. This result provides a key foundation for future exploration of the Alba proteins in relation to abiotic stress.

Abiotic stresses have negative effects on plants growth and development. Plants, being sessile, have developed specific adaptive strategies that allow them to rapidly detect and respond to abiotic stress factors. The detoxification efflux carriers (DTX)/multidrug and toxic compound extrusion (MATE) transporters are of significance in the translocation of abscisic acid (ABA), a phytohormone with profound role in plants under various abiotic stress conditions. The ABA signaling cascades are the core regulators of abiotic stress responses in plants, triggering major changes in gene expression and adaptive physiological responses. We therefore carried out genome-wide analysis of the DTX/MATE gene family, transformed a DTX/MATE gene in Arabidopsis and carried out functional analysis under drought, salt, and cold stress conditions. We identified 128, 70, and 72 DTX/MATE genes in Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii, respectively. The proteins encoded by the DTX/MATE genes showed varied physiochemical properties but they all were hydrophobic. The Gh_D06G0281 (DTX/MATE) over-expressing Arabidopsis lines were highly tolerant under drought, salt, and cold stress with high production of antioxidant enzymes and significantly reduced levels of oxidants. Lipid peroxidation, as measured by the level of malondialdehyde concentrations was relatively low in transgenic lines compared to wild types, an indication of reduced oxidative stress levels in the transgenic plants. Based on physiological measurements, the transgenic plants exhibited significantly higher relative leaf water content, reduced excised leaf water loss and a significant reduction in ion leakage as a measure of the cell membrane stability compared to the wild types. Abiotic stress responsive genes, ABF4, CBL1, SOS1, and RD29B were highly expressed in the transgenic lines compared to the non-transformed wild type plants. The protein encoded by the Gh_D06G0281 (DTX/MATE) gene was predicted to be located within the plasma membrane. Since signals from extracellular stimuli are transmitted through the plasma membrane most of which are conducted by plasma membrane proteins it is possible the Gh_D06G0281 (DTX/MATE) gene product could be important for this process.

Plant height, which shows dynamic development and heterosis, is a major trait affecting plant architecture and has an indirect influence on economic yield related to biological yield in cotton. In the present study, we carried out dynamic analysis for plant height and its heterosis by quantitative trait loci (QTL) mapping at multiple developmental stages using two recombinant inbred lines (RILs) and their backcross progeny. At the single-locus level, 47 QTL were identified at five developmental stages in two hybrids. In backcross populations, QTL identified at an early stage mainly showed partial effects and QTL detected at a later stage mostly displayed overdominance effects. At the two-locus level, we found that main effect QTL played a more important role than epistatic QTL in the expression of heterosis in backcross populations. Therefore, this study implies that the genetic basis of plant height heterosis shows dynamic character and main effect QTL with dominance determines heterosis for plant height in Upland cotton.

Cotton fiber, a raw natural fiber material, is widely used in the textile industry. Understanding the genetic mechanism of fiber traits is helpful for fiber quality improvement. In the present study, the genetic basis of fiber quality traits was explored using two recombinant inbred lines (RILs) and corresponding backcross (BC) populations under multiple environments in Upland cotton based on marker analysis. In backcross populations, no significant correlation was observed between marker heterozygosity and fiber quality performance and it suggested that heterozygosity was not always necessarily advantageous for the high fiber quality. In two hybrids, 111 quantitative trait loci (QTL) for fiber quality were detected using composite interval mapping, in which 62 new stable QTL were simultaneously identified in more than one environment or population. QTL detected at the single-locus level mainly showed additive effect. In addition, a total of 286 digenic interactions (E-QTL) and their environmental interactions [QTL × environment interactions (QEs)] were detected for fiber quality traits by inclusive composite interval mapping. QE effects should be considered in molecular marker-assisted selection breeding. On average, the E-QTL explained a larger proportion of the phenotypic variation than the main-effect QTL did. It is concluded that the additive effect of single-locus and epistasis with few detectable main effects play an important role in controlling fiber quality traits in Upland cotton.

Wild cotton species have significant agronomic traits that can be introgressed into elite cultivated varieties. The use of a genetic map is important in exploring, identification and mining genes which carry significant traits. In this study, 188 F2mapping individuals were developed from Gossypium thurberi (female) and Gossypium trilobum (male), and were genotyped by using simple sequence repeat (SSR) markers. A total of 12,560 simple sequence repeat (SSR) markers, developed by Southwest University, thus coded SWU were screened out of which only 994 were found to be polymorphic, and 849 markers were linked in all the 13 chromosomes. The map had a length of 1,012.458 cM with an average marker distance of 1.193 cM. Segregation distortion regions (SDRs) were observed on Chr01, Chr02, Chr06, Chr07 Chr09, Chr10 and Chr11 with a large proportion of the SDR regions segregating towards the heterozygous allele. There was good syntenic block formation that revealed good collinearity between the genetic and physical map of G. raimondii, compared to the Dt_sub genome of the G. hirsutum and G. barbadense. A total of 2,496 genes were mined within the SSR related regions. The proteins encoding the mined genes within the SDR had varied physiochemical properties; their molecular weights ranged from 6.586 to 252.737 kDa, charge range of -39.5 to 52, grand hydropathy value (GRAVY) of -1.177 to 0.936 and isoelectric (pI) value of 4.087 to 12.206. The low GRAVY values detected showed that the proteins encoding these genes were hydrophilic in nature, a property common among the stress responsive genes. The RNA sequence analysis revealed more of the genes were highly upregulated in various stages of fiber development for instance; Gorai.002G241300 was highly up regulated at 5, 10, 20 and 25 day post anthesis (DPA). Validation through RT-qPCR further revealed that these genes mined within the SDR regions might be playing a significant role under fiber development stages, therefore we infer that Gorai.007G347600 (TFCA), Gorai.012G141600 (FOLB1), Gorai.006G024500 (NMD3), Gorai.002G229900 (LST8) and Gorai.002G235200 (NSA2) are significantly important in fiber development and in turn the quality, and further researches needed to be done to elucidate their exact roles in the fiber development process. The construction of the genetic map between the two wild species paves away for the mapping of quantitative trait loci (QTLs) since the average distance between the markers is small, and mining of genes on the SSR regions will provide an insight in identifying key genes that can be introgressed into the cultivated cotton cultivars.

A bacterial artificial chromosome (BAC) library for G. mustelinum Miers ex G. Watt (AD4) was constructed. Intact nuclei from G. mustelinum (AD4) were used to isolate high molecular weight DNA, which was partially cleaved with Hind III and cloned into pSMART BAC (Hind III) vectors. The BAC library consisted of 208,182 clones arrayed in 542 384-microtiter plates, with an average insert size of 121.72 kb ranging from 100 to 150 kb. About 2% of the clones did not contain inserts. Based on an estimated genome size of 2372 Mb for G. mustelinum, the BAC library was estimated to have a total coverage of 10.50 × genome equivalents. The high capacity library of G. mustelinum will serve as a giant gene resource for map-based cloning of quantitative trait loci or genes associated with important agronomic traits or resistance to Verticillium wilt, physical mapping and comparative genome analysis.

Tea (Camellia sinensis L.) is a perennial woody plant that is widely cultivated to produce a popular non-alcoholic beverage; this beverage has received much attention due to its pleasant flavor and bioactive ingredients, particularly several important secondary metabolites. Due to the significant changes in the metabolite contents of the buds and the young expanding leaves of tea plants, high-performance liquid chromatography (HPLC) analysis and isobaric tags for relative and absolute quantitation (iTRAQ) analysis were performed. A total of 233 differentially expressed proteins were identified. Among these, 116 proteins were up-regulated and 117 proteins were down-regulated in the young expanding leaves compared with the buds. A large array of diverse functions was revealed, including roles in energy and carbohydrate metabolism, secondary metabolite metabolism, nucleic acid and protein metabolism, and photosynthesis- and defense-related processes. These results suggest that polyphenol biosynthesis- and photosynthesis-related proteins regulate the secondary metabolite content of tea plants. The energy and antioxidant metabolism-related proteins may promote tea leaf development. However, reverse transcription quantitative real-time PCR (RT-qPCR) showed that the protein expression levels were not well correlated with the gene expression levels. These findings improve our understanding of the molecular mechanism of the changes in the metabolite content of the buds and the young expanding leaves of tea plants.

Cotton is a pioneer of saline land crop, while salt stress still causes its growth inhibition and fiber production decrease. Phenotype identification showed better salt tolerance of a wild diploid cotton species Gossypium klotzschianum. To elucidate the salt-tolerant mechanisms in G. klotzschianum, we firstly detected the changes in hormones, H2O2 and glutathione (GSSH and GSH), then investigated the gene expression pattern of roots and leaves treated with 300 mM NaCl for 0, 3, 12, 48 h, and each time control by RNA-seq on the Illumina-Solexa platform. Physiological determination proved that the significant increase in hormone ABA at 48 h, while that in H2O2 was at 12 h, likewise, the GSH content decrease at 48 h and the GSSH content increase at 48 h, under salt stress. In total, 37,278 unigenes were identified from the transcriptome data, 8,312 and 6,732 differentially expressed genes (DEGs) were discovered to be involved in salt stress tolerance in roots and leaves, respectively. Gene function annotation and expression analysis elucidated hormone biosynthesis and signal transduction, reactive oxygen species (ROS), and salt overly sensitive (SOS) signal transduction related genes revealed the important roles of them in signal transmission, oxidation balance and ion homeostasis in response to salinity stress. This is a report which focuses on primary response to highly salty stress (upto 300 mM NaCl) in cotton using a wild diploid Gossypium species, broadening our understanding of the salt tolerance mechanism in cotton and laying a solid foundation of salt resistant for the genetic improvement of upland cotton with the resistance to salt stress.