Tailoring Galactose Oxidase for Self-Powered Benzyl Alcohol Sensing.

Benzyl alcohol (BnOH) is a widely-used preservative in a variety of cosmetics, but the excess addition (≥1.0 %) may cause strong symptoms such as nausea, gastrointestinal irritation, convulsion, even death, making it crucial to monitor and control the addition quantity. Herein, we have developed a test-strip-like BnOH detection method via tailoring a galactose oxidase (GOase) towards BnOH oxidation and preparing a self-powered electrochromic strip for BnOH concentration visualization. A double-substituted GOase variant (Y329S/R330F), on the basis of the reported GOase M1 , has been obtained by semi-rational design with a 24.6-fold improved activity towards BnOH compared to GOase M1 . The GOase Y329S/R330F electrode has a response to BnOH with a linear range of 0.04 to 3.25 mM (R2 =0.9985), a sensitivity of 122.78 µA mM-1 cm-2 , and a detection limit of 0.03 mM (S/N=3). Coupling an electrochromic Prussian blue (PB) cathode helps the successful sensing visualization without any further power supply. The present sensing is more convenient and user-friendly than the generally used gas chromatography (GC) and high performance liquid chromatography (HPLC), and brings a more accessible solution to the field of quality controlling.

Methyl jasmonate elicits distinctive hydrolyzable tannin, flavonoid, and phyto-oxylipin responses in pomegranate (Punica granatum L.) leaves.

MAIN CONCLUSION: Transcriptome and biochemical analyses suggested that, while suppression of multiple flavonoids and anthocyanins occurs at least partially at the transcriptional level, increased biosynthesis of non-jasmonate phyto-oxylipins is likely controlled non-transcriptionally. Methyl jasmonate (MeJA) produced in plants can mediate their response to environmental stresses. Exogenous application of MeJA has also shown to activate signaling pathways and induce phytoalexin accumulation in many plant species. To understand how pomegranate plants respond biochemically to environmental stresses, metabolite analysis was conducted in pomegranate leaves subjected to MeJA application and revealed unique changes in hydrolyzable tannins, flavonoids, and phyto-oxylipins. Additionally, transcriptome and real-time qPCR analyses of mock- and MeJA-treated pomegranate leaves identified differentially expressed metabolic genes and transcription factors that are potentially involved in the control of hydrolyzable tannin, flavonoid, and phyto-oxylipin pathways. Molecular, biochemical, and bioinformatic characterization of the only lipoxygenase with sustained, MeJA-induced expression showed that it is capable of oxidizing polyunsaturated fatty acids, though not located in the subcellular compartment where non-jasmonate (non-JA) phyto-oxylipins were produced. These results collectively suggested that while the broad suppression of flavonoids and anthocyanins is at least partially controlled at the transcriptional level, the induced biosynthesis of non-JA phyto-oxylipins is likely not regulated transcriptionally. Overall, a better understanding of how pomegranate leaves respond to environmental stresses will not only promote plant health and productivity, but also have an impact on human health as fruits produced by pomegranate plants are a rich source of nutritional compounds.

Regulation of photosystem I-light-harvesting complex I from a red alga Cyanidioschyzon merolae in response to light intensities.

Photosynthetic organisms use different means to regulate their photosynthetic activity in respond to different light conditions under which they grow. In this study, we analyzed changes in the photosystem I (PSI) light-harvesting complex I (LHCI) supercomplex from a red alga Cyanidioschyzon merolae, upon growing under three different light intensities, low light (LL), medium light (ML), and high light (HL). The results showed that the red algal PSI-LHCI is separated into two bands on blue-native PAGE, which are designated PSI-LHCI-A and PSI-LHCI-B, respectively, from cells grown under LL and ML. The former has a higher molecular weight and binds more Lhcr subunits than the latter. They are considered to correspond to the two types of PSI-LHCI identified by cryo-electron microscopic analysis recently, namely, the former with five Lhcrs and the latter with three Lhcrs. The amount of PSI-LHCI-A is higher in the LL-grown cells than that in the ML-grown cells. In the HL-grown cells, PSI-LHCI-A completely disappeared and only PSI-LHCI-B was observed. Furthermore, PSI core complexes without Lhcr attached also appeared in the HL cells. Fluorescence decay kinetics measurement showed that Lhcrs are functionally connected with the PSI core in both PSI-LHCI-A and PSI-LHCI-B obtained from LL and ML cells; however, Lhcrs in the PSI-LHCI-B fraction from the HL cells are not coupled with the PSI core. These results indicate that the red algal PSI not only regulates its antenna size but also adjusts the functional connection of Lhcrs with the PSI core in response to different light intensities.

LMI1-like and KNOX1 genes coordinately regulate plant leaf development in dicotyledons.

KEY MESSAGE: This report reveals that the LMI1-like and KNOX1 genes coordinately control the leaf development and different combinations of those genes which produce diverse leaf shapes including broad, lobed and compound leaves. Class I KNOTTED1-like homeobox (KNOX1) genes are involved in compound leaf development and are repressed by the ASYMMETRIC LEAVES1 (AS1)-AS2 complex. Cotton plants have a variety of leaf shapes, including broad leaves and lobed leaves. GhOKRA, a LATE MERISTEM IDENTITY 1 (LMI1)-like gene, controls the development of an okra leaf shape. We cloned the corresponding cotton homologs of Arabidopsis thaliana AS1 and AS2 and seven KNOX1 genes. Through virus-induced gene silencing technology, we found that either GhAS1 or GhAS2-silenced cotton plants showed a great change in leaf shape from okra leaves to trifoliolate dissected leaves. In the shoot tips of these plants, the expression of the cotton ortholog of Knotted in A. thaliana 1 (KNAT1), GhKNOTTED1-LIKE2/3/4 (GhKNL2/3/4), was increased. However, GhKNOX1s-silenced plants maintained the wild-type okra leaves. A novel dissected-like leaf in A. thaliana was further generated by crossing plants constitutively expressing GhOKRA with either as1-101 or as2-101 mutant plants. The dissected-like leaves showed two different leaf vein patterns. This report reveals that the LMI1-like and KNOX1 genes coordinately control leaf development, and different combinations of these genes produce diverse leaf shapes including broad leaves, lobed leaves and compound leaves. This is the first report on the artificial generation of compound leaves from simple leaves in cotton.

Exploring the Phytochemical Landscape of the Early-Diverging Flowering Plant Amborella trichopoda Baill.

Although the evolutionary significance of the early-diverging flowering plant Amborella (Amborella trichopoda Baill.) is widely recognized, its metabolic landscape, particularly specialized metabolites, is currently underexplored. In this work, we analyzed the metabolomes of Amborella tissues using liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS). By matching the mass spectra of Amborella metabolites with those of authentic phytochemical standards in the publicly accessible libraries, 63, 39, and 21 compounds were tentatively identified in leaves, stems, and roots, respectively. Free amino acids, organic acids, simple sugars, cofactors, as well as abundant glycosylated and/or methylated phenolic specialized metabolites were observed in Amborella leaves. Diverse metabolites were also detected in stems and roots, including those that were not identified in leaves. To understand the biosynthesis of specialized metabolites with glycosyl and methyl modifications, families of small molecule UDP-dependent glycosyltransferases (UGTs) and O-methyltransferases (OMTs) were identified in the Amborella genome and the InterPro database based on conserved functional domains. Of the 17 phylogenetic groups of plant UGTs (A-Q) defined to date, Amborella UGTs are absent from groups B, N, and P, but they are highly abundant in group L. Among the 25 Amborella OMTs, 7 cluster with caffeoyl-coenzyme A (CCoA) OMTs involved in lignin and phenolic metabolism, whereas 18 form a clade with plant OMTs that methylate hydroxycinnamic acids, flavonoids, or alkaloids. Overall, this first report of metabolomes and candidate metabolic genes in Amborella provides a starting point to a better understanding of specialized metabolites and biosynthetic enzymes in this basal lineage of flowering plants.

Mutation of SELF-PRUNING homologs in cotton promotes short-branching plant architecture.

In cotton, the formation of fruiting branches affects both plant architecture and fiber yield. Here, we report map-based cloning of the axillary flowering mutation gene (GbAF) that causes bolls to be borne directly on the main plant stem in Gossypium barbadense, and of the clustered boll mutation gene (cl1) in G. hirsutum. Both mutant alleles were found to represent point mutations at the Cl1 locus. Therefore, we propose that the GbAF mutation be referred to as cl1b. These Cl1 loci correspond to homologs of tomato SELF-PRUNING (SP), i.e. Gossypium spp. SP (GoSP) genes. In tetraploid cottons, single monogenic mutation of either duplicate GoSP gene (one in the A and one in the D subgenome) is associated with the axillary cluster flowering phenotype, although the shoot-indeterminate state of the inflorescence is maintained. By contrast, silencing of both GoSPs leads to the termination of flowering or determinate plants. The architecture of axillary flowering cotton allows higher planting density, contributing to increased fiber yield. Taken together the results provide new insights into the underlying mechanism of branching in cotton species, and characterization of GoSP genes may promote the development of compact cultivars to increase global cotton production.

Isolation and characterization of PSI-LHCI super-complex and their sub-complexes from a red alga Cyanidioschyzon merolae.

Photosystem I (PSI)-light-harvesting complex I (LHCI) super-complex and its sub-complexes PSI core and LHCI, were purified from a unicellular red alga Cyanidioschyzon merolae and characterized. PSI-LHCI of C. merolae existed as a monomer with a molecular mass of 580 kDa. Mass spectrometry analysis identified 11 subunits (PsaA, B, C, D, E, F, I, J, K, L, O) in the core complex and three LHCI subunits, CMQ142C, CMN234C, and CMN235C in LHCI, indicating that at least three Lhcr subunits associate with the red algal PSI core. PsaG was not found in the red algae PSI-LHCI, and we suggest that the position corresponding to Lhca1 in higher plant PSI-LHCI is empty in the red algal PSI-LHCI. The PSI-LHCI complex was separated into two bands on native PAGE, suggesting that two different complexes may be present with slightly different protein compositions probably with respective to the numbers of Lhcr subunits. Based on the results obtained, a structural model was proposed for the red algal PSI-LHCI. Furthermore, pigment analysis revealed that the C. merolae PSI-LHCI contained a large amount of zeaxanthin, which is mainly associated with the LHCI complex whereas little zeaxanthin was found in the PSI core. This indicates a unique feature of the carotenoid composition of the Lhcr proteins and may suggest an important role of Zea in the light-harvesting and photoprotection of the red algal PSI-LHCI complex.

Isolation and characterization of a PSI-LHCI super-complex and its sub-complexes from a siphonaceous marine green alga, Bryopsis Corticulans.

A novel super-complex of photosystem I (PSI)-light-harvesting complex I (LHCI) was isolated from a siphonaceous marine green alga, Bryopsis corticulans. The super-complex contained 9-10 Lhca antennas as external LHCI bound to the core complex. The super-complex was further disintegrated into PSI core and LHCI sub-complexes, and analysis of the pigment compositions by high-performance liquid chromatography revealed unique characteristics of the B. corticulans PSI in that one PSI core contained around 14 α-carotenes and 1-2 ε-carotenes. This is in sharp contrast to the PSI core from higher plants and most cyanobacteria where only ß-carotenes were present, and is the first report for an α-carotene-type PSI core complex among photosynthetic eukaryotes, suggesting a structural flexibility of the PSI core. Lhca antennas from B. corticulans contained seven kinds of carotenoids (siphonaxanthin, all-trans neoxanthin, 9'-cis neoxanthin, violaxanthin, siphonein, ε-carotene, and α-carotene) and showed a high carotenoid:chlorophyll ratio of around 7.5:13. PSI-LHCI super-complex and PSI core showed fluorescence emission peaks at 716 and 718 nm at 77 K, respectively; whereas two Lhca oligomers had fluorescence peaks at 681 and 684 nm, respectively. By comparison with spinach PSI preparations, it was found that B. corticulans PSI had less red chlorophylls, most of them are present in the core complex but not in the outer light-harvesting systems. These characteristics may contribute to the fine tuning of the energy transfer network, and to acclimate to the ever-changing light conditions under which the unique green alga inhabits.

ATP regeneration by ATPases for in vitro biotransformation.

Adenosine triphosphate (ATP) regeneration is a significant step in both living cells and in vitro biotransformation (ivBT). Rotary motor ATP synthases (ATPases), which regenerate ATP in living cells, have been widely assembled in biomimetic structures for in vitro ATP synthesis. In this review, we present a comprehensive overview of ATPases, including the working principle, orientation and distribution density properties of ATPases, as well as the assembly strategies and applications of ATPase-based ATP regeneration modules. The original sources of ATPases for in vitro ATP regeneration include chromatophores, chloroplasts, mitochondria, and inverted Escherichia coli (E. coli) vesicles, which are readily accessible but unstable. Although significant advances have been made in the assembly methods for ATPase-artificial membranes in recent decades, it remains challenging to replicate the high density and orientation of ATPases observed in vivo using in vitro assembly methods. The use of bioproton pumps or chemicals for constructing proton motive forces (PMF) enables the versatility and potential of ATPase-based ATP regeneration modules. Additionally, overall robustness can be achieved via membrane component selection, such as polymers offering great mechanical stability, or by constructing a solid supporting matrix through layer-by-layer assembly techniques. Finally, the prospects of ATPase-based ATP regeneration modules can be expected with the technological development of ATPases and artificial membranes.

SbMYB3 transcription factor promotes root-specific flavone biosynthesis in Scutellaria baicalensis.

Scutellaria baicalensis Georgi produces abundant root-specific flavones (RSFs), which provide various benefits to human health. We have elucidated the complete biosynthetic pathways of baicalein and wogonin. However, the transcriptional regulation of flavone biosynthesis in S. baicalensis remains unclear. We show that the SbMYB3 transcription factor functions as a transcriptional activator involved in the biosynthesis of RSFs in S. baicalensis. Yeast one-hybrid and transcriptional activation assays showed that SbMYB3 binds to the promoter of flavone synthase II-2 (SbFNSII-2) and enhances its transcription. In S. baicalensis hairy roots, RNAi of SbMYB3 reduced the accumulation of baicalin and wogonoside, and SbMYB3 knockout decreased the biosynthesis of baicalein, baicalin, wogonin, and wogonoside, whereas SbMYB3 overexpression enhanced the contents of baicalein, baicalin, wogonin, and wogonoside. Transcript profiling by qRT-PCR demonstrated that SbMYB3 activates SbFNSII-2 expression directly, thus leading to more abundant accumulation of RSFs. This study provides a potential target for metabolic engineering of RSFs.

Identification and Characterization of Two Regiospecific Tricetin UDP-Dependent Glycosyltransferases from Pomegranate (Punica granatum L.).

Tricetin (5,7,3',4',5'-pentahydroxyflavone) is a dietary flavone from flowers of Myrtales plants with demonstrated functions in promoting human health. By contrast, the bioactivity of its glucosylated derivative tricetin 4'-O-glucoside has not been extensively explored. We conducted metabolite profiling analysis of pomegranate (a Myrtales plant) floral tissues and revealed that tricetin and tricetin 4'-O-glucoside accumulate in anthers, but not petals. In addition, the comparative analysis of anther and petal transcriptomes identified 10 UGTs that are more highly expressed in anthers than petals. Of the 10 UGTs, PgUGT76Z1 and PgUGT73AL1 glucosylated specifically at the 4'-O position of tricetin to form tricetin 4'-O-glucoside. The phylogenetic analysis indicated that PgUGT76Z1 and PgUGT73AL1 belong to different plant UGT groups, suggesting a convergent evolution of these tricetin UGTs. Overall, identification and characterization of PgUGT76Z1 and PgUGT73AL1 not only provides evolutionary insights into tricetin glucosylation, but also offers an opportunity to produce tricetin 4'-O-glucoside in large quantities through microbial biotransformation or plant metabolic engineering, thus facilitating the investigation of tricetin 4'-O-glucoside bioactivities.

Electrochromic properties of pyrene conductive polymers modified by chemical polymerization.

Pyrene is composed of four benzene rings and has a unique planar melting ring structure. Pyrene is the smallest condensed polycyclic aromatic hydrocarbon, and its unique structural properties have been extensively studied. Pyrene has excellent properties such as thermal stability, high fluorescence quantum efficiency and high carrier mobility. This paper mainly used thiophene, EDOT and triphenylamine groups to enhance the pyrene based π-conjugated system and control the molecular accumulation of organic semiconductors, and improve their charge transport performances. Five kinds of polymer were synthesized and correspondingly characterized. The five kinds of pyrene conductive polymer had outstanding properties in terms of solubility, fluorescence intensity and thermal stability, good film-forming properties, stable electrochromic properties and high coloring efficiency. The coloration efficiency (CE) of PPYTP was as high as 277 cm2 C-1, and the switching response time was short. The coloring time of PPYEDOT was 1.3 s and the bleaching time was 3.2 s. The lower impedance will also provide the possibility of such polymers being incorporated into electrochromic devices in the future. In short, the synthesized new pyrene conductive polymers will have wide application prospects in the field of electrochromic materials.

Effective genome editing and identification of a regiospecific gallic acid 4-O-glycosyltransferase in pomegranate (Punica granatum L.).

Pomegranate (Punica granatum L.) trees are woody perennials that bear colorful and nutritious fruits rich in phenolic metabolites, e.g., hydrolyzable tannins (HTs) and flavonoids. We here report genome editing and gene discovery in pomegranate hairy roots using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), coupled with transcriptome and biochemical analyses. Single guide RNAs (sgRNAs) were designed to target two UDP-dependent glycosyltransferases (UGTs), PgUGT84A23 and PgUGT84A24, which possess overlapping activities in ß-glucogallin (a galloylglucose ester; biosynthetic precursor of HTs) biosynthesis. A unique accumulation of gallic acid 3-O- and 4-O-glucosides (galloylglucose ethers) was observed in the PgUGT84A23 and PgUGT84A24 dual CRISPR/Cas9-edited lines (i.e., ugt84a23 ugt84a24) but not the control (empty vector) or PgUGT84A23/PgUGT84A24 single edited lines (ugt84a23 or ugt84a24). Transcriptome and real-time qPCR analyses identified 11 UGTs with increased expression in the ugt84a23 ugt84a24 hairy roots compared to the controls. Of the 11 candidate UGTs, only PgUGT72BD1 used gallic acid as substrate and produced a regiospecific product gallic acid 4-O-glucoside. This work demonstrates that the CRISPR/Cas9 method can facilitate functional genomics studies in pomegranate and shows promise for capitalizing on the metabolic potential of pomegranate for germplasm improvement.

[Effects of tongue acupuncture combined with rehabilitation training group of dysarthria on speech function in post-stroke dysarthria patients].

OBJECTIVE: To observe the effects of tongue and nape acupuncture combined with rehabilitation training group of dysarthria on speech function in post-stroke dysarthria patients, and to explore the treatment of dysarthria. METHODS: Eighty patients with dysarthria were randomly divided into an observation group (40 cases) and a control group (40 cases). The patients in the two groups were treated with conventional treatment. The patients in the control group were treated with the acupuncture combined with rehabilitation training group of dysarthria; the patients in the observation group were treated with the control group treatment and tongue acupuncture, once a day, 6 days per week for 2 weeks. The patients were evaluated with general dysarthria scale and dysarthria checklist of Chinese Rehabilitation Study Center before and after 2-week treatment. RESULTS: After treatment, the total score and each item score of general dysarthria scale were reduced (all P<0.05); all the score in the observation group was lower than those in the control group (all P<0.05), except the score of jaw which had no significant difference between the two groups. After treatment, the dysarthria checklist of Chinese Rehabilitation Study Center in the observation group was superior to that in the control group (P<0.05). The total effective rate was 85.0% (34/40) in the observation group, which was higher than 67.5% (27/40) in the control group (P<0.05). CONCLUSION: Tongue acupuncture, nape acupuncture and rehabilitation training group of dysarthria could effectively improve the speech function of post-stroke dysarthria patients.

Genomic insights into divergence and dual domestication of cultivated allotetraploid cottons.

BACKGROUND: Cotton has been cultivated and used to make fabrics for at least 7000 years. Two allotetraploid species of great commercial importance, Gossypium hirsutum and Gossypium barbadense, were domesticated after polyploidization and are cultivated worldwide. Although the overall genetic diversity between these two cultivated species has been studied with limited accessions, their population structure and genetic variations remain largely unknown. RESULTS: We resequence the genomes of 147 cotton accessions, including diverse wild relatives, landraces, and modern cultivars, and construct a comprehensive variation map to provide genomic insights into the divergence and dual domestication of these two important cultivated tetraploid cotton species. Phylogenetic analysis shows two divergent groups for G. hirsutum and G. barbadense, suggesting a dual domestication processes in tetraploid cottons. In spite of the strong genetic divergence, a small number of interspecific reciprocal introgression events are found between these species and the introgression pattern is significantly biased towards the gene flow from G. hirsutum into G. barbadense. We identify selective sweeps, some of which are associated with relatively highly expressed genes for fiber development and seed germination. CONCLUSIONS: We report a comprehensive analysis of the evolution and domestication history of allotetraploid cottons based on the whole genomic variation between G. hirsutum and G. barbadense and between wild accessions and modern cultivars. These results provide genomic bases for improving cotton production and for further evolution analysis of polyploid crops.

[Effect of early rehabilitation combined with abdomen needle therapy for motor function and psychological obstacle of stroke].

OBJECTIVE: To study the clinical effect of early rehabilitation combined with abdomen needle therapy for the motor function and psychological obstacle of stroke based on the western medical treatment. METHODS: Ninety patients with acute stroke were randomly divided into an observation group A,an observation group B and a control group, 30 cases in each group. Conventional western medical treatment and early rehabilitation for the affected limbs wete applied in the three groups for one month. Besides,abdomen needle therapy was used on Qihai(CV 6),Guanyuan(CV 4),Tianshu(ST 25),Daheng(SP 15),Huaroumen(ST 24),Wailing(ST 26),Shangfengshidian,Shangfengshiwaidian,Xiafengshidian and Xiafengshixiadian in the observation group A. The needles were retained for 20 min without activity of the affected limbs. Based on the treatment as the group A,early rehabilitation was adopted in the observation group B. All treatment was given once a day,and 10 times were taken as a course. Three courses were required with two days at the interval. Fugl-Meyer assessment(FMA),Hamilton anxiety scale(HAMA),Hamilton depression scale(HAMD) and China stroke scale(CSS) were used before and after treatment. RESULTS: Scores of FMA、HAMA、HAMD and CSS after treatment were improved than those before treatment in the three groups(all P<0.05). All the above scores of the two observation groups were better than those of the control group(all P<0.05). The scores of HAMA and HAMD of the observation group B were superior to those of the observation group A(both P<0.05). CONCLUSIONS: Conventional treatment combined with abdomen needle therapy can improve the motor function and the psychological obstacle of stroke,and the effect is better than that of the conventional treatment. Early rehabilitation based on the two therapeutic methods can help relieve psychological status.

Insights into Interspecific Hybridization Events in Allotetraploid Cotton Formation from Characterization of a Gene-Regulating Leaf Shape.

The morphology of cotton leaves varies considerably. Phenotypes, including okra, sea-island, super-okra, and broad leaf, are controlled by a multiple allele locus, L2 Okra leaf (L2°) is an incomplete mutation that alters leaf shape by increasing the length of lobes with deeper sinuses. Using a map-based cloning strategy, we cloned the L2 locus gene, which encodes a LATE MERISTEM IDENTITY 1 (LMI1)-like transcription factor (GhOKRA). Silencing GhOKRA leads to a change in phenotype from okra to broad leaf. Overexpression of GhOKRA in Arabidopsis thaliana greatly increases the degree of the leaf lobes and changes the leaf shape. Premature termination of translation in GhOKRA results in the production of broad leaves. The sequences of OKRA from diploid progenitor D-genome species, and wild races and domesticated allotetraploid cottons in Gossypium hirsutum show that a premature termination mutation occurred before and after the formation of tetraploid cotton, respectively. This study provides genomic insights into the two interspecific hybridization events: one produced the present broad leaf and another formed okra leaf phenotype with complete OKRA, that occurred during allotetraploid cotton formation.

Transcriptome Analysis of Short Fiber Mutant Ligon lintless-1 (Li1) Reveals Critical Genes and Key Pathways in Cotton Fiber Elongation and Leaf Development.

For efficient spinning and superior fabric production, long fiber length is a desired trait for cotton production. To unveil the molecular basis of the cotton fiber length regulation, a short fiber mutant, Ligon lintless-1 (Li1), is selected to compare with its corresponding wild type (WT). Li1 is a monogenic dominant cotton mutant causing extremely short fibers (<6mm) on mature seeds with visible pleiotropic effects on vegetative growth and development. In this research, we compared the transcriptome of fiber bearing ovules at 1 DPA, 3 DPA, 8 DPA and leaf between Li1 mutant and WT. A total of 7,852 differentially expressed genes (DEGs) were detected in ovules and leaves, which mainly participated in sugar, secondary metabolite and lipid metabolism pathways based on KEGG analysis. The common DEGs at 1 DPA and 3 DPA were involved in the responses to endogenous stimulus, signal transduction and long-chain fatty acid biosynthesis. For 3 DPA, 8 DPA and leaf, the common DEGs were involved in the responses to auxin and receptor kinases related pathway. Further analysis showed that 37 genes involved in very-long-chain fatty acid biosynthesis were suppressed in Li1 mutant during fiber fast elongation development. Most of the DEGs involved in cell wall metabolism, such cellulose synthase, expansin family, and glycosyl hydrolase were differentially expressed at 3 DPA and 8 DPA. Our results provide new insights into the mechanisms of fiber elongation, and offer novel genes as potential objects for fiber length improvement.

Genome-wide mining, characterization, and development of microsatellite markers in gossypium species.

Although much research has been conducted to characterize microsatellites and develop markers, the distribution of microsatellites remains ambiguous and the use of microsatellite markers in genomic studies and marker-assisted selection is limited. To identify microsatellites for cotton research, we mined 100,290, 83,160, and 56,937 microsatellites with frequencies of 41.2, 49.1, and 74.8 microsatellites per Mb in the recently sequenced Gossypium species: G. hirsutum, G. arboreum, and G. raimondii, respectively. The distributions of microsatellites in their genomes were non-random and were positively and negatively correlated with genes and transposable elements, respectively. Of the 77,996 developed microsatellite markers, 65,498 were physically anchored to the 26 chromosomes of G. hirsutum with an average marker density of 34 markers per Mb. We confirmed 67,880 (87%) universal and 7,705 (9.9%) new genic microsatellite markers. The polymorphism was estimated in above three species by in silico PCR and validated with 505 markers in G. hirsutum. We further predicted 8,825 polymorphic microsatellite markers within G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. In our study, genome-wide mining and characterization of microsatellites, and marker development were very useful for the saturation of the allotetraploid genetic linkage map, genome evolution studies and comparative genome mapping.

Sequence-based ultra-dense genetic and physical maps reveal structural variations of allopolyploid cotton genomes.

BACKGROUND: SNPs are the most abundant polymorphism type, and have been explored in many crop genomic studies, including rice and maize. SNP discovery in allotetraploid cotton genomes has lagged behind that of other crops due to their complexity and polyploidy. In this study, genome-wide SNPs are detected systematically using next-generation sequencing and efficient SNP genotyping methods, and used to construct a linkage map and characterize the structural variations in polyploid cotton genomes. RESULTS: We construct an ultra-dense inter-specific genetic map comprising 4,999,048 SNP loci distributed unevenly in 26 allotetraploid cotton linkage groups and covering 4,042 cM. The map is used to order tetraploid cotton genome scaffolds for accurate assembly of G. hirsutum acc. TM-1. Recombination rates and hotspots are identified across the cotton genome by comparing the assembled draft sequence and the genetic map. Using this map, genome rearrangements and centromeric regions are identified in tetraploid cotton by combining information from the publicly-available G. raimondii genome with fluorescent in situ hybridization analysis. CONCLUSIONS: We report the genotype-by-sequencing method used to identify millions of SNPs between G. hirsutum and G. barbadense. We construct and use an ultra-dense SNP map to correct sequence mis-assemblies, merge scaffolds into pseudomolecules corresponding to chromosomes, detect genome rearrangements, and identify centromeric regions in allotetraploid cottons. We find that the centromeric retro-element sequence of tetraploid cotton derived from the D subgenome progenitor might have invaded the A subgenome centromeres after allotetrapolyploid formation. This study serves as a valuable genomic resource for genetic research and breeding of cotton.