The independent variable of this research was information technology , whereas the dependent variable was competitive advantage. The study found that IT had a significant impact on the competitive advantage of logistics SMEs operating in Pakistan. All the variables related to IT had a significant impact on competitive advantage, which included IT valence, IT resource commitment, IT managerial commitment, and IT competency. This study helps managers and owners of logistics SMEs in decision-making, who can understand how much IT can enhance their performance and reduce their risks. This study has been specifically conducted with logistics SMEs operating in Pakistan, which means that there is much scope to be worked upon, i.e., by selecting companies operating in other countries and comparing them with current findings. This study observes the impact of information technology  on competitive advantage, and other independent variables can be studied to find the impact on competitive advantage.The aim of this study was to examine the importance of information technology for logistics Small Medium Entreprise (SMEs) in Pakistan. It is the time of technological rapidness; especially after COVID-19, the word business has majorly transformed into a digital business. If an organization did not shift toward technology, it would be hard for it to even sustain in this rapid era. This study adopts the questionnaire after extensive literature review. A quantitative study was conducted among logistics SMEs in Pakistan to empirically verify what competitive advantages they are leading and gaining from information technology and how much information technology is important for their sustainability. The literature lacks information about the ways in which information technology has been integrated into logistics SMEs operating model, and more specifically, there is no information about IT valence, IT resource commitment, IT managerial commitment, and IT competency. The research is primarily quantitative in nature, where the data were collected  The logistics industry has developed essentially while logistics has turned into a significant piece of the business financial framework and major worldwide economic activity lately. Logistics has quickened financial development and efficiency development. Productive logistics is a significant determinant of a nation’s intensity . The mosCompetitive advantage is the reason for greater execution. Understanding the life structures of competitive advantage is of vital significance to senior supervisors who bear a definitive obligation regarding an association’s long-haul endurance and achievement. Dissecting the reasons for competitive advantage enables an SME to make and pick up an advantage . In the Given the case of the logistics industry in Pakistan, the CPEC project has been acknowledged as a game changer for not only the logistics SMEs but also for its complement for the economies that are directly or indirectly related to Pakistan . This can be attributed to the fact that this project is intended to focus on the development of a national logistics network that would result in the expansion of distribution and warehousing networks; however, the lack of sufficient logistics and processing facilities is expected to result in a challenging situation for the agricultural products . HoweverThe term competitive advantage refers to the conditions or circumstances that put companies in a superior business position . This coIn the technologically advanced era, information technology has become a necessity for SMEs operating in different fields and domains, which means that the logistics industry is no exception to it. Considering this aspect, in the studies conducted by Information technology commitment has been defined as the commitment to work on the improvement of IT, which is not only related to resources but also to its components including equipment, budget, and even personnel, whereas studies have also integrated managerial commitment within it as well . In specH1a: IT resources commitment has a positive impact on competitive advantages.Companies, irrespective of their scope and nature of operations, operate in a technologically advanced era, and this has made it exceptionally important for companies to support their operational procedures and processes through technological integration . This caH2a: IT competency has a positive impact on competitive advantage.From an organizational perspective, IT valence reflects the beliefs of the top management within an organization with respect to IT. In this specific context, studies have reflected that it serves as a sign of the managerial community about the importance that must be placed on the information technology . When thH3a: IT valence has a positive impact on competitive advantage.Organizations operate in an intensely competitive business landscape, and this has made the companies shift their attention from traditional operations to IT-supported operations . WithoutH4a: IT managerial commitment has a positive impact on competitive advantage.The study framework being worked upon is related to the influence of IT on the competitive advantage of logistics SMEs. As guided by the literature, the variables being investigated include IT resource commitment, IT competency, IT valence, and IT managerial commitment with respect to competitive advantage. This framework is shown in The information presented within the previous sections has led to the development of the following hypotheses:H1a: IT resources commitment has a positive impact on competitive advantages.H2a: IT competency has a positive impact on competitive advantage.H3a: IT valence has a positive impact on competitive advantage.H4a: IT managerial commitment has a positive impact on competitive advantage.This section has described and explained the materials being used in this study, followed by the methodology being used in it. The section has been developed in accordance with the research questions and entails information related to design and procedures adopted. For this section, There are 1,130 logistics SMEs operating in Pakistan. This number of listed logistics SMEs operating in Karachi was available in the The first general question asked by the logistics SMEs operating in Karachi was related to the years of operations in the field refer to . This leThe next general question asked by the logistics SMEs was related to the total number of employees refer to , and it For this study, the data have been collected from the website of the Once the questionnaire was finalized, it was reviewed by the industry and academic experts, who highlighted some issues within the questionnaire. Based on their review and feedback, the questionnaire was revised.A Cronbach’s alpha test was conducted on the questionnaire to see whether the findings from the instrument were reliable or not. The findings for the entire questionnaire are represented in This section of the study has specifically focused on testing the hypothesis, but before doing so, it is important to highlight some general information that was collected through the questionnaire.R-Square value is 0.452, it can be argued that the predictors can only explain 45.2% variation in the competitive advantage of logistics SMEs. This suggests that apart from information technology, there are several other factors that can influence the competitive advantage of logistics SMEs, which the study has not taken into account because of its exclusive focus on IT.Regression analysis was conducted in this study to identify whether the IT-related variables had an impact on the competitive advantage of the logistics SMEs or not. Considering this particular purpose, this statistical test was conducted, and it helped in either accepting or rejecting the hypotheses. The first results were related to IT valence, which was tested for its impact on the CA of logistics SMEs. The Sig. value of the ITV was 0.002, which was less than 0.05; hence the null hypothesis has been rejected and the alternate hypothesis has been accepted, which means that IT valence has a significantly positive impact on the competitive advantage of logistics SMEs. Another important aspect that must be looked into is the beta value, which was 0.120 for this variable, and this identified that any change in the IT valence by the logistics SMEs could result in a 12% change in their competitive advantage.The results related to IT competency, which was tested for its impact on the CA of logistics SMEs. The Sig. value of the ITC was 0.019, which was less than 0.05; hence the null hypothesis has been rejected and the alternate hypothesis has been accepted, which means that IT competency has a significantly positive impact on the competitive advantage of logistics SMEs. The beta value was 0.085 for this variable, and this helped to understand that any change in the IT competency by the logistics SMEs could result in a 8.5% change in their competitive advantage.The results related to IT resource commitment, which was tested for its impact on the CA of logistics SMEs. The Sig. value of the ITC was 0.000, which was less than 0.05; hence the null hypothesis has been rejected and the alternate hypothesis has been accepted, which means that IT resource commitment has a significantly positive impact on the competitive advantage of logistics SMEs. Another important aspect that must be looked into is the beta value, which was 0.197 for this variable, and this identified that any change in the IT resource commitment by the logistics SMEs could result in a 19.7% change in their competitive advantage.The results related to IT managerial commitment, which was tested for its impact on the CA of logistics SMEs. The Sig. value of the ME was 0.027, which was less than 0.05; hence the null hypothesis has been rejected and the alternate hypothesis has been accepted, which means that IT managerial commitment has a significantly positive impact on the competitive advantage of logistics SMEs. Another important aspect that must be looked into is the beta value, which was 0.099 for this variable, and this identified that any change in the IT managerial commitment by the logistics SMEs could result in a 9.9% change in their competitive advantage.When IT resource commitment was tested for its impact on competitive advantage, it was found that it had a significant impact, which is similar to the studies conducted previously. For instance, When IT valence was tested for its impact on competitive advantage, it was found that it had a significant impact, which is similar to the studies conducted previously. This can be confirmed through earlier conducted studies, which suggested that valence and its underlying beliefs offer guidance to the managers and the internal stakeholders about the risks and opportunities associated with the application of information technology. When the managers are informed about the beliefs and the opportunities that may be available for the SMEs, it can influence them to develop specific behavior toward IT integration within the company . In specThe study has contributed to the literature by adding sufficient and relevant information related to IT and its impact on competitive advantage of logistics SMEs. Since there is no particular knowledge and information related to the Karachi-based logistics SMEs, this study and its findings have filled the void in the literature. Managers employed in different logistics SMEs in Karachi and all over Pakistan can take advantage of this study and its findings, since they can enhance their knowledge related to IT and the ways in which it can improve organizational performance and competitive advantage. The findings from this research can change the ways in which the logistics SMEs have been operating for several years; for instance, the findings can help logistics SMEs to comprehend IT as a necessity instead of being an option for these SMEs and their operations. This can bring a radical change in managerial commitment and behavior and would ensure that IT is integrated within all operations to enhance the likelihood of achieving and improving competitive advantage.The key recommendations to further add value and to bring improvement in competitive advantage are as follows. Logistics SMEs are being recommended to understand the value and significance of IT integration and the ways in which this can help in bringing improvement in performance and productivity, hence leading to an overall improvement in competitive advantage. Even when IT resource commitment had a low significant impact on competitive advantage compared to other variables, it is being recommended that this factor should not be underestimated since it can serve as a platform through which managers can change their opinions and beliefs about information technology and can help in learning about the opportunities and benefits that IT can offer in the future.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.DL: conceived idea and introduction. MS: introduction and literature review. MQ: research methodology. MA: data collection. FM: data analysis. HK: results and discussion. JM: supervision, proofread, and addressed remarks of the reviewers. All authors contributed to the article and approved the submitted version.


An intercalary segmental allograft is an option for limb salvage in bone tumours. Stable and congruent intercalary reconstructions are a prerequisite for achieving host-graft union. However, a too rigid fixation could increase the risk of late complications correlated with negative bone remodelling. This study compared the reconstruction stiffness achieved by three different host-graft junctions, namely, end-to-end, modified step-cut, and taper. A low-stiffness bone plate was used as the fixation method, except for the taper junction where a low-stiffness intramedullary nail was also used to investigate the effects of different types of fixation on construct stiffness. Composite femora were tested under four loading conditions to determine coronal and sagittal bending stiffness, as well as torsional stiffness in opposite directions. Stiffness values were expressed as a percentage of intact host bone stiffness (%IBS). While a reduction of coronal bending stiffness was found with taper junctions (76%IBS) compared with the high values ensured by end-to-end (96%IBS) and modified step-cut junctions (92%IBS), taper junctions significantly increased stiffness under sagittal bending and torsion in intra- and extra-direction: end-to-end 29%IBS, 7%IBS, 7%IBS, modified step-cut 38%IBS, 20%IBS, 21%IBS, and taper junction 52%IBS, 55%IBS, 56%IBS, respectively. Construct stiffness with taper junctions was decreased by 11–41%IBS by replacing the bone plate with an intramedullary nail. Taper junctions can be an alternative to achieve intercalary reconstructions with more homogeneous and, in three out of four loading conditions, significantly higher construct stability without increasing bone plate stiffness. The risk of instability under high torsional loads increases when taper junctions are associated with a low-stiffness intramedullary nail. Intercalary reconstruction surgery allows limb preservation after the removal of bone tumours located in long-bone diaphysis and metaphysis. Among the reconstructive options, surgeons can choose to perform an intercalary massive allograft, possibly combined with a vascularised fibular autograft , 2.Besides infection, the two other major complications associated with an intercalary massive allograft are non-union and late intercalary reconstruction fracture –5.In general, patients’ habits and health status have a significant impact on the risk for non-union –11. For Late intercalary reconstruction fractures have been observed in physically active patients or due to local stress concentration in the allograft area , 19–22.a priori. Since this might contribute to the occurrence of intercalary reconstruction fracture occurring in long-term follow-up  The bending and torsional stability of intercalary reconstructions with three different host-graft junctions, namely, end-to-end, step-cut, and taper. All intercalary reconstructions were fixed with a low-stiffness bone plate [unicortical fixation ], to det(ii) The bending and torsional stability of an intercalary reconstruction fixed with two different types of fixation, namely, low-stiffness bone plate and intramedullary nail. Both intercalary reconstructions included taper junctions to evaluate the effects of the types of fixation on the construct stiffness.All intercalary reconstructions were performed using composite femora, which are suitable for comparative biomechanical studies. A medium-size femur  was chosen as a model of the diseased femur, hereinafter referred to as “host bone”, of a 15-year-old boy. This model was selected using (i) the 50th percentile for height equal to 170 cm  and (ii)A reference system was marked on all the femora prior to osteotomy , 33. TheTwo types of fixation devices were used to stabilise the intercalary reconstruction, namely, bone plate and intramedullary nail. Low stiffness implants  were used to decrease the mechanical contribution of the fixation device and, at the same time, enhance the contribution of the shape of the two host-graft junctions to the intercalary reconstruction stiffness.The length of diaphysis resection in the host bone was determined by considering that the whole intercalary reconstruction had to undergo uniform bending and torsional moment. The longitudinal length of the resection had to be shorter than the distance between the inner loading rollers 200 mm,  in the fAdditionally, the longitudinal length of diaphysis resection had to be a multiple of the centre-to-centre distance between holes of the bone plate to achieve a 9-mm longitudinal shift of the screw axis with respect to the two diaphyseal osteotomies. To satisfy these requirements, the length of diaphysis resection was set at 72 mm. Therefore, the two osteotomies were performed 36 mm apart from the midpoint of the biomechanical length of all host bones.The massive graft was extracted from the diaphysis of 12 donor segments. Two osteotomies were performed 36 mm apart from the midpoint of the biomechanical length to extract the grafts for the intercalary reconstruction with the end-to-end junctions. The aforementioned distance was increased to 54 mm to provide an 18-mm overlap of the massive graft for intercalary reconstruction with the step-cut or taper junctions on each side . Prior tWe investigated three different intercalary reconstruction techniques. They differed in the shape of the following two host-graft junctions: (i) two end-to-end junctions, achieved by cutting both the host bone and the graft orthogonally to the longitudinal axis  and (ii)The reference system marked on each femur was used to align the two host bone stumps and the massive graft properly and to restore the anteroversion angle of the femoral neck. The host bone stumps were kept in the desired spatial position, i.e., aligned with the lateral side oriented upward to make plate or nail fixation easier, by means of two-part rigid moulds clamping the proximal and distal epiphysis. The massive graft was medially supported, except for those with taper junction that was self-sustaining once assembled.Bridging plate or nail fixation was performed following the operative technique provided by the manufacturer.The bone plate was placed on the lateral side of the diaphysis with the midplate aligned with the midpoint of the massive graft. It was not necessary to bend the plate to match the lateral contour of the intercalary reconstruction. Starting from the most proximal and distal hole and proceeding toward the middle of the plate, (i) a 3.2-mm hole was drilled using the LCP drill guide in a neutral position except for the most proximal hole, which was instead drilled with the drill guide in eccentric position to achieve dynamic compression; (ii) the screw length was determined using a depth gauge; (iii) the hole was tapped for a 4.5-mm cortex screw; (iv) the screw of appropriate length was manually inserted and tightened to 5 Nm. Screws were inserted on the three most proximal and distal holes of the plate , except The intramedullary nail was inserted on the great trochanter. A 10-mm hole was drilled on the tip of the great trochanter and manually reamed using the specific tool. The inner part of the distal stump was reamed using an 11-mm flexible reamer, i.e., 1 mm larger than the diameter of the nail. Following nail insertion, the lateral guide was used to drill a transversal pre-hole and insert the most proximal 5-mm self-tapping screw. The nail was locked distally with two transversal 5-mm self-tapping screws inserted after having drilled two pre-holes using the targetting device. Slightly longitudinal compaction was achieved by tightening the proximal axial screw to 3 Nm. Finally, a second proximal hole was drilled using the targetting device, and the second proximal 5-mm self-tapping screw was inserted .Bending and torsion testing was performed to determine the stiffness of the 12 intercalary reconstructions and three intact host bones, whose average value was used as a reference to express the stiffness of each intercalary reconstruction as a percentage of intact host bone stiffness (%IBS).All femora were tested under four-point loading conditions. The support and loading spans were 300 and 200 mm, respectively . The heiA preload of 0.05 kN was applied to the femur using a material testing machine . Then, a monotonic ramp was applied in load control at a rate of 0.1 kN/s up to 1.0 kN, corresponding to a bending moment of 25 Nm. This value roughly simulates the bending moment occurring in the femur of a 60 kg active patient during daily activity . The verAll femora underwent torsional testing allowing free motion of the femur in the transversal plane. Before testing, each femur was then oriented vertically in an upside-down position using a three-degree of freedom clamping device. The femoral head was embedded in acrylic resin into a metal holder, leaving 150 mm between the mould surface and the midpoint of the femoral biomechanical length. The metal holder was fixed to a rail bearing and mounted orthogonally to the actuator of a biaxial testing machine . The femoral axis was aligned with the load cell axis. Finally, the distal condyles were embedded in acrylic resin into a metal holder mounted onto the load cell, leaving a free length of 300 mm of the femur .A pretorque of 1 Nm was applied to the femur. Then, a monotonic ramp was applied in torque control at a rate of 3 Nm/s up to 25 Nm, i.e., 0.042 BWm , except The slope, i.e., the stiffness, of each load-displacement or torque-rotation curve was calculated to determine the slope of the best-fit line for each dataset. An iterative algorithm, which essentially consisted in discharging the first pair of load and displacement values of the dataset and repeating the fitting process, was performed until the coefficient of determination (R2) of the line of best fit reached the value of 0.995. Therefore, the non-linear response (if any) of the intercalary reconstruction at very-low load values was neglected.The differences, in terms of bending or torsional stiffness, between different intercalary reconstructions were evaluated using the Friedman test. A non-parametric multiple comparison test (Conover’s test), based on the median rank differences of the groups, was used to analyse stiffness values determined for the three intercalary reconstructions stabilised with bone plates differing in junction geometry .A total of 75 monotonic tests were carried out. The mechanical response to bending or torsional load of the intercalary reconstructions, including different junction geometries, was extremely linear . The onlP < 0.01 in all four cases). The junction geometry impacted the intercalary reconstruction stiffness in different ways. The intercalary reconstruction with the end-to-end or modified step-cut junctions showed a bending stiffness in the coronal plane significantly higher compared with the intercalary reconstruction with taper junctions . The type of fixation consistently impacted the intercalary reconstruction stiffness: the fixation with bone plate provided higher construct stiffness than the intramedullary nail, regardless of the loading configuration  the use of synthetic femora neglects any tissue and/or anatomical difference among patients; (ii) intercalary reconstructions were performed under optimal conditions, i.e., achieving a perfect match at the host-graft interface and neglecting the presence of surrounding soft tissues; (iii) monotonic mechanical tests were performed aiming at investigating the postoperative mechanical condition and neglecting mid- and long-term changes in stiffness of the intercalary reconstructions due to biological processes.Nevertheless, this proof-of-concept study provides experimental evidence that the host-graft junction has a significant impact on the bending or torsional stiffness of the intercalary reconstruction. The taper junction, fixed with the bone plate, works on the same principle of a one-piece setscrew collar combined with lateral reinforcement. This solution increases the load-carrying capacity of the host-graft junction, especially the torsional one. In fact, the largest improvements were found in torsional stiffness values. Replacing the bone plate with an intramedullary nail changes the load transfer mechanism, especially the torsional one . In factBending stiffness can be divided into sagittal and coronal bending stiffness. Regardless of the type of fixation, replacing end-to-end or step-cut junctions with conical ones, the sagittal bending stiffness of the intercalary reconstruction is increased. This is hardly surprising, since, under sagittal bending moments, both bone plate and intramedullary nail are roughly aligned with the neutral axis, and therefore, their contribution to the second moment of inertia of the construct is small. Under this condition, the conical junction contributes significantly to the sagittal bending stiffness by ensuring load transfer further away from the neutral axis compared with both fixation types. This argument still applies to the intercalary construction that is fixed using the intramedullary nail when the construct undergoes coronal bending moments. Conversely, the bone plate is distant, on the tension side, from the neutral axis during the coronal bending moment. Therefore, it counteracts the lateral tensile load while the host-graft interface undergoes non-uniform compressive stress, creating a pivot point at the far cortex. Replacing the end-to-end with a step-cut junction determines a small medial shift of the pivot point with negligible effects on bending stiffness. Conversely, the taper junction changes the interface geometry leading to a more marked medial shift of the pivot point, resulting in a decrease in bending stiffness. However, the coronal bending stiffness still remains high, the reconstruction providing a median stiffness value of 76% IBS.It must be acknowledged that the aforementioned load transfer mechanisms are greatly affected by the stiffness of the fixation devices: the stronger the fixation device, the stiffer the intercalary reconstruction . Increasin vivo. It could be argued that the need to reaming both stump and graft ends further complicates an already complex surgery. However, the use of conical reamers would make the additional step easier (Regardless of the load transfer mechanism, the taper junction allows to achieve an intercalary reconstruction with a more homogeneous response to different loading conditions, which should ensure a more stable reconstruction despite the complex loading condition it undergoes p easier , ensurinp easier , 56, 57.The findings of this study show that intercalary reconstructions with taper junctions, fixed with a low-stiffness bone plate, demonstrate, under four different loading conditions, greater homogeneity and, in three out of four conditions, higher mechanical stiffness compared with intercalary reconstruction with end-to-end or modified step-cut junctions. Replacing the bone plate with an intramedullary nail decreases the stiffness of the intercalary reconstruction, especially in terms of torsional stability. Regardless of the type of fixation, tapering of the stump and graft ends increases the contact between the graft and viable host bone tissue, with a potential beneficial effect on cellular colonisation of the graft ends.The taper junction with low-stiffness fixation could be a useful alternative to achieve intercalary reconstructions with stiff responses to different loading conditions and reduce the risk of late cortical atrophy.The raw data supporting the conclusions of this article will be made available by the corresponding author upon request, without undue reservation.MBa and MM contributed to the conception and design of the study and supervised all project tasks. MP performed the surgical tasks. PE, MBl, and RF prepared specimens, performed mechanical tests, and collected and analysed the data. MBa wrote the first draft of the manuscript. MM wrote clinical sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


Acinetobacter sp. 1H8 and Acinetobacter indicus 3B2) were inoculated into CTLs. The quality of CTLs were significantly improved after fermentation. The content of solanone, 6-methyl-5-hepten-2-one, benzeneacetic acid, ethyl ester, cyclohexanone, octanal, acetophenone, and 3,5,5-trimethyl-2-cyclohexen-1-one were significantly increased after inoculated Acinetobacter sp. 1H8. The inoculation of Acinetobacter sp. 1H8 enhanced the normal evolutionary trend of bacterial community. The content of trimethyl-pyrazine, 2,6-dimethyl-pyrazine, and megastigmatrienone were significantly increased after inoculated Acinetobacter indicus 3B2. The inoculation of Acinetobacter indicus 3B2 completely changed the original bacterial community. Network analysis revealed that Acinetobacter was negatively correlated with Aquabacterium, positively correlated with Bacillus, and had significant correlations with many volatile flavor compounds. This work may be helpful for improving fermentation product quality by regulating microbial community, and gain insight into the microbial ecosystem.Metabolic activity of the microbial community greatly affects the quality of cigar tobacco leaves (CTLs). To improve the quality of CTLs, two extrinsic microbes ( Debaryomyces hansenii and Yarrowia lipolytica were incorporated into the cheese as part of the starter, the cheese could significantly shorten the ripening time while maintaining a good strong flavor  and the native microbes (intrinsic) lead to incomplete structure and function of microbial communities. Microbial interactions, such as symbiotic, synergistic, predation, parasitic, and competitive, can greatly affect the metabolic activity of the microbial communities . RationaAcinetobacter were the important producers of the aldehydes and ketones. And we isolated two flavor producers, Acinetobacter sp. 1H8 and Acinetobacter indicus 3B2 from high-quality cigar tobacco leaves (CTLs). We speculated that their addition could improve the quality of some ordinary CTLs. In this study, we investigated whether these two extrinsic stains could improve the quality and flavor of the CTLs and their effects on the structure and function of bacterial communities.Cigars are one of the oldest traditional tobacco fermented products . Flavor-Acinetobacter sp. 1H8 CGMCC NO.23678 and Acinetobacter indicus 3B2 CGMCC NO.23679.The strains used in this study were isolated from the CTLs and deposited in the China General Microbiological Culture Collection Center (CGMCC): 2HPO4 1.5 g/l NaH2PO4 3 g/l, and autoclaved at 121°C for 15 min. Strains were inoculated by sterile loops in 250-ml flasks with 50 ml fermentation medium, and cultured at 220 rpm 30°C for 36 h. Then the fermentation broth was inoculated into CTLs with 30% inoculation amount (v/g) and fermented in a biochemical incubator. All fermentations were conducted without agitation at 30°C for 15 days. The unfermented CTLs and uninoculated CTLs were prepared as Control 1 and Control 2, respectively. All experiments were performed in triplicate. The quality of CTLs was blindly assessed by three professional tasters. With 10–20 years of testing experience, these tobacco tasters have conducted sensory evaluation on more than 2,000 cigar samples, and can accurately, consistently, and repeatedly evaluate cigars.The fermentation medium for culturing strains was prepared with sucrose 20 g/l, peptone 20 g/l, yeast powder 15 g/l, KVolatile flavor compounds (VFCs) in CTLs were analyzed by headspace solid phase microextraction-gas chromatography–mass spectrometry (HS-SPME-GC–MS). CTLs were dried at 40°C and pulverized by a grinder. 1.5-g powder was placed in a 10 ml glass vial and extracted by headspace solid-phase microextraction  at 60°C for 30 min. After extraction, volatile flavor compounds (VCFs) were identified using a Pegasus BT GC-TOFMS , with a DB-5MS column (60 m × 0.25 mm id × 0.25 μm film thickness). Helium C-60 was used as a carrier gas with a flow rate of 1 ml/min, and the injector port was heated to 250°C. The oven temperature was fixed at 40°C for 2 min, increased to 250°C at a rate 10°C/min, and then held for 5 min. Meanwhile, the transfer line and ion source temperatures were maintained at 280°C and 210°C, respectively. Electron impact (EI) was used as the ionization mode, with an EI voltage of 70 eV, and a mass scan range of 33–400 m/z was used for full-scan mode with an acquisition rate of 10 scans/s.The acquired GC–MS raw data from the QC sample was analyzed with the Automatic Mass Spectral Deconvolution and Identification System (AMDIS) to verify individual analytes’ presence and deconvolute the co-eluting peaks as previously described. The parameters for the deconvolution were set as follows: component width = 20, adjacent peaks subtraction = 2, resolution = high, sensitivity = low, and shape requirements = medium. The parameters for peak detection were set at the default values. The detected compounds with an abundance of <1,000 and a signal-to-noise value of <50 were removed to discard identification errors or duplication. Compounds were identified by comparing the mass spectra and retention index with those in the NIST08 Mass Spectral Database, the Agilent Fiehn Metabolomics Retention Time Locked (RTL) Library, and an in-house library focused on tobacco metabolites. Standards for constructing the in-house library were prepared as described above. A SCAN + SIM method and an in-house automatic integration method were then established in the Agilent MSD Chem Station and applied to quantify the selective ion traces as previously described. Manual corrections were performed to guarantee the accuracy of the integration. A three-dimensional matrix was generated, including the sample information, peak retention time, and relative peak intensities. Internal standards and any known artificial peaks, such as peaks caused by noise, column bleed, and derivatization procedures, were removed from the matrix.g for 15 min. Total microbial genomic DNA was extracted using the DNeasy PowerSoil Kit , following the manufacturer’s instructions, and stored at −20°C until further analysis. The quality of extracted DNA was measured by agarose gel electrophoresis. PCR amplification of the bacterial and archaeal 16S rRNA genes V4–V5 region was performed using the forward primer 515F  and oscillated at 4°C, 220 rpm for 4 h. CTLs were removed by gauze, and microbes were collected by centrifuging at 7,000 × The 16S rRNA gene sequences were processed using QIIME 2 . Brieflyp < 0.05, |r| > 0.3), network analysis was performed by using Gephi software.Heat maps and cluster analyses were performed in the statistical environment R v. 4.0.0. The GalaxyAcinetobacter on original bacterial community, bacteria in CTLs with different treatments were sequenced. The high-throughput sequencing generated 642,151 sequence reads from sequence reads from 12 CTLs. After the quality control processing, including filtered, denoised, merged, and nonchimeric, there were 483,897 high-quality sequences, with an average of 40,324 sequences, and obtained 1,914 ASVs. The abundance of bacterial taxa was shown in Proteobacteria, Firmicutes, and Bacteroidetes were predominant phyla, Aquabacterium, Bacillus, Acinetobacter, Muribaculaceae, and Pseudomonas were predominant genera. There was no doubt that fermentation changed the composition of bacterial communities. At phyla level, the abundance of Proteobacteria and Firmicutes increased from 59.9%5 and 9.98% to 77.59% and 15.58% after fermentation, respectively, while the abundance of Bacteroidetes decreased from 23.10% to 2.16%. At genera level, the abundance of Aquabacterium increased from 14.26% to 41.08%, while the abundance of Muribaculaceae and Pseudomonas decreased from 17% and 14.04% to <0.01%. Similarly, external microorganisms also changed the composition of bacterial communities. At phyla level, the abundance of Proteobacteria increased from 59.95% to 95.61% after inoculation with Acinetobacter sp. 1H8 and decreased to 37.63% after inoculation with Acinetobacter indicus 3B2, while the abundance of Bacteroidetes decreased from 15.58% to 3.1% after inoculation with Acinetobacter sp. 1H8 and increased to 37.63% after inoculation with Acinetobacter indicus 3B2. At genera level, the abundance of Aquabacterium increased from 41.08% to 59.01% after inoculation with Acinetobacter sp. 1H8 and decreased to 27.61% after inoculation with Acinetobacter indicus 3B2. The abundance of Bacillus and Acinetobacter increased from 1.83% and 0.62% to 33.68% and 22.43% after inoculation with Acinetobacter indicus 3B2, respectively.To explore the influence of the inoculation of Acinetobacter indisputably altered the structure of bacterial community.The alpha diversity of the bacterial community was evaluated by indices including Chao1 index, Shannon index, and Simpson index, in which the first and the latter two represent richness and diversity, respectively . They alp < 0.05), which consist of 5 phyla, 4 classes, 18 orders, 25 families, and 29 genera. In detail, 17 genera were significantly enriched in uninoculated CTLs, such as Rhodococcus, Blastococcus, Promicromonospora, and Sanguibacter. Eight genera were significantly enriched in unfermented CTLs, such as Lactobacillus, Sphingobium, and Rhizopus. Two genera were significantly enriched in CTLs inoculated Acinetobacter indicus 3B2, such as Bacillus and Acinetobacter. Only Aquabacterium was significantly enriched in CTLs inoculated Acinetobacter sp. 1H8.To explore the different bacteria among CTLs with different treatments, LEfSe analysis was conducted to reveal the significant differences below the level of phylum . The cirAcinetobacter sp. IH8 and uninoculated CTLs were classified into one cluster with the similar abundance of bacterial metabolic pathway, this may be because they have similar bacterial community. The addition of external microorganisms not only changed the structure of bacterial community, but also changed the function of bacterial community. The abundance of a large number of bacterial metabolic pathways decreased significantly after inoculation. LEfSe analysis was conducted to identify the significant different bacterial metabolic pathways in CTLs with different treatments -6,10-dimethyl-5,9-undecadien-2-one, and (E)-3,7-dimethyl-2,6-octadienal were decreased after fermentation. VFCs of inoculated CTLs varied significantly compared to uninoculated CTLs.p < 0.05). In detail, six VFCs were significantly enriched in uninoculated CTLs, such as acetic acid, ethyl ester, benzeneacetaldehyde, and benzeneethanol. Eight VFCs were significantly enriched in unfermented CTLs, such as (E)-6,10-dimethyl-5,9-undecadien-2-one, acetic acid, and 6-methyl-3,5-heptadiene-2-one. Four VFCs were significantly enriched in CTLs inoculated Acinetobacter indicus 3B2, including neophytadiene, tetramethyl-pyrazine, 2,6-dimethyl-pyrazine, and megastigmatrienone. Seven VFCs were significantly enriched in CTLs inoculated Acinetobacter sp. 1H8, such as solanone, 6-methyl-5-hepten-2-one, and benzeneacetic acid, ethyl ester.LEfSe analysis was used to identify the significant different VFCs in CTLs with different treatments . Among tn:37) and representative bacteria (n: 20) were analyzed by Spearman’s correlation coefficients and visualized by Gephi. As shown in Acinetobacter were positively related to acetic acid, ethyl ester, 2-ethyl-1-hexanol, and megastigmatrienone, and negatively related to 6-methyl-5-hepten-2-one, hexanal, 3-methyl−butanal, acetophenone, benzeneacetic acid, ethyl ester, octanal, 3,5-octadien-2-one, and 2,6-dimethyl-2,6-octadiene. Aquabacterium and bacillus, which were significantly associated with Acinetobacter, were also related to many volatile flavor compounds. Aquabacterium was positively related to most volatile flavor compounds, while Bacillus was negatively related to most volatile flavor compounds.The changes of volatile flavor compounds were mainly caused by microbial metabolism, so it was very important to determine the relationship between bacteria and VFCs. The relationships between VFCs  were inoculated into CTLs, they exerted different effects on the original microbial community. The abundance of Acinetobacter remained stable in uninoculated CTLs after fermentation. When Acinetobacter sp. 1H8 was inoculated in CTLs, this strain was completely inhibited by the native microbiotas, the abundance of Acinetobacter was significantly reduced after fermentation. The inoculation of Acinetobacter sp. 1H8 caused the endogenous microbes to turn from competition to cooperation to compete together against the extrinsic microbes to suppress the extrinsic perturbation. Normally, the microbial community remains stable due to the complete suppression of extrinsic microbes by endogenous microbes  improved the quality and flavor of CTLs. Inoculated microbes can not only exert their own metabolic ability, but also affect the structure and function of native microbial community. We revealed that the interaction between exogenous microorganisms and native microbes, the different effects of exogenous microorganisms on original microbial community, the association between microbes and VFCs, and the formation mechanism of tobacco flavor. Collectively, our present work has proved the effect of inoculated microorganisms in traditional fermentation industry and elucidated changes in the overall structure and function of microbial communities after inoculation. These results suggest that controlling the microbial community could significantly improve the quality and safety of fermentation products, and this work may provide a good way to gain insight into the microbial ecosystem of traditional fermentation.When some traditional fermented products that are produced by spontaneous fermentation unable to meet the demands of consumers, inoculating extrinsic microbes may improve these products. In this work, we demonstrate that the inoculation of two extrinsic microbes (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI SRA BioProject, accession no: PRJNA813020.TZ: conceptualization, data curation, formal analysis, methodology, software, and writing—original draft. QiZ, QW, and PL: investigation, methodology, and resources. XW, QuZ, and WC: methodology, resources, and project administration. JZ, GD, and DL: funding acquisition, supervision, and writing—review and editing. All authors contributed to the article and approved the submitted version.This work was supported by the National Key Research and Development Program of China (2019YFC1605800), the China National Tobacco Corporation 2020 Major Science and Technology Project 110202001040(XJ-02), and the Major projects on constructing the “mellow and sweet fragrance styles” of Chinese-Stylistic Tobacco (ctx201905).QiZ, DL, PL, QuZ, and WC were employed by China Tobacco Sichuan Industrial Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This December issue of the JGH Open marks the swansong of my tenure as Editor in Chief (EiC). We launched the journal in September 2017 [One of our greatest concern was the article processing charge (APC) which is levied for all “open” journals. To encourage submissions the Journal of Gastroenterology and Hepatology Foundation  subsidised the APC until 2018. Professor Ian Roberts Thomson, a very close friend, was JGHF chairman and was involved in the discussions in setting up the journal and also took on the role as an associate editor. Ian had also deputize for me in late 2021 and early 2022. His friendship and support throughout my tenure has been invaluable and reassuring.We started by publishing monthly issues but submissions were few in the beginning and we had to change to bimonthly issues. Slowly, however submissions have increased and in 2021 we reverted back to publishing monthly. The JGH Open is now indexed in the Directory of Open Access Journals (DOAJ), Embase (Elsevier), EBSCO, and PubMed via PMC deposit (NLM) and in the Emerging Sources Citation Index (ESCI) [I have been ably supported by a team of associate editors, many of whom are friends. Some have left the position over the years but we have had new faces stepping in to replace them. I am truly grateful to each and every one of them for their help and immeasurable contributions to the journal. Professor Vineet Ahuja from the All‐India Institute of Medical Sciences, Delhi takes over the position of EiC from first January 2023. Vineet has been in the editorial board form the beginning. In Vineet, we have a well‐regarded academician who has published widely. He will no doubt bring the journal up to the next level.The journal has been fortunate in being managed by a very efficient team from Wiley in the day‐to‐day running of the journal. The publication office is in Melbourne with Tamara D'Mello as our Senior Publishing Manager over the past 1 year. The help from Tamara, her predecessors and the publication managers have been tremendous. The editors are truly grateful to be supported by a “professional” team from Wiley.


In persons with multiple sclerosis, nerve conductivity can be reduced. The assessment is generally performed via motor evoked potentials (MEP). So far, a strongly associated motor performance surrogate for changes in the extracted central motor conduction time (CMCT) is missing.CMCT and performance in the nine-hole peg test and maximum thumb tapping frequencies over 10 s of 12 persons with multiple sclerosis were measured prior to and after training over 5 consecutive days. Each training consisted of 10,000 thumb taps at maximum effort with the dominant upper limb.d = 0.79) and 10,000 taps (d = 1.04), the nine-hole peg test (d = 0.60), and CMCT (d = 0.52). The nondominant upper limb only improved in the nine-hole peg test (d = 0.38). Models of multiple linear regression predicted 0.78  and 0.87  of the variance in CMCT changes.The dominant upper limb improved in maximum tapping frequency over 10 s  or rather describing a potential of change (model 2). However, we were able to show moderate changes in CMCT within 5 days. Tapping tasks have been shown to be a valid tool to assess disease severity and progression in multiple sclerosis (MS) –8 (expecCMCT can be impaired (slowed) by a loss of myelin due to MS  and sincIf changes in CMCT can be assessed by tapping tasks, it would allow a quick, temporally dense, and economic assessment of a myelination surrogate in MS patients and could therefore support medical and pharmacological research that aims to foster remyelination or improve motor conduction. A prior study observed slowing of CMCT and disability progression in persons with progressive MS but did not correlate CMCT and motor performance . Mamoei Our objective was to examine whether changes in CMCT of MS patients are reflected by the performance of a simple thumb tapping task. We hypothesized that highly repetitive (Gulde 2020), maximum  thumb taα of 0.05, and a power of 0.80, using G*Power (version 3.1.9.7)  was computed on the basis of MEPs by a transcranial magnetic stimulator , which is part of the clinic’s standard protocol for MS. The musculus adductor pollicis was used as the target muscle, and CMCT was calculated as the mean difference between central (M1) and peripheral MEP latencies of three trials . A single coil  was used, and patients were seated in a chair with arm and leg support. The EMG signal was derived from surface electrodes and using a Neuropack μ device . For the central stimulation, we asked patients to slightly contract their muscles (pinching index finger and thumb together “like holding a fly between your fingers”). For the peripheral stimulation, we asked patients to relax their muscles. The intensity of the stimulations was set by increasing them step by step until a slight muscle contraction became visible. We decided against the protocol of 5/10 visible EMG reactions to keep the number of stimulations for patients at a minimum. As mentioned before, for the central and the peripheral stimulations, the mean of three successful stimulations was used to derive the CMCT. We decided to not include the MEP amplitude due to its reported very low intraclass correlation of 0.01 to 0.34 . For eacPatients were asked to perform one trial of 10,000 thumb taps as fast as possible with their dominant hand  on each of 5 consecutive days. We decided to use the dominant (or in case of strong impairments of the dominant upper-limb the better-functioning thumb) in order to ensure an already well-developed cortical representation and the motor and motivational capacity to execute the task. The experiment was conducted with custom software  on a Lumia 550 smartphone (Microsoft Corp.), showing the participant the number of taps , the duration in minutes and seconds , the mean frequency of the last 50 taps and the mean frequency of all taps. Additionally, a graph was showing the course of the mean of 50 taps (from left to right). Patients held the smartphone in landscape orientation in both their hands (like a joypad or Gameboy).Patients were assessed with the nine-hole peg test (PEG), a maximum thumb tapping task over 10 s (10 s TAP), and their CMCT was computed by MEPs on days 1 and 6 . On day 1, they also started with their first training of 10,000 taps with the thumb of their dominant hand right after the assessment. The 10-s maximum tapping rate (10 s TAP) of both thumbs was assessed on all days but day 3 and day 4 (weekend), when patients were training in an unsupervised manor. Testing of sensorimotor performance and assessments of CMCT were performed on the same day, in the same room, and by the same unblinded investigator.One patient reported the development of pain in both upper limbs directly and progressively after an unrelated session of physical therapy (therapeutic climbing). One patient reported muscle soreness of the finger extensors the day after the first training session. One patient revealed a CMCT of 31 ms at the beginning and 28 ms at the end of the study and was removed as an outlier (a relapse occurred approx. 3 months prior to the study).t-tests with a Bonferroni α correction to 0.05/4 = 0.0125 (one sided for the dominant upper limb). Further, a linear mixed-effects model , and proportional variance (linear mixed effects). α was set to 0.05.The trained and untrained upper limbs’ performance and CMCT, as well as changes in CMCT, were compared using paired lmerTest –34) was p = 0.036)). CMCT only improved on the dominant side and was faster in 11 of 12 participants (92%)  nor the nondominant upper limb (p = 0.576). Conduction times were 14.16 ms ± 1.41 ms (pretest) and 14.19 ms ± 1.27 ms (posttest) for the dominant and 14.30 ms ± 1.79 ms (pretest) and 14.44 ms ± 1.46 ms (posttest) for the nondominant upper-limb. The intraclass correlation coefficients for the peripheral conduction times were 0.92  and 0.92 . 10,000 TAP performance improved over the course of 5 days (by 4:29 min ± 0:58 min from 39:42 min ± 4:44 min to 35:13 min ± 4:48 min). All data are given in Table The 10 s TAP and PEG performance improved on the dominant side as well as the nondominant (except 10 s TAP of the nondominant side with a trend p = 0.06). CMCT R2adjusted of 0.78 .There were no significant random effects for changes in CMCT, so we applied models of multiple linear regression. One resulting model (model 1) was significant with an R2adjusted of 0.87  for changes in CMCT did not include any parameters of changes in performance . It resulted in an  for chanR2 = 0.37, p = 0.035, greater improvements predicting greater reductions in CMCT). Second, changes in CMCT and FM (time since patient reported first manifestation) . Third, changes in CMCT and BMI . Changes in 10 s TAP or PEG performance were not associated with changes in CMCT .Further, single regressions showed three strong significant associations: First, changes in CMCT and changes in 10,000 TAP (p = 0.012). CMCT changes of both sides were not associated (p = 0.424), even after adjusting the CMCT of the dominant upper limb by the regression models .The intraclass correlation for the CMCT of the nondominant upper limb was 0.61  in the 10,000 TAP task, and interaction of the 10,000 TAP absolute improvement and the difference in CMCT between dominant and nondominant upper-limb at the beginning of the experiment. A second model, not including parameters of changes of performance, even resulted in an R2adjusted of 0.87.In the reported study, we examined if changes in CMCT are reflected by the performance of a thumb tapping task in a sample of 12 MS patients. In order to evoke changes in CMCT, we introduced a highly repetitive thumb tapping over the course of 5 days. CMCT of the trained upper limb did significantly improve by an average of 1.2 ms in 11 of 12 patients. Although a quick tapping assessment of 10 s (10 s TAP) was not associated with those changes, changes in tapping performance over 10,000 repetitions  were able to explain 37% of the variance in changes in CMCT alone. A model of multiple linear regression of changes in CMCT even resulted in an p = 0.181–0.424), even after training adjustment of the dominant upper limb, so the chance of a systematic change could be expected very low  of 0.61, which is in line with reports on the reliability of CMCT . Changesformance ).We were able to predict changes in CMCT, but the prediction needed factors, which’ assessment would exceed the assessment time of CMCT by transcranial magnetic stimulation by far  or did not include any parameter of performance changes (model 2 and BMI or FM). Therefore, we are not able to present an economic or feasible surrogate. However, we were able to observe a set of interesting peculiarities:less better or even worse CMCT of the dominant side predicting a greater reduction in CMCT). If this training effect holds true in further studies, the wish for promotion of improving conductivity [First, CMCT strongly improved over the course of five days with an average of approx. 3 h of training volume (186 min ± 25 min). An underlying mechanism could be a restored conductivity of impaired nervous pathways or axonal sections , 36 or auctivity , 18, 19 uctivity  observedThe more is broken, the more can be restored would be supported by the impact of starting performance in model 1  and in model 2 (10 s TAP). However, this seems to be contradicted by the impact of the EDSS in model 2, being in line with previously published evidence from inpatient rehabilitation (lower EDSS grades predicting better improvements in sensorimotor performance) [unhealthy lifestyle (from a perspective of neuroplasticity) [the more is broken, the more can be restored and would underline the impact of the central nervous milieu as will be discussed in the following. Longer disease duration has been linked to normal levels of central nervous inflammation [Second, changes in CMCT were significantly associated with FM. The longer the timespan since the notice of  MS symptoms, the stronger the reduction in CMCT by training. An odd addition is missing associations with patients’ age, intake of DMT, EDSS, and type of MS or an impact of current smoking. Patrikios et al.  did, forormance) . The EDSsticity)  and the ammation , so a ceammation  and as Fammation  observedOf particular interest for rehabilitation purposes is the observation of an improvement of motor performance on the nondominant side in 10 s TAP and PEG. A post hoc correlational analysis revealed no significant association between dominant and nondominant performance gains, so, alternatively to a skill transfer (Andree 2002), learning the task would be a potential explanation  , so a skOne strong limitation of the current study should be mentioned. The investigator was not blinded. This should not strongly affect the derived CMCT, but could potentially have an effect on the sensorimotor performance. However, we argue that the internal consistency of our data indicates that a potential bias would be neglectable. All analyses were run after completing the dataset.R2adjusted of 0.78 and 0.87). However, CMCT assessment by transcranial magnetic stimulation remains to be not replaceable by motor performance markers.CMCT was significantly reduced by 5 consecutive days of intensive and highly repetitive training. However, we were not able to present an economic (model 1) and feasible (model 2) surrogate for future studies on improvements of CMCT. The first model would take more effort than the TMS procedure, while the second model rather estimated the potential of CMCT change of the individual. The extent of changes in the CMCT appeared to be affected by (course) indicators of an inflammatory milieu  and the extent of demyelination . The performance of other tasks (10 s TAP and PEG) appeared to profit from training as well. An association between the amplitude of changes in CMCT and motor performance was visible in 10,000 TAP (but not 10 s TAP or PEG) and models of multiple linear regression revealed strong predictive capacity (


Compared with the wild type , the branching degree of starch in sub-1 mutant was significantly increased by 19.6%. Chain length DP 6–12, DP 25–36 and DP > 36 of amylopectin significantly decreased, while chain length DP 13–24 significantly increased. Average chain length of wild-type and sub-1 mutant starches were greater than DP 22. Moreover, the crystal structure and physical properties of starch have changed markedly in sub-1 mutant. For example, the starch crystallinity of sub-1 mutant was only 8.94%, while that of wild-type was 22.3%. Compared with wild type, water solubility of starch was significantly reduced by 29.42%, whereas swelling power significantly increased by 97.07% in sub-1 mutant. In order to further analyze the molecular mechanism of efficient accumulation of amylopectin in sub-1 mutant, metabolome and transcriptome were performed. The results showed that glucose accumulated in sub-1 mutant, then degradation of starch to glucose mainly depends on α-amylase. At night, the down-regulated β-amylase gene resulted in the inhibition of starch degradation. The starch and sucrose metabolism pathways were significantly enriched. Up-regulated expression of SUS, AGPase2, AGPase3, PYG, GPI and GYS provide sufficient substrate for starch synthesis in sub-1 mutant. From the 0H to 16H light treatment, granule-bound starch synthase (GBSS1) gene was inhibited, on the contrary, the starch branching enzyme (SBE) gene was induced. Differential expression of GBSS1 and SBE may be an important reason for the decrease ratio of amylose/amylopectin in sub-1 mutant. Taken together, our results indicated that the sub-1 mutant can accumulate the amylopectin efficiently, potentially through altering the differential expression of AGPase, GBSS1, SBE, and BAM. This study also provides theoretical guidance for creating crop germplasm with high amylopectin by means of synthetic biology in the future.Large-scale use of fossil fuels has brought about increasingly serious problems of environmental pollution, development and utilization of renewable energy is one of the effective solutions. Duckweed has the advantages of fast growth, high starch content and no occupation of arable land, so it is a promising starchy energy plant. A new submerged duckweed mutant ( Lemna aequinoctialis is symmetrical and is nearly round or obovate in shape. Frond is the main organ where carbon dioxide (CO2) in the atmosphere is absorbed to synthesize carbohydrates and store energy in the form of starch. However, the storage site of starch in frond is not well understood.Duckweed is one kind of monocot aquatic plant that grows on the water surface. It has the characteristics of fast reproduction, high starch content and low lignin content . Therefo8 Da, while that of amylose was 106 Da [Spirodela oligorrhiza and Lemna minor. We found that the amylopectin content was 79.15% and 72.77%, respectively, and the average chain length was higher than DP 28 [Spirodela oligorrhiza and Lemna minor were detected by wide-angle XRD [sub-1) was obtained in our laboratory. The total starch content of sub-1 mutant was twice that of the wild type, and the released glucose and ethanol yield were significantly increased [sub-1).Starch is an important natural glucose polymer, consisting of amylose and amylopectin. In general, the former is connected by α-1,4) glycosidic bonds, and also linked by α- glycosidic bond within the side chains, however the side chains are connected to the main chain or intercepted by α-1,6) glycosidic bond. The latter is connected end to end by α- glycosidic bonds, and at the branch chain connected by α- glycosidic bonds. Amylopectin is the main type of polysaccharide, and its content varies greatly, generally ranging from 72% to 85% in starch granule . Amylope glycosid glycosids 106 Da . From thncreased . HoweverIn plant, starch metabolism has been relatively well understood, which involve four types of enzymes ,13. AGPasub-1 mutant [sub-1 is a quality raw material for bioethanol production. In this study, we analyzed the internal structure and physiochemical characteristics of starch particles in sub-1 mutant, including chain length distribution, branching degree, crystallinity, XRD diffraction and so on. Metabolome and transcriptome investigate the differentially accumulated metabolites and differentially expressed genes in the starch metabolism pathway and explore the transcription factors that may affect starch accumulation. This study laid a theoretical foundation for the development and utilization of sub-1 mutant in the future. A new duckweed variety with high starch was screened from the heavy ion radiation mutagenic mutant library. As it accumulated a large amount of starch and sank to the bottom of water under oligotrophic or crowded conditions, we named it 1 mutant . There iL. aequinoctialis 6002) appeared to be obovate .Starch is a polysaccharide formed by the polymerization of glucose molecules. The number of glucose molecules involved in the composition of starch is called the degree of polymerization (DP). With the value of DP as the horizontal coordinate and the relative peak area of DP as the vertical coordinate, the distribution curves of chain lengths can be obtained. According to the structure model of amylopectin, the relative number of amylopectin chain lengths are normalized and divided into four parts: DP 6–12, DP 13–24, DP 25–36 and DP > 36, corresponding to A, B1, B2, and B3 chains . In the ectively . Compareectively . The degby 19.6% . This resub-1 mutant was only 8.94% (sub-1 mutant. It has been reported that B-type starch has the peaks at diffraction angle (2θ) of 5.6°, 17°, 22°, and 24°. V-type starch has diffraction peaks at 7.8°, 12.5° and 19.5°, which is composed of a single helical structure and co-exist with other crystal types [sub-1 mutant, starch granules showed diffraction peaks at 5° (0.6), 15° (0.6), 17.5° (5.6), 20° (2.8) and 23.5° (3.3), reflecting the typical characteristics of B-type starch. The long chain of amylopectin maintains the rigidity of starch granule and also maintain the long-range order of starch granule [sub-1 mutant.The crystallinity and crystal morphology of starch were analyzed by X-ray diffraction. The results showed that the starch crystallinity of wild-type was 22.3%, while that of the ly 8.94% a. Previo−1 to 800 cm−1 are mostly from the elastic vibrations of C-O and C-C, and this range is sensitive to the physiological state of starch [−1 and 1022 cm−1 represent the crystalline and amorphous regions, respectively, and the absorption peak at 995 cm−1 corresponds to the bending vibration of the hydroxyl group [sub-1 mutant . The principal component analysis (PCA) of differentially accumulated metabolites showed that the value of PC1 was 59.32%, and PC2 was 10.65%. The value of PC1 was much higher than PC2, indicating that genetic background plays an important role in metabolite accumulation, followed by light treatment , including photosynthesis (42), phenylpropanoid biosynthesis (61), carbon fixation in photosynthetic organisms (56), starch and sucrose metabolism (83) and so on (In order to investigate the molecular mechanism of starch accumulation in 6–55.00% . Therefo6–55.00% . 3297 up6–55.00% . 31 meta6–55.00% . In the 6–55.00% . 24 metand so on . In the Transcription factors (TFs) play an important role in gene expression regulation. It reported that WRKY, AP2, NAC and bZIP were involved in starch metabolism ,28. In tsub-1 mutant were extracted and then reverse transcribed into cDNA. The transcriptional levels of genes were analyzed using 18S RNA as an internal reference gene. Those detected genes contain sucrose synthase (SUS), glucose-6-phosphate isomerase (GPI), glycogen synthase (GYS), granule-bound starch synthase 1 (GBSS1), ADP-glucose pyrophosphorylase 2 (AGPase2), ADP-glucose pyrophosphorylase 3 (AGPase3), starch branching enzyme (SBE), isoamylase1 (ISA1), α-amylase (AMY), β-amylase (BAM), phosphoglucan-water dikinase (PWD) and glycogen phosphorylase (PYG). In the 0H group, SUS, AGPase2, AGPase3, AMY and PYG genes were up-regulated, while GBSS1, ISO1 and BAM genes were down-regulated  in the sucrose and starch metabolism were differentially expressed in sub-1 mutant. This study provided a new idea for the creation of crop germplasm with high-amylopectin. At present, wild-type duckweed with high starch is relatively few, and it is particularly important to create high-starch germplasm by radiation mutagenesis or genetic engineering. In the previous study, we screened a new high-starch variety L. aequinoctialis 6002) used in this study are inverted oval  and 79.15% (Spirodela oligorrhiza), respectively [sub-1 mutant is a new germplasm resource with high amylopectin. The fronds of duckweed  and L. minor (DP 28.2) [sub-1 mutant starch was significantly increased by 97.07%, and water solubility was decreased by 29.42%  . The proDP 28.2) . StudiesDP 28.2) . SwellinDP 28.2) . In thisy 29.42% . Overall1 mutant .sub-1 mutant showed B-type structural characteristics. It has been reported that the crystal region of starch was composed of double helices wrapped by short chains amylopectin and small crystals packed into them [sub-1 mutant starch was only 8.94%, which was much lower than that of the wild type (22.3%)  , probablsub-1 mutant differ in their response to circadian rhythm. Treatment with 0H and 16H light exposure, the samples were collected for transcriptome and metabolome analysis. Principal component analysis (PCA) showed that the value of PC1 (59.32%) was much higher than that of PC2 (10.65%)  a. In add(10.65%) c. Based  glucose ,37. In tt groups . While t6H group . The exp1 mutant . Under p.65% a. I1 mutant .sub-1 mutant, SUS, PYG, GPI, GYS and PWD were up-regulated, and these genes enhance glycogen synthesis and accumulation  of starch and sucrose metabolic pathways were significantly enriched . Therefomulation . At the staining . ADP-gluynthesis . Granule1 mutant . Starch 1 mutant . L. aequinoctialis 6002) used in this study was collected from Weishan Lake, Shandong, China . A new duckweed variety sub-1 was obtained by differentiation and screening of callus  induced by heavy ion radiation [sub-1 plants were cultured in liquid SH medium with 10 g/L sucrose. The parameters of culture room were set as follows: 25 ± 1 °C temperature, 16 h/8 h (day/night), 80 μmol m−2s−1 light intensity, 50–60% relative humidity.The wild-type duckweed .Duckweed was placed in 4% paraformaldehyde solution, then dehydrated, impregnated and fixed with a series of concentrations of ethanol. The sample was impregnated with different proportions of xylene and absolute ethanol for 45 min at each concentration. Impregnated with xylene: Paraffin  for 12 h. Paraffin and sample were added to the embedding molds and removed after cooling on ice. The thickness of slice was set to 8 um, and slices were cut and placed in water at 42 °C. Slices were fully unfolded and placed on the slide. The dried slides were placed in a dye tank filled with 100% xylene for 30 min, and deparaffinized with serial concentrations of alcohol. The plant materials were stained with 1% I2/KI staining. Then the slices were stained in solution B (0.5% periodate acid) for 15 min, and washed by purified water for 10 s. Then slices were stained with solution A (Schiff reagent) in the dark for 30 min and washed with purified water for 5 min. Finally, slices were stained with solution C (Hematosylin) for 30 s and washed with water. Microscope  observed the slices and photographed.Slices were deparaffinized as described in the Ig for 5 min and discarded the supernatant. The starch granules were dried at 37 °C. It can be used for the determination of amylose content, branching degree, chain length distribution, swelling power, X-ray differaction (XRD) and fourier transformed infrared spectrometry (FTIR) analysis.Duckweed was inoculated into 40 cm × 60 cm iron plates and cultured for 2 weeks. The plant materials were broken using blender, and the residue was filtered through filter cloth. The mixture of water and starch granules were centrifuged at 5000× About 10 mg starch granules were taken and added 100 μL 95% alcohol and 900 μL NaOH solution. The mixture was boiled for 13 min, then the volume was fixed to 10 mL. Mix 0.5 mL supernatant, 0.1 mL acetic acid and 0.2 mL potassium iodide solution, and the volume was fixed to 10 mL and placed in the dark for 10 min. The absorption value was measured at 620 nm . AccordiAbout 5 mg starch granules were taken, and added 1 mL DMSO, heated at 80 °C overnight. After centrifugation at 12,000 rpm for 10 min, the supernatant was added to the tube for nuclear magnetic analysis. The instrument used in this experiment was Bruker BioSpin GmbH. The scanning times were 32, resonant RF was 500.23 MHz, and NMR spectrum was 1 H .TM CarboPacTM PA200  chromatographic column were used. Mobile phase A: 0.2 M NaOH, mobile phase B: 0.2 M NaOH/0.2 M NaAC, the flow rate was 0.4 mL/min, column temperature was 30 °C. The composition of enzymatic hydrolysate was analyzed by electrochemical detector.The starch granules were digested with isoamylase , and the supernatant of the sample was taken and used for the analysis of chain length distribution of amylopectin . Thermo After water balance, starch granules were screened with 100 mesh, placed on the loading platform, spread evenly, pressed tightly, and tested on the machine. X’Pert Pro X-ray diffractometer  was used in this experiment. The parameters: copper target Cu Kα (λ = 0.15406 nm), power 1600 W (40 kV × 40 mA), NaI scintillation counter. The diffraction angle (2θ) was in the range of 4°–60° with a scanning rate of 4°/min and step size 0.02° . DS-SS-R−1 to 4000 cm−1, the number of scans was 32, and the resolution was 4.00 cm−1 [First, starch granules were screened with 100 mesh. A small amount of sample was mixed with potassium bromide and pressed into tablets. Fourier transformed infrared spectrometry Nicolet iZ-10 was used . The spectra were set from 400 cm.00 cm−1 .g for 20 min. The tube with precipitation was dried at 80 °C for 20 min, and put into a incubator for 20 min, marked as M2. The weight of aluminum drum was marked M3. The supernatant in the aluminum drum was placed in the oven at 80 °C to a constant weight, marked as M4 [Mix 0.1 g starch and 10 mL deionized water in the tube (M1), water bath at 95 °C for 60 min. Cool the tube to room temperature and centrifuge at 8000× ed as M4 . The calsub-1 mutant were exposed to light for 0H and 16H, respectively, and total RNA was extracted from 12 samples. The cDNA library was constructed with high quality RNA and quantified by Qubit 2.0. The insert size of the cDNA library was detected by Agilent 2100. The effective concentration of the library was quantitatively detected by Q-PCR. After being qualified, sequencing was performed on the illumina HisSeq platform. Comparison of clean data with rice genome, the mapped data was obtained. Based on the number of reads assembled genome, the FPKM value and differential expression of each gene were calculated. The filter condition of differentially expressed genes (DEGs) were as follows: |log2Fold Change| ≥ 1 and FDR < 0.05. Finally, the enrichment analysis of DEGs were carried out, such as Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and Clusters of Orthologous Groups (KOG).Wild type and Similar to the transcriptome sampling, a total of 12 samples were used for metabolome and divided into 4 groups with 3 biological replicates. Metabolite detection was based on UPLC-MS/MS  data collection system. The original data was compressed into multiple principal components to describe the features of original data. PC1 represents the most obvious features, PC2 represents the most significant features except for PC1. The heatmap was drawn by the pheatmap package of the R software, which exhibits the accumulation pattern of metabolites among 12 samples. The screening conditions of differentially accumulated metabolites (DAMs) were as follows: Fold change ≥ 2 or fold change ≤ 0.5, meanwhile, VIP ≥ 1.2O. The procedure of the Q-PCR detection system  was as follows: 98 °C predenature, 94 °C denature, 74 °C anneal and extend. 18S rRNA was used as the housekeeping gene. The expression of candidate genes was obtained by calculating the 2−ΔΔCT value.Total RNA was extracted after light treatment for 0H and 16H, respectively. The mRNA was reverse transcribed into cDNA by First-Strand cDNA Synthesis SuperMix , and the product could be used as the template of Q-PCR. The reaction system was as follows: 1 μL template, 0.5 μL forward primer, 0.5 μL reverse primer, 10 μL SYBR Green, 8 μL ddHp < 0.05) and data processing system  were used. The asterisk means that there is a significant difference between wild type and sub-1 mutant . The letter in sub-1 mutant. At least 3 biological replicates were used for statistical analysis, and the results were expressed as mean ± standard deviation. Duncan’s multiple test of SPSS 17.0  (sub-1 mutant is a new duckweed variety with high amylopectin, and its amylopectin content is up to 84.04%. At the same time, the degree of starch branching increased by 19.6% and the number of branched chain increased. These traits are benefit for the enzymatic hydrolysis of starch and bioethanol conversion. Up-regulated expression of AGPase2, AGPase3 and SBE and down-regulated GBSS1 are the important reasons for the decreased ratio of amylose/amylopectin (sub-1 mutant implies that the up-regulated AMY gene plays a major role in the starch degradation (BAM gene at night leads to a decrease in starch degradation and the underlying molecular mechanism needs further investigation. This study laid a foundation for the development and utilization of the sub-1 mutant starch and provided theoretical guidance for the germplasm creation of high amylopectin crops.The lopectin . In addiradation . While t


FtNAC70, FtPUL, and FtGBSS1-3 in the lightcyan module and FtbHLH5, C3H, FtBE2, FtISA3, FtSS3-5, and FtSS1 in the brown. All the above genes were preferentially expressed in seeds, further suggesting their role in seed starch biosynthesis. These results provide crucial guidance for further research on starch biosynthesis and its regulatory network in Tartary buckwheat.Starch is a major component of crop grains, and its content affects food quality and taste. Tartary buckwheat is a traditional pseudo-cereal used in food as well as medicine. Starch content, granule morphology, and physicochemical properties have been extensively studied in Tartary buckwheat. However, the complex regulatory network related to its starch biosynthesis needs to be elucidated. Here, we performed RNA-seq analyses using seven Tartary buckwheat varieties differing in starch content and combined the RNA-seq data with starch content by weighted correlation network analysis (WGCNA). As a result, 10,873 differentially expressed genes (DEGs) were identified and were functionally clustered to six hierarchical clusters. Fifteen starch biosynthesis genes had higher expression level in seeds. Four trait-specific modules and 3131 hub genes were identified by WGCNA, with the lightcyan and brown modules positively correlated with starch-related traits. Furthermore, two potential gene regulatory networks were proposed, including the co-expression of  Fagopyrum tataricum, also called Tartary buckwheat, belongs to the Fagopyrum genus of Polygonaceae Mill and is a type of traditional pseudo-cereal for both medicine and food. Tartary buckwheat is famous for its high nutritional value, i.e., balanced amino acids, vitamins, minerals, and flavonoids  [ercetin) ,2,3. Theercetin) .F. esculentum), wheat (Triticum aestivum) and barley (Hordeum vulgare), with 19.3–20.2% iodine affinity, 1.36–1.48 blue values, and 645–657 nm maximum absorption wavelength [Starch, comprised of amylose and amylopectin, is the major component of Tartary buckwheat seeds, accounting for the highest proportion of their dry weight . Starch velength . The stavelength . Meanwhivelength .In endosperm, starch biosynthesis is a complex process controlled by the synergistic action of five starch synthetases, namely ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE) ,15. AGPaOryza sativa), maize (Zea mays), wheat, and barley [ZmNAC128 and ZmNAC130, which can bind to the prompters of Brittle 2 (BT2), Zpu1 (encoding DBE), GBSSI, Sh2 (encoding AGPL), SSV, ISA2, and SSIIa, and thus positively affect starch synthesis in the endosperm of maize [Genes encoding the starch biosynthesis enzymes have been massively characterized and functionally analyzed in cereals, such as rice , has been isolated [In Tartary buckwheat, starch-related research has focused on the starch content, granule morphology, and physicochemical properties ,11,12,13isolated . Meanwhiisolated . These wAmylose and amylopectin contents were measured in the matured seeds of 20 Tartary buckwheat varieties (lines), which presented obvious characteristics in their seed size, morphology, shell color, and shell thickness. The results showed that the amylose content ranged from 17.61% to 21.00%, with an average of 19.24%. The amylopectin content ranged from 19.67% to 28.49%, with an average of 23.63%. The ratio of amylopectin/amylose ranged from 1.05 to 1.43, with an average of 1.23 . We subsTo discover the global changes in gene expression level, high-throughput RNA-seq was carried out for late-filling-stage seeds of seven selected varieties , with three biological replicates for each variety. In each sample, 40,261,724–102,065,872 clean reads and 6.04–15.31 G clean bases were obtained. Q30 ranged from 88.86% to 89.77% and GC content ranged from 47.64% to 50.03% . The cleUsing PK1 as a control, 10,873 DEGs were identified, among which 9573, 3490, 1655, 977, 909, and 906 were identified in the comparisons of M11_vs._PK1, JQ2_vs._PK1, DMQ_vs._PK1, BK2_vs._PK1, QK2_vs._PK1, and XMQ_vs._PK1, respectively a. Of theThe metabolic pathways that the DEGs were related to were analyzed based on KEGG annotation. Among the top 20 enriched pathways in the six comparisons, four metabolic pathways, namely “starch and sucrose metabolism”, “biosynthesis of secondary metabolites”, “circadian rhythm—plant”, and “metabolic pathways” were enriched in all six comparisons . InteresMeanwhile, the identified DEGs in each comparison were annotated to a KOG category. As a result, “carbohydrate transport and metabolism”, “secondary metabolite biosynthesis”, “posttranslational modification, protein turnover, chaperones”, “transport and catabolism”, “signal transduction mechanisms”, and “general function prediction only” were listed in the top annotated categories in all comparisons . The catWe also performed GO analysis for the DEGs and the top 50 enriched items related to biological processes, cellular components, and molecular functions were identified . All comFtAGPL1~FtAGPL4 and FtAGPS1~FtAGPS2), 5 GBSS encoding genes (FtGBSS1~FtGBSS5), 12 SS encoding genes , 4 BE encoding genes (FtBE1~FtBE4), and 4 DBE encoding genes (FtISA1~FtISA3 and FtPUL) , 3 GBSS encoding genes , 5 SS encoding genes , 2 BE encoding genes (FtBE1 and FtBE2), and 2 DBE encoding genes (FtISA3 and FtPUL) had higher expression levels, suggesting they had potential functions in the starch biosynthesis of Tartary buckwheat seeds. In addition, 20 out of the 31 genes in the starch biosynthesis pathway were identified as DEGs, including 12 in the hierarchical cluster of C2, 4 in C4, and 4 in C6  were more highly expressed in the varieties with higher starch content  . Their ed FtPUL) . Among t 4 in C6 . It is w content c.p value < 0.05 level. The greenyellow and pink modules were negatively correlated to the amylose content, amylopectin content, and amylose/amylopectin, but positively correlated to amylopectin/amylose. On the other hand, the lightcyan module was positively correlated to amylose content, amylopectin content, and amylose/amylopectin, but negatively correlated to amylopectin/amylose. The brown module was positively correlated to amylose content, amylopectin content, and amylose/amylopectin. Gene expression patterns of four trait-specific modules are shown in WGCNA was performed using 22,191 identified genes whose average count number was equal to or larger than 10 in at least one variety. As a result, nine modules were identified based on the association of gene expression levels with starch-related traits a. Among The hub genes were then identified using the threshold of the top 30% in intramodule connectivity, absolute KME (module eigengene-based connectivity) value ≥0.8, and absolute GS (gene trait significance) value ≥0.2, resulting in a total of 3131 hub genes in four trait-specific modules, including 2409, 217, 415, and 90 hub genes in greenyellow, pink, brown, and lightcyan, respectively .p-value < 0.05 level, resulting in 43, 15, 2, and 5 pathways, which were enriched in the greenyellow, pink, lightcyan, and brown modules, respectively  and carbohydrate metabolism (B09101) were listed on the top two enriched pathways  [TFs in the hub genes of four trait-specific modules were screened; the resulting 60, 1, 4, and 36 TFs were enriched in the greenyellow, pink, lightcyan, and brown modules, respectively a. In theAC77/15) ,34,35, oFtNAC70 and FtNAC15, one AP2/ERF-ERF (FtPinG0002070100.01), and one RWP-RK (FtPinG0002032100.01), were clustered to C6 based on their gene expression patterns; meanwhile, all five TFs in the brown module, namely one E2F-DP (FtPinG0001244300.01), two bHLH  [FtWRKY39) [TFs are often in the upstream of a regulatory network, so we identified the TFs in the hub genes of the top 50 in intramodule connectivity, resulting in nine TFs in total, including four TFs in the lightcyan module and five TFs in the brown module . InterestbHLH91) , one WRKtWRKY39) , and oneFtNAC70, was co-expressed with two starch biosynthesis genes, FtPUL and FtGBSS1-3, in the lightcyan module  but negatively correlated to four starch biosynthesis genes, FtBE2, FtISA3, FtSS3-5, and FtSS1 (Pearson R ≤ −0.91), at the significance level of 0.01  seed kernels, genes involved in starch biosynthesis, including AGPase, GBSS, SS, SBE, and ISA encoding genes, were also enriched in the developing seed [RNA-seq is a well-established and widely used method for identifying global gene regulation at a transcriptional level. We carried out RNA-seq analyses for seven varieties that differed in their starch content, resulting in 29,978 mapped genes and 10,873 DEGs, using PK1 as a control. Through metabolic pathway enrichment analysis, the “starch and sucrose metabolism” pathway was found to be significantly enriched and was positively related to starch content. Meanwhile, “carbohydrate transport and metabolism”, which included the process of starch biosynthesis and metabolism, was listed in the top annotated KOG categories. The KOG categories result was consistent with the KEGG enrichment result, suggesting the starch biosynthesis pathway differed in the seven varieties. In recent transcriptome analyses of developing Chinese chestnut , has been isolated and involved in amylose biosynthesis [FtGBSS1-2 and was highly expressed in the seeds in our study. Another reported starch-related gene in F. esculentum, namely FeGBSS (Genbank accession: HW041459.1), was highly homologous to FtGBSS1-1; however, FtGBSS1-1 was not preferentially expressed in the seeds in our study, suggesting it might participate in amylose biosynthesis in other tissues than the seeds of Tartary buckwheat. In addition, we previously performed a transcriptome analysis of three seed developmental stages in Tartary buckwheat and identified 23 DEGs corresponding to starch biosynthesis, among which six DEGs were expressed positively with seed development and showed higher expression in the later developmental seeds [Based on our RNA-seq data, we identified 31 genes related to the starch biosynthesis pathway, including ynthesis . This geFtGBSS1-3 and FtPUL were listed in the lightcyan module and FtBE2, FtSS3-5, FtISA3, and FtSS1 were listed in the brown module.With the combination of the four starch-related traits and the gene expression data achieved through WGCNA analysis, nine modules were identified, of which four modules  were identified as trait-specific modules. Though the greenyellow and pink modules included more hub genes and more enriched pathways, the starch-related pathway was absent in them but significantly enriched in the lightcyan and brown modules, which were positively correlated to amylose content, amylopectin content, and amylose/amylopectin based on the module-trait relationship. Among the starch biosynthesis genes, ZmNAC128 and ZmNAC130 could bind to the prompters of the starch biosynthesis genes, including Brittle 2 (BT2), Zpu1 (encoding DBE in maize), GBSSI, Sh2 (encoding AGPL), SSV, ISA2, and SSIIa, and thus positively affect starch synthesis in the endosperm of maize [TaNAC019 was reported to regulate starch biosynthesis by the regulation of SSIIa and Susy1, and thus accelerate starch accumulation in wheat [FtNAC70 and FtNAC15, were identified as top-50 hub genes. Both of their expression patterns have been reported to be increasing with the seed development of Tartary buckwheat [FtNAC70 showed that it was a seed-specific NAC TF. Meanwhile, its expression was significantly positively correlated to FtGBSS1-3 (Pearson R = 0.84) and FtPUL (Pearson R = 0.79), suggesting that FtNAC70 should be a key positive regulator in regulating starch biosynthesis in Tartary buckwheat.According to the results of TF analysis, 4 and 36 TFs were identified in the lightcyan and brown modules, respectively, among which nine TFs were in hub genes of the top 50 in intramodule connectivity. Some of the above TFs, such as NAC, AP2/ERF-ERF, bHLH, and WRKY, were homologous to the functionally characterized regulators related to starch metabolism in cereal crops. For example, NAC family genes are involved in starch biosynthesis in rice and maize. of maize . TaNAC01in wheat . In our uckwheat ,34. It iFtbHLH5 and FtbHLH91, were identified as top-50 hub genes. FtbHLH5 was more highly expressed in seeds and stems, whereas FtbHLH91 was more highly expressed in leaves rather than other tissues [FtbHLH5 might be involved in regulating starch biosynthesis in seeds and stems, whereas FtbHLH91 might be involved in regulating starch biosynthesis in the stem. The expression of FtbHLH5 was significantly negatively correlated to FtBE2 (Pearson R = −0.96), FtSS3-5 (Pearson R = −0.93), FtSS1 (Pearson R = −0.93), and FtISA3 (Pearson R = −0.92), suggesting that FtbHLH5 should be a key negative regulator in regulating starch biosynthesis in Tartary buckwheat.TFs in the bHLH family could regulate starch biosynthesis in an indirect manner. A novel FLOURY ENDOSPERM2 (FLO2) could interact with the bHLH proteins in a transcriptional complex that could regulate starch storage by influencing the development of endosperm in rice . OPAQUE1 tissues . In addi tissues , suggestZmEREB156, was sucrose- and ABA-inducible and positively regulated starch synthesis by directly binding to the promoter of ZmSSIIIa in maize [Sugar Signaling in Barley2 (SUSIBA2), a plant-specific WRKY TF, could bind to the promoters of BEIIb; the heterologous expression of it in rice produced a rice variety with a high starch content [OS1, encoding an RWP-RK TF, downregulated certain genes in specific cell types, including a majority of zein- and starch-related genes in central starch endosperm cells [FtWRKY39), and an RWP-RK TF (FtPinG0002032100.01), were identified as top-50 hub genes, suggesting they might be involved in starch biosynthesis in Tartary buckwheat. The expression level of FtWRKY39 increased gradually with the development of seeds [An AP2/ERF-ERF family gene, in maize . Sugar S content . A mutanrm cells . In our of seeds , suggestFtbHLH5, significantly positively correlated to FtbHLH5 (Pearson R = 1.00) and significantly negatively correlated to FtBE2, FtSS3-5, FtSS1, and FtISA3. In addition, C3H was preferentially expressed in seeds rather than other tissues. These suggested that C3H might interact with FtbHLH5 and be another key negative regulator in regulating starch biosynthesis in Tartary buckwheat.Though no evidence has been shown at present, an E2F-DP TF (FtPinG0001244300.01) and a C3H-type Zn-finger protein (FtPinG0003310400.01) were also identified as top-50 hub genes, suggesting they might be involved in starch biosynthesis in Tartary buckwheat. Notably, the C3H-type Zn-finger protein was in the co-expression network of A total of 20 Tartary buckwheat varieties/lines were used in this study, namely Biku 2 (BK2), Damiqiao (DMQ), Jinkuqiao (JKQ), Jinqiao 2 (JQ2), Jiujiangkuqiao (JJ), Miku 11 (M11), Miku 127 (M127), Miku 13 (M13), Miku 18 (M18), Miku 5 (M5), Miku 55 (M55), Pinku 1 (PK1), Qianheiqiao 1 (QHQ1), Qianku 2 (QK2), Sanya A5 (SYA5), Sanya B60 (SYB60), Sanya B68 (SYB68), Sanya B69 (SYB69), Sanya C28 (SYC28), and Xiaomiqiao (XMQ). In the early August of 2020, their seeds were sowed in the Anshun experimental field of our laboratory and grew with normal field management. When they were nearly matured, seeds at late filling stage were taken with three replicates, immediately frozen in liquid nitrogen, shelled on ice, and stored in an ultra-low-temperature freezer for starch measurement and RNA-seq. When the plants were matured, the ripened seeds were harvested and dried for starch measurement. Then the ripened seeds of all 20 varieties/lines and the late-filling-stage seeds of seven varieties, namely PK1, BK2, QK2, XMQ, DMQ, JQ2, and M11 were used for starch measurement, with three biological replicates and two (or three) technical replicates. Meanwhile, the late-filling-stage seeds of the seven mentioned varieties were used for RNA-seq analysis, with three biological replicates.(597 nm–480 nm) and OD(541 nm–700 nm), respectively. The equation of the standard curve for amylose wasAmylose and amylopectin content were measured in accordance with a previous report , with so2O was added. Then, 200 µL solution was placed into a new 2 mL centrifuge tube and 1 mL ddH2O was added. The pH was adjusted to 3.0 using 2 mol/L HCl. Then, 20 µL iodine reagent was added and mixed. The volume was titrated to 2 mL with ddH2O. After standing at room temperature for 25 min, the mixtures were applied to a spectrophotometer for amylose measurement at 597 nm and 480 nm and amylopectin measurement at 541 nm and 700 nm. Then, the amylose and amylopectin content were calculated using Equations (1) and (2), respectively.For each variety/line of Tartary buckwheat, 0.1000 g ground powder of the seeds was accurately weighed and placed in a 50 mL centrifuge tube. Then, 10 mL absolute ethanol was added and mixed. The amylose and amylopectin were extracted in a water bath at 80 °C for 30 min. After cooling, the samples were centrifuged at 12,000 rpm at 25 °C for 5 min. The supernatant was discarded, and 10 mL KOH (1 mol/L) was added to the retained precipitate. The sample was placed into a water bath at 100 °C for 15 min, followed by being transferred to a freezer at 4 °C. Afterward, a 1 mL sample was transferred into a new 50 mL centrifuge tube and 10 mL ddHhttp://www.mbkbase.org/Pinku1/ (accessed on 8 September 2022) and the chromosomal location of the mapped genes were obtained by HISAT2 with default parameters [ftp://ftp.ncbi.nih.gov/blast/db (accessed on 8 September 2022), KEGG https://www.genome.jp/kegg (accessed on 8 September 2022), KOG https://www.ncbi.nlm.nih.gov/COG/ (accessed on 8 September 2022), and GO http://geneontology.org/ (accessed on 8 September 2022) using the BLAST program . Gene expression was quantified by FPKM (fragments per kilobase of transcript per million fragments mapped) as the following equation:RNA was extracted using the RNAprep Pure Plant Plus Kit produced by TIANGEN . Library construction was carried out by Metware Biotechnology Co., Ltd. . After that, the library was sequenced on an Illumina NovaSeq 6000 platform  and 150 bp paired-end reads were generated. Data filtering and quality control were also completed by this company using fastp, resulting in more than 6 G clean data with Q20 > 95% and Q30 > 85% for each sample ,43. Aftehttps://cran.r-project.org/web/packages/corrplot/index.html (accessed on 8 September 2022).Pearson correlation was analyzed and visualized using “corrplot” in R language 2(FoldChange) ≥1 and padj  < 0.05 [https://cran.r-project.org/web/packages/available_packages_by_name.html#available-packages-C (accessed on 8 September 2022). For the hierarchical cluster of all identified DEGs, the scale was set as “row”. For the hierarchical cluster of partial analyzed DEGs, the scale was set as “none”. KEGG pathway and GO item enrichment were performed by TBtools [https://cran.r-project.org/web/packages/ggplot2/index.html (accessed on 8 September 2022).DEGs were identified by “DESeq2” in R language with default parameters, using absolute value of Log) < 0.05 . The ups) < 0.05 . The hie TBtools . The topFor tissue-specific analysis, gene expression data of four different tissues  were obtained from the published genome data for Tartary buckwheat , whereashttp://itak.feilab.net/cgi-bin/itak/online_itak.cgi (accessed on 8 September 2022). The gene co-expression networks of the starch biosynthesis genes and TFs were visualized using Cytoscape [WGCNA was performed using “WGCNA” in R language . After fytoscape .FtAGPL2, FtAGPL4, FtAGPS1, FtGBSS1-2, FtGBSS1-3, FtGBSS1-5, FtSS1, FtSS2-1, FtSS3-2, FtSS3-5, FtSS4-3, FtBE1 FtBE2, FtISA3 and FtPUL, had a higher expression level in seeds. In addition, four trait-specific modules  and 3131 hub genes in these modules were identified by WGCNA, among which the lightcyan and brown modules were positively correlated to amylose content, amylopectin content, and amylose/amylopectin. Starch and sucrose metabolism and carbohydrate metabolism pathways were enriched in both the lightcyan and brown modules. Based on TF analysis in trait-specific modules, FtNAC70 was co-expressed with FtPUL and FtGBSS1-3 in the lightcyan module; FtbHLH5 and C3H were co-expressed with FtBE2, FtISA3, FtSS3-5, and FtSS1 in the brown module. All nine genes in the co-expression networks were preferentially expressed in seeds rather than other tissues, suggesting they might be responsible for starch biosynthesis in the seeds. These results would not only enrich the gene resources related to starch, but also provide a molecular insight into the starch biosynthesis regulatory mechanism in Tartary buckwheat. The results here appear to be an excellent starting point for further research on Tartary buckwheat. This work will be of benefit in the future for a complementary theoretical study using quantum chemical topology to identify the vdW interactions that play a role in the RNA-seq analysis. The estimation of non-covalent interaction energies present in the structures of RNA presented here could help to better guide further research on starch biosynthesis and its regulatory network in Tartary buckwheat.In this work, we performed RNA-seq analyses using seven Tartary buckwheat varieties that differed in starch content and combined their RNA-seq data with starch content using WGCNA. As a result, 10, 873 DEGs were identified and functionally clustered to six hierarchical clusters based on their expression patterns. Subsequently, starch biosynthesis genes were identified, and 15 of them, including