Large-scale field phenotyping using backpack LiDAR and CropQuant-3D to measure structural variation in wheat.

Plant phenomics bridges the gap between traits of agricultural importance and genomic information. Limitations of current field-based phenotyping solutions include mobility, affordability, throughput, accuracy, scalability, and the ability to analyze big data collected. Here, we present a large-scale phenotyping solution that combines a commercial backpack Light Detection and Ranging (LiDAR) device and our analytic software, CropQuant-3D, which have been applied jointly to phenotype wheat (Triticum aestivum) and associated 3D trait analysis. The use of LiDAR can acquire millions of 3D points to represent spatial features of crops, and CropQuant-3D can extract meaningful traits from large, complex point clouds. In a case study examining the response of wheat varieties to three different levels of nitrogen fertilization in field experiments, the combined solution differentiated significant genotype and treatment effects on crop growth and structural variation in the canopy, with strong correlations with manual measurements. Hence, we demonstrate that this system could consistently perform 3D trait analysis at a larger scale and more quickly than heretofore possible and addresses challenges in mobility, throughput, and scalability. To ensure our work could reach non-expert users, we developed an open-source graphical user interface for CropQuant-3D. We, therefore, believe that the combined system is easy-to-use and could be used as a reliable research tool in multi-location phenotyping for both crop research and breeding. Furthermore, together with the fast maturity of LiDAR technologies, the system has the potential for further development in accuracy and affordability, contributing to the resolution of the phenotyping bottleneck and exploiting available genomic resources more effectively.

Establishment of a hepatocellular carcinoma patient-derived xenograft platform and its application in biomarker identification.

Using a method optimized in hepatocellular carcinoma (HCC), we established patient-derived xenograft (PDX) models with an increased take rate (42.2%) and demonstrated that FBS +10% dimethyl sulfoxide exhibited the highest tumor take rate efficacy. Among 254 HCC patients, 103 stably transplantable xenograft lines that could be serially passaged, cryopreserved and revived were established. These lines maintained the diversity of HCC and the essential features of the original specimens at the histological, transcriptome, proteomic and genomic levels. Tumor engraftment was associated with lack of encapsulation, poor tumor differentiation, large size and overexpression of cancer stem cell biomarkers, and was an independent predictor for overall survival and tumor recurrence after resection. To confirm the preclinical value of the PDX model in HCC treatment, several antitumor agents were tested in 16 selected PDX models. The results revealed a high degree of pharmacologic heterogeneity in the cohort, as well as heterogeneity to different agents in the same individual. The sorafenib responses observed between HCC patients and the corresponding PDXs were also consistent. After molecular characterization of the PDX models, we explored the predictive markers for sorafenib response and found that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) might play an important role in sorafenib resistance and sorafenib response is impaired in patients with MAP3K1 downexpression. Our results indicated that PDX models could accurately reproduce patient tumors biology and could aid in the discovery of new treatments to advance in precision medicine.

Rapid evolution of a retro-transposable hotspot of ovine genome underlies the alteration of BMP2 expression and development of fat tails.

BACKGROUND: Sheep have developed the ability to store fat in their tails, which is a unique way of reserving energy to survive a harsh environment. However, the mechanism underlying this adaptive trait remains largely unsolved. RESULTS: In the present study, we provide evidence for the genetic determinants of fat tails, based on whole genome sequences of 89 individual sheep. A genome-wide scan of selective sweep identified several candidate loci including a region at chromosome 13, a haplotype of which underwent rapid evolution and spread through fat-tailed populations in China and the Middle East. Sequence analysis revealed an inter-genic origin of this locus, which later became a hotspot of ruminant-specific retro-transposon named BovB. Additionally, the candidate locus was validated based on a fat- and thin-tailed cross population. The expression of an upstream gene BMP2 was differentially regulated between fat-tailed and thin-tailed individuals in tail adipose and several other tissue types. CONCLUSIONS: Our findings suggest the fixation of fat tails in domestic sheep is caused by a selective sweep near a retro-transposable hotspot at chromosome 13, the diversity of which specifically affects the expression of BMP2. The present study has shed light onto the understanding of fat metabolism.

Whole-genome sequencing of six dog breeds from continuous altitudes reveals adaptation to high-altitude hypoxia.

The hypoxic environment imposes severe selective pressure on species living at high altitude. To understand the genetic bases of adaptation to high altitude in dogs, we performed whole-genome sequencing of 60 dogs including five breeds living at continuous altitudes along the Tibetan Plateau from 800 to 5100 m as well as one European breed. More than 150× sequencing coverage for each breed provides us with a comprehensive assessment of the genetic polymorphisms of the dogs, including Tibetan Mastiffs. Comparison of the breeds from different altitudes reveals strong signals of population differentiation at the locus of hypoxia-related genes including endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) and beta hemoglobin cluster. Notably, four novel nonsynonymous mutations specific to high-altitude dogs are identified at EPAS1, one of which occurred at a quite conserved site in the PAS domain. The association testing between EPAS1 genotypes and blood-related phenotypes on additional high-altitude dogs reveals that the homozygous mutation is associated with decreased blood flow resistance, which may help to improve hemorheologic fitness. Interestingly, EPAS1 was also identified as a selective target in Tibetan highlanders, though no amino acid changes were found. Thus, our results not only indicate parallel evolution of humans and dogs in adaptation to high-altitude hypoxia, but also provide a new opportunity to study the role of EPAS1 in the adaptive processes.

Fibroblast-derived tenascin-C promotes Schwann cell migration through ß1-integrin dependent pathway during peripheral nerve regeneration.

Peripheral nerve regeneration requires precise coordination and dynamic interaction among various types of cells in the tissue. It remains unclear, however, whether the cellular crosstalk between fibroblasts and Schwann cells (SCs) is related to phenotype modulation of SCs, a critical cellular process after peripheral nerve injury. In this study, microarray analysis revealed that a total of 6,046 genes were differentially expressed in the proximal nerve segment after sciatic nerve transection in rats, and bioinformatics analysis further identified tenascin-C (TNC), an extracellular matrix (ECM) protein, as a key gene regulator. TNC was abundantly produced by nerve fibroblasts accumulating at the lesion site, rather than by SCs as usually expected. TNC significantly promoted SC migration without effects on SC proliferation in primary culture. In co-culture of fibroblasts and SCs, inhibition of TNC expression either by siRNA transfection or antibody blockade could suppress SC migration, while TNC-stimulated SC migration was mediated by TNC binding to ß1-integrin receptor in SCs through activation of Rac1 effectors. The in vivo evidence showed that exogenous TNC protein enhanced SC migration and axonal regrowth. Our results highlight that TNC-mediated cellular interaction between fibroblasts and SCs may regulate SC migration through ß1-integrin-dependent pathway during peripheral nerve regeneration.

MiR-9 inhibits Schwann cell migration by targeting Cthrc1 following sciatic nerve injury.

The regulative effects of microRNAs (miRNAs) on responses of Schwann cells to a nerve injury stimulus are not yet clear. In this study, we noted that the expression of eight miRNAs was downregulated at different time points following rat sciatic nerve transection, and found that 368 potential targets of these eight miRNAs were mainly involved in phenotypic modulation of Schwann cells. Of these miRNAs, miR-9 was identified as an important functional regulator of Schwann cell migration that was a crucial regenerative response of Schwann cells to nerve injury. In vitro, upregulated expression of miR-9 inhibited Schwann cell migration, whereas silencing of miR-9 promoted Schwann cell migration. Intriguingly, miR-9 exerted this regulative function by directly targeting collagen triple helix repeat containing protein 1 (CTHRC1), which in turn inactivated downstream Rac1 GTPase. Rac1 inhibitor reduced the promotive effects of anti-miR-9 on Schwann cell migration. In vivo, high expression of miR-9 reduced Schwann cell migration within a regenerative nerve microenvironment. Collectively, our results confirmed the role of miR-9 in regulating Schwann cell migration after nerve injury, thus offering a new approach to peripheral nerve repair.

Noninvasive diagnosis and monitoring of mutations by deep sequencing of circulating tumor DNA in esophageal squamous cell carcinoma.

Circulating tumor DNA (ctDNA) is becoming an important biomarker in noninvasive diagnosis and monitoring of tumor dynamics. This study tested the feasibility of plasma ctDNA for the non-invasive analysis of tumor mutations in esophageal squamous cell carcinoma (ESCC) by sequencing of tumor, tumor-adjacent, and normal tissue, as well as pre-surgery and post-surgery plasma. Exome sequencing of eight patients identified between 29 and 134 somatic mutations in ESCCs, many of which were also determined in ctDNA. Comparison of pre-surgery and post-surgery plasma has shown that mutations had reduced frequency or disappeared after surgery treatment. We further evaluated the TruSight Cancer sequencing panel by using it to detect mutations in the plasma of three patients. Tumor mutations were only found in one of them. To design a sequencing panel with improved targeting, we identified significantly mutated genes by meta-analysis of 532 ESCC genomes. Our results confirmed the well-known driver genes and found several uncharacterized genes. The new panel consisted of 90 recurrent genes, which theoretically achieved 94% and 75% of sensitivity when detecting at least 1 and 2 mutant genes in ESCC patients, respectively. Our results demonstrate the feasibility of using ctDNA to detect ESCCs and monitor treatment effect. The low-cost and sensitive target panel could facilitate clinical usage of ctDNA as a noninvasive biomarker.

The 3DGD: a database of genome 3D structure.

UNLABELLED: The studies of chromatin 3D structure help us to understand its formation and function. Techniques combining chromosome conformation capture and next generation sequencing can capture chromatin structure information and has been applied to several different species and cell lines. We built 3DGD (3D Genome Database), a database that currently collected Hi-C data on four species, for easy accessing and visualization of chromatin 3D structure data. With the integration of other omics data such as genome-wide protein-DNA-binding data, this data source would be useful for researchers interested in chromatin structure and its biological functions. AVAILABILITY AND IMPLEMENTATION: The 3DGD v1.1, data browser, downloadable files and documentation are available at: http://3dgd.biosino.org/.

Charge transport and ac response under light illumination in gate-modulated DNA molecular junctions.

Using a two-strand tight-binding model and within nonequilibrium Green's function approach, we study charge transport through DNA sequences (GC)NGC and (GC)1(TA)NTA (GC)3 sandwiched between two Pt electrodes. We show that at low temperature DNA sequence (GC)NGC exhibits coherent charge carrier transport at very small bias, since the highest occupied molecular orbital in the GC base pair can be aligned with the Fermi energy of the metallic electrodes by a gate voltage. A weak distance dependent conductance is found in DNA sequence (GC)1(TA)NTA (GC)3 with large NTA. Different from the mechanism of thermally induced hopping of charges proposed by the previous experiments, we find that this phenomenon is dominated by quantum tunnelling through discrete quantum well states in the TA base pairs. In addition, ac response of this DNA junction under light illumination is also investigated. The suppression of ac conductances of the left and right lead of DNA sequences at some particular frequencies is attributed to the excitation of electrons in the DNA to the lead Fermi surface by ac potential, or the excitation of electrons in deep DNA energy levels to partially occupied energy levels in the transport window. Therefore, measuring ac response of DNA junctions can reveal a wealth of information about the intrinsic dynamics of DNA molecules.

Genome sequence of Anopheles sinensis provides insight into genetics basis of mosquito competence for malaria parasites.

BACKGROUND: Anopheles sinensis is an important mosquito vector of Plasmodium vivax, which is the most frequent and widely distributed cause of recurring malaria throughout Asia, and particularly in China, Korea, and Japan. RESULTS: We performed 454 next-generation sequencing and obtained a draft sequence of A. sinensis assembled into scaffolds spanning 220.8 million base pairs. Analysis of this genome sequence, we observed expansion and contraction of several immune-related gene families in anopheline relative to culicine mosquito species. These differences suggest that species-specific immune responses to Plasmodium invasion underpin the biological differences in susceptibility to Plasmodium infection that characterize these two mosquito subfamilies. CONCLUSIONS: The A. sinensis genome produced in this study, provides an important resource for analyzing the genetic basis of susceptibility and resistance of mosquitoes to Plasmodium parasites research which will ultimately facilitate the design of urgently needed interventions against this debilitating mosquito-borne disease.

miR-221 and miR-222 promote Schwann cell proliferation and migration by targeting LASS2 after sciatic nerve injury.

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Their roles in regulating the responses of Schwann cells (SCs) to injury stimuli remain unexplored. Here we report dynamic alteration of miRNA expression following rat sciatic nerve injury using microarray analysis. We harvested the proximal nerve stumps and identified 77 miRNAs that showed significant changes at four time points after nerve transection. Subsequently, we analyzed the expression pattern of miRNA, selected one significant profile, and then integrated putative miRNA targets with differentially expressed mRNA yielding 274 potential targets. The 274 targets were mainly involved in cell proliferation, cell locomotion and cellular homeostasis that were known to play important roles in modulating cell phenotype. The upregulation of the miR-221 and miR-222 cluster (miR-221/222) was found to correlate with the injury-induced SC phenotypic modulation. Enhanced expression of miR-221/222 could promote SC proliferation and migration in vitro, whereas silencing their expression resulted in a reduced proliferation and migration. Further studies revealed that longevity assurance homologue 2 (LASS2) was a direct target of miR-221/222 in SCs because miR-221/222 bound directly to the 3'-untranslated region of LASS2, thus reducing both mRNA and protein levels of LASS2. Silencing of LASS2 recapitulated the effects of miR-221/222 mimics, whereas enforced knockdown of LASS2 reversed the suppressive effects of miR-221/222 inhibitors. Our findings indicate that injury promotes SC proliferation and migration through the regulation of miR-221/222 by targeting LASS2, and provide new insights into the role of miRNAs in nerve regeneration.

Negative differential conductance and hysteretic current switching of benzene molecular junction in a transverse electric field.

We study charge transport through single benzene molecular junction (BMJ) directly sandwiched between two platinum electrodes by using a tight-binding model and the non-equilibrium Green's function approach. Pronounced negative differential conductance is observed at finite bias voltage, resulting from charge redistribution in BMJ and a Coulomb blockade effect at the interface of molecule-electrode contacts. In the presence of a transverse electric field, hysteretic switching behavior and large spin-polarization of current are obtained, indicating the potential application of BMJ for acting as a nanoscale current modulator or spintronic molecular device.

miR-182 inhibits Schwann cell proliferation and migration by targeting FGF9 and NTM, respectively at an early stage following sciatic nerve injury.

The regulation of Schwann cell (SC) responses to injury stimuli by microRNAs (miRNAs) remains to be explored. Here, we identified 17 miRNAs that showed dynamic expression alterations at five early time points following rat sciatic nerve resection. Then we analyzed the expression pattern of 17 miRNAs, and integrated their putative targets with differentially expressed mRNAs. The resulting 222 potential targets were mainly involved in cell phenotype modulation, including immune response, cell death and cell locomotion. Among 17 miRNAs, miR-182 expression was up-regulated. The enhanced expression of miR-182 was correlated with nerve injury-induced phenotype modulation of SCs. Further investigation revealed that fibroblast growth factor 9 (FGF9) and neurotrimin (NTM) were two direct targets of miR-182 in SCs, with miR-182 binding to the 3'-untranslated region of FGF9 and NTM. Silencing of FGF9 and NTM recapitulated the inhibiting effect of miR-182 mimics on SC proliferation and migration, respectively, whereas enforced knockdown of FGF9 and NTM reversed the promoting effect of miR-182 inhibitor on SC proliferation and migration, respectively. Our data indicate that nerve injury inhibits SC proliferation and migration through rapid regulation of miR-182 by targeting FGF9 and NTM, providing novel insights into the roles of miRNAs in nerve injury and repair.

Evaluating coverage of exons by HapMap SNPs.

Genome-wide association (GWA) studies are currently one of the most powerful tools in identifying disease-associated genes or variants. In typical GWA studies, single-nucleotide polymorphisms (SNPs) are often used as genetic makers. Therefore, it is critical to estimate the percentage of genetic variations which can be covered by SNPs through linkage disequilibrium (LD). In this study, we use the concept of haplotype blocks to evaluate the coverage of five SNP sets including the HapMap and four commercial arrays, for every exon in the human genome. We show that although some Chips can reach similar coverage as the HapMap, only about 50% of exons are completely covered by haplotype blocks of HapMap SNPs. We suggest further high-resolution genotyping methods are required, to provide adequate genome-wide power for identifying variants.

External Validation of the Kidney Failure Risk Equation Among Urban Community-Based Chinese Patients With CKD.

Rationale & Objective: The Kidney Failure Risk Equations have been proven to perform well in multinational databases, whereas validation in Asian populations is lacking. This study sought to externally validate the equations in a community-based chronic kidney disease cohort in China. Study Design: A retrospective cohort study. Setting & Participants: Patients with and estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 dwelling in an industrialized coastal city of China. Exposure: Age, sex, eGFR, and albuminuria were included in the 4-variable model, whereas serum calcium, phosphate, bicarbonate, and albumin levels were added to the previously noted variables in the 8-variable model. Outcome: Initiation of long-term dialysis treatment. Analytical Approach: Model discrimination, calibration, and clinical utility were evaluated by Harrell's C statistic, calibration plots, and decision curve analysis, respectively. Results: A total of 4,587 participants were enrolled for validation of the 4-variable model, whereas 1,414 were enrolled for the 8-variable model. The median times of follow-up were 4.0 (interquartile range: 2.6-6.3) years for the 4-variable model and 3.4 (2.2-5.6) years for the 8-variable model. For the 4-variable model, the C statistics were 0.750 (95% CI: 0.615-0.885) for the 2-year model and 0.766 (0.625-0.907) for the 5-year model, whereas the values were 0.756 (0.629-0.883) and 0.774 (0.641-0.907), respectively, for the 8-variable model. Calibration was acceptable for both the 4-variable and 8-variable models. Decision curve analysis for the models at the 5-year scale performed better throughout different net benefit thresholds than the eGFR-based (<30 mL/min/1.73 m2) strategy. Limitations: A large proportion of patients lack albuminuria measurements, and only a subset of population could provide complete data for the 8-variable equation. Conclusions: The kidney failure risk equations showed acceptable discrimination and calibration and better clinical utility than the eGFR-based strategy for incidence of kidney failure among community-based urban Chinese patients with chronic kidney disease.

Accurate and reliable risk evaluation of chronic kidney disease (CKD) prognosis can be helpful for physicians to make decisions concerning treatment opportunity and therapeutic strategy. The kidney failure risk equation is an outstanding model for predicting risk of kidney failure among patients with CKD. However, the equation is lacking validation among Chinese populations. In the current study, we demonstrated that the equation had good discrimination among an urban community-based cohort of patients with CKD in China. The calibration was also acceptable. Decision curve analysis also showed that the equation performed better than a traditional kidney function-based strategy. The results provide the basis for using predictions derived from the kidney failure risk equation to improve the management of patients with CKD in community settings in China.

The de novo sequence origin of two long non-coding genes from an inter-genic region.

BACKGROUND: The gene Polymorphic derived intron-containing, known as Pldi, is a long non-coding RNA (lncRNA) first discovered in mouse. Although parts of its sequence were reported to be conserved in rat and human, it can only be expressed in mouse testis with a mouse-specific transcription start site. The consensus sequence of Pldi is also part of an antisense transcript AK158810 expressed in a wide range of mouse tissues. RESULT: We focused on sequence origin of Pldi and Ak158810. We demonstrated that their sequence was originated from an inter-genic region and is only presented in mammalians. Transposable events and chromosome rearrangements were involved in the evolution of ancestral sequence. Moreover, we discovered high conservation in part of this region was correlated with chromosome rearrangements, CpG demethylation and transcriptional factor binding motif. These results demonstrated that multiple factors contributed to the sequence origin of Pldi. CONCLUSIONS: We comprehensively analyzed the sequence origin of Pldi-Ak158810 loci. We provided various factors, including rearrangement, transposable elements, contributed to the formation of the sequence.

The Dissection of Nitrogen Response Traits Using Drone Phenotyping and Dynamic Phenotypic Analysis to Explore N Responsiveness and Associated Genetic Loci in Wheat.

Inefficient nitrogen (N) utilization in agricultural production has led to many negative impacts such as excessive use of N fertilizers, redundant plant growth, greenhouse gases, long-lasting toxicity in ecosystem, and even effect on human health, indicating the importance to optimize N applications in cropping systems. Here, we present a multiseasonal study that focused on measuring phenotypic changes in wheat plants when they were responding to different N treatments under field conditions. Powered by drone-based aerial phenotyping and the AirMeasurer platform, we first quantified 6 N response-related traits as targets using plot-based morphological, spectral, and textural signals collected from 54 winter wheat varieties. Then, we developed dynamic phenotypic analysis using curve fitting to establish profile curves of the traits during the season, which enabled us to compute static phenotypes at key growth stages and dynamic phenotypes (i.e., phenotypic changes) during N response. After that, we combine 12 yield production and N-utilization indices manually measured to produce N efficiency comprehensive scores (NECS), based on which we classified the varieties into 4 N responsiveness (i.e., N-dependent yield increase) groups. The NECS ranking facilitated us to establish a tailored machine learning model for N responsiveness-related varietal classification just using N-response phenotypes with high accuracies. Finally, we employed the Wheat55K SNP Array to map single-nucleotide polymorphisms using N response-related static and dynamic phenotypes, helping us explore genetic components underlying N responsiveness in wheat. In summary, we believe that our work demonstrates valuable advances in N response-related plant research, which could have major implications for improving N sustainability in wheat breeding and production.

Detecting the borders between coding and non-coding DNA regions in prokaryotes based on recursive segmentation and nucleotide doublets statistics.

BACKGROUND: Detecting the borders between coding and non-coding regions is an essential step in the genome annotation. And information entropy measures are useful for describing the signals in genome sequence. However, the accuracies of previous methods of finding borders based on entropy segmentation method still need to be improved. METHODS: In this study, we first applied a new recursive entropic segmentation method on DNA sequences to get preliminary significant cuts. A 22-symbol alphabet is used to capture the differential composition of nucleotide doublets and stop codon patterns along three phases in both DNA strands. This process requires no prior training datasets. RESULTS: Comparing with the previous segmentation methods, the experimental results on three bacteria genomes, Rickettsia prowazekii, Borrelia burgdorferi and E.coli, show that our approach improves the accuracy for finding the borders between coding and non-coding regions in DNA sequences. CONCLUSIONS: This paper presents a new segmentation method in prokaryotes based on Jensen-Rényi divergence with a 22-symbol alphabet. For three bacteria genomes, comparing to A12_JR method, our method raised the accuracy of finding the borders between protein coding and non-coding regions in DNA sequences.

Towards biological characters of interactions between transcription factors and their DNA targets in mammals.

BACKGROUND: In post-genomic era, the study of transcriptional regulation is pivotal to decode genetic information. Transcription factors (TFs) are central proteins for transcriptional regulation, and interactions between TFs and their DNA targets (TFBSs) are important for downstream genes' expression. However, the lack of knowledge about interactions between TFs and TFBSs is still baffling people to investigate the mechanism of transcription. RESULTS: To expand the knowledge about interactions between TFs and TFBSs, three biological features (sequence feature, structure feature, and evolution feature) were utilized to build TFBS identification models for studying binding preference between TFs and their DNA targets in mammals. Results show that each feature does have fairly well performance to capture TFBSs, and the hybrid model combined all three features is more robust for TFBS identification. Subsequently, correspondence between TFs and their TFBSs was investigated to explore interactions among them in mammals. Results indicate that TFs and TFBSs are reciprocal in sequence, structure, and evolution level. CONCLUSIONS: Our work demonstrates that, to some extent, TFs and TFBSs have developed a coevolutionary relationship in order to keep their physical binding and maintain their regulatory functions. In summary, our work will help understand transcriptional regulation and interpret binding mechanism between proteins and DNAs.

Evolution of protein phosphorylation for distinct functional modules in vertebrate genomes.

Recent publications have revealed that the evolution of phosphosites is influenced by the local protein structures and whether the phosphosites have characterized functions or not. With knowledge of the wide functional range of phosphorylation, we attempted to clarify whether the evolutionary conservation of phosphosites is different among distinct functional modules. We grouped the phosphosites in the human genome into the modules according to the functional categories of KEGG (Kyoto Encyclopedia of Genes and Genomes) and investigated their evolutionary conservation in vertebrate genomes from mouse to zebrafish. We have found that the phosphosites in the vertebrate-specific functional modules (VFMs), such as cellular signaling processes and responses to stimuli, are evolutionarily more conserved than those in the basic functional modules (BFMs), such as metabolic and genetic processes. The phosphosites in the VFMs are also significantly more conserved than their flanking regions, whereas those in the BFMs are not. These results hold for both serine/threonine and tyrosine residues, although the fraction of phosphorylated tyrosine residues is increased in the VFMs. Moreover, the difference in the evolutionary conservation of the phosphosites between the VFMs and BFMs could not be explained by the difference in the local protein structures. There is also a higher fraction of phosphosites with known functions in the VFMs than BFMs. Based on these findings, we have concluded that protein phosphorylation may play more dominant roles for the VFMs than BFMs during the vertebrate evolution. As phosphorylation is a quite rapid biological reaction, the VFMs that quickly respond to outer stimuli and inner signals might heavily depend on this regulatory mechanism. Our results imply that phosphorylation may have an essential role in the evolution of vertebrates.