Plexin B3 guides axons to cross the midline in vivo.

During the development of neural circuits, axons are guided by a variety of molecular cues to navigate through the brain and establish precise connections with correct partners at the right time and place. Many axon guidance cues have been identified and they play pleiotropic roles in not only axon guidance but also axon fasciculation, axon pruning, and synaptogenesis as well as cell migration, angiogenesis, and bone formation. In search of receptors for Sema3E in axon guidance, we unexpectedly found that Plexin B3 is highly expressed in retinal ganglion cells of zebrafish embryos when retinal axons are crossing the midline to form the chiasm. Plexin B3 has been characterized to be related to neurodevelopmental disorders. However, the investigation of its pathological mechanisms is hampered by the lack of appropriate animal model. We provide evidence that Plexin B3 is critical for axon guidance in vivo. Plexin B3 might function as a receptor for Sema3E while Neuropilin1 could be a co-receptor. The intracellular domain of Plexin B3 is required for Semaphorin signaling transduction. Our data suggest that zebrafish could be an ideal animal model for investigating the role and mechanisms of Sema3E and Plexin B3 in vivo.

Microbial cell factories using Paenibacillus: status and perspectives.

Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus has been widely applied in: agriculture, medicine, industry, and environmental remediation. Paenibacillus species not only accelerate plant growth and degrade toxic substances in wastewater and soil but also produce industrially-relevant enzymes and antimicrobial peptides. Due to a lack of genetic manipulation tools and methods, exploitation of the bioresources of naturally isolated Paenibacillus species has long been limited. Genetic manipulation tools and methods continue to improve in Paenibacillus, such as shuttle plasmids, promoters, and genetic tools of CRISPR. Furthermore, genetic transformation systems develop gradually, including: penicillin-mediated transformation, electroporation, and magnesium amino acid-mediated transformation. As genetic manipulation methods of homologous recombination and CRISPR-mediated editing system have developed gradually, Paenibacillus has come to be regarded as a promising microbial chassis for biomanufacturing, expanding its application scope, such as: industrial enzymes, bioremediation and bioadsorption, surfactants, and antibacterial agents. In this review, we describe the applications of Paenibacillus bioproducts, and then discuss recent advances and future challenges in the development of genetic manipulation systems in this genus. This work highlights the potential of Paenibacillus as a new microbial chassis for mining bioresources.

[Prevention and control of antimicrobial resistance using CRISPR-Cas system: a review].

Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.

Cloning and characterization of nicotinic acetylcholine receptor γ-like gene in adult transparent Pristella maxillaris.

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating the development and function of nervous system. The muscle AChR is composed of four homologous glycoprotein subunits with a stoichiometry α2ßγδ in fetal or α2ßεδ in adult. But the mechanism controlling the transition of fetal AChR γ-subunit to adult AChR ε is still unknown. Here a gene annoted AChR γ-like in Pristella maxillaris was first cloned by rapid amplification of cDNA ends (RACE) based on a transcriptome of dorsal fins. The full length of AChR γ-like was 1984 bp and it encoded 518 amino acids from 100 bp to 1653 bp. The multiple alignment analysis showed that AChR γ-like had 98% protein identity to AChR γ-like in Astyanax mexicanus. Then an 11647 bp DNA from 5'-UTR to 3'-UTR was cloned based on gene structure of AChR γ-like in A.mexicanus. Additionally a 2768 bp DNA upstream 5'-UTR was cloned by chromosome walking method. Furthermore, the results from semi-quantitative PCR showed that AChR γ-like was highly expressed in embryo and adult tissues, such as the muscle, eye, heart and intestine. While it showed low expression in the brain and gill. Significantly, the results of in situ hybridization showed strong diffused expression of AChR γ-like in the muscle of 1 dpf (day post-fertilization) embryo. And weak signal was observed in the muscle of 2-4 dpf embryos. All these data indicated that AChR γ-like could be one subunit of AChRs in the muscle and it could be used to study the development of the neuromuscular junction in adult transparent Pristella maxillaris. Thus our work will lay the foundation for using Pristella maxillaris to analyze the in vivo function of the nAChRs in adult vertebrate.

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish.

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Aggressive-like behavior and increased glycine transporters in a zebrafish model of CHARGE syndrome.

Coloboma, heart defects, choanal atresia, restricted growth and development, genital hypoplasia, ear abnormalities and/or hearing loss (CHARGE) syndrome is a congenital disorder that is mainly caused by mutations within chromodomain helicase DNA-binding protein 7 (chd7). Behavioral abnormalities have been addressed in CHARGE syndrome, but the underlying mechanisms are still poorly understood. Here, we performed four behavioral tests-including the open-field test, novel-tank test, shoaling test and mirror-induced attack test-in chd7 heterozygous zebrafish mutants in order to characterize the behavioral abnormalities in a zebrafish model of CHARGE syndrome. We found that chd7 heterozygous mutants exhibited anxious-like behavior and aggressive-like behavior in the open-field test and in the mirror-induced attack test, respectively, which resembled the reported behavioral abnormalities in CHARGE syndrome in humans. Moreover, we found that glycine and D-cycloserine treatment rescued the aggressive behavior of chd7 heterozygous zebrafish mutants, indicating that the excitation and inhibition balance might be disrupted in the brains of chd7 heterozygous zebrafish mutants. Further analysis showed that the expression of glycine transporters was dramatically increased in the brains of chd7 heterozygous zebrafish mutants. Treatment with an inhibitor of glycine transporter 1, sarcosine, partially rescued the aggressive-like behavior of chd7 heterozygous zebrafish mutants. Taken together, our data suggest that the aggressive behavior in CHARGE syndrome may be due to the increased expression of glycine transporters, and inhibition of the activity of glycine transporters may be an approach to treat the behavioral abnormalities in CHARGE syndrome.

Volatile composition and classification of Lilium flower aroma types and identification, polymorphisms, and alternative splicing of their monoterpene synthase genes.

Lily is a well-known ornamental plant with a diversity of fragrant types. Basic information on lily floral scent compounds has been obtained for only a few accessions, and little is known about Lilium aroma types, the terpene synthase genes that may play roles in the production of key volatiles, or the range of monoterpenes that these genes produce. In this study, 41 cultivars were analyzed for volatile emissions, and a total of 46 individual volatile compounds were identified, 16 for the first time in lilies. Lily accessions were classified into six groups according to the composition of major scent components: faint-scented, cool, fruity, musky, fruity-honey, and lily. Monoterpenes were one of the main groups of volatiles identified, and attention was focused on terpene synthase (TPS) genes, which encode enzymes that catalyze the last steps in monoterpene synthesis. Thirty-two candidate monoterpene synthase cDNAs were obtained from 66 lily cultivars, and 64 SNPs were identified. Two InDels were also shown to result from variable splicing, and sequence analysis suggested that different transcripts arose from the same gene. All identified nucleotide substitution sites were highly correlated with the amounts of myrcene emitted, and InDel site 230 was highly correlated with the emission of all major monoterpenoid components, especially (E)-ß-ocimene. Heterologous expression of five cDNAs cloned from faint-scented and strong-scented lilies showed that their corresponding enzymes could convert geranyl diphosphate to (E)-ß-ocimene, α-pinene, and limonene. The findings from this study provide a major resource for the assessment of lily scent volatiles and will be helpful in breeding of improved volatile components.

Sema3E is required for migration of cranial neural crest cells in zebrafish: Implications for the pathogenesis of CHARGE syndrome.

CHARGE syndrome is a congenital disorder with multiple malformations in the craniofacial structures, and cardiovascular and genital systems, which are mainly affected by neural crest defects caused by loss-of-function mutations within chromodomain helicase DNA-binding protein 7 (CHD7). However, many patients with CHARGE syndrome test negative for CHD7. Semaphorin 3E (sema3E) is a gene reported to be mutated in patients with CHARGE syndrome. However, its role in the pathogenesis of CHARGE syndrome has not been verified experimentally. Here, we report that the knockdown of sema3E results in severe craniofacial malformations, including small eyes, defective cartilage and an abnormal number of otoliths in zebrafish embryos, which resemble the major features of CHARGE syndrome. Further analysis reveals that the migratory cranial neural crest cells are scattered in the region of the hindbrain, and the postmigratory neural crest cells are reduced in the pharyngeal arches upon sema3E knockdown. Notably, immunostaining and time-lapse imaging analyses of a neural crest cell-labelled transgenic fish line, sox10:EGFP, show that the migration of cranial neural crest cells is severely impaired, and many of these cells are misrouted upon sema3E knockdown. Furthermore, the sox10-expressing cranial neural crest cells are scattered in chd7 homozygous mutants, which phenocopied the phenotype in sema3E morphants. Overexpression of sema3E rescues the phenotype of scattered cranial neural crest cells in chd7 homozygotes, indicating that chd7 may control the expression of sema3E to regulate cranial neural crest cell migration. Collectively, our data demonstrate that sema3E is involved in the pathogenesis of CHARGE syndrome by modulating cranial neural crest cell migration.

Complete mitochondrial genome and phylogenic analysis of Rhinogobius cliffordpopei (Perciformes, Gobiidae).

The Rhinogobius (Perciformes, Gobiidae), is a species-rich freshwater gobiid genus. In this article, the complete mitochondrial genome (mtDNA) of the Rhinogobius cliffordpopei was first determined. The mtDNA of R. cliffordpopei consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. In the phylogenetic tree, R. cliffordpopei firstly joined with R. brunneu s, and next clustered with R. giurinus. Then, they constituted Gobiidae clade with the rest other six species. This relationship consists of taxonomic status. The results would provide basal molecular data for the future research of adaptive evolution in Rhinogobius.

Effect of Resveratrol on Sirtuins, OPA1, and Fis1 Expression in Adult Zebrafish Retina.

Purpose: We determined whether sirtuins (SIRT1-SIRT7) are expressed in the zebrafish retina, evaluated the modulatory effect of resveratrol in the normal retina, and examined N-Methyl-D-aspartic acid (NMDA)-induced zebrafish retinal damage associated with mitochondrial sirtuins and mitochondrial fusion and fission mediators, OPA1 and Fis1. Methods: Sirtuins, OPA1, and Fis1 mRNA expression was analyzed by RT-PCR and quantitative real time PCR (qPCR) in adult zebrafish (AB type) retina and liver. qPCR showed an effect of resveratrol on SIRTs (SIRT1, 3, 4, 5) and OPA1 and Fis1 in low and high concentrations (5 and 50 mg/L) at different time points (0, 1, 24, and 48 hours) in the retina. Western blots were performed to examine the expression of SIRTs and OPA1 proteins under high concentrations of resveratrol for 24 hours. Hematoxylin and eosin staining, qPCR and mitochondrial copy number, and DNA damage assays then were used to confirm the protective effects of resveratrol on NMDA-induced retinal damage. Results: The seven sirtuins and OPA1 were highly expressed in zebrafish retina compared to the liver. Treatment with resveratrol promoted SIRT1, mitochondrial sirtuins, and OPA1 gene and protein expression, and improved mitochondrial DNA repair in adult zebrafish retina. Interestingly, the effect of resveratrol on SIRT4 gene and protein expression was significantly higher in the zebrafish retina. Importantly, resveratrol offered protection against NMDA-induced retinal damage by activating the SIRT1 gene and subsequent protein expression. Mitochondrial sirtuins and OPA1 genes likely had a role in regulating mitochondrial dynamics. Conclusions: To our knowledge, our study is the first composite analysis of sirtuins in adult zebrafish retina and provides sufficient evidence that resveratrol, as an activator of SIRT1, protects NMDA-induced zebrafish retinal damage by potentially mediating mitochondrial sirtuins and OPA1 genes.

CRMP2 and CRMP4 Are Differentially Required for Axon Guidance and Growth in Zebrafish Retinal Neurons.

Axons are directed to their correct targets by guidance cues during neurodevelopment. Many axon guidance cues have been discovered; however, much less known is about how the growth cones transduce the extracellular guidance cues to intracellular responses. Collapsin response mediator proteins (CRMPs) are a family of intracellular proteins that have been found to mediate growth cone behavior in vitro; however, their roles in vivo in axon development are much less explored. In zebrafish embryos, we find that CRMP2 and CRMP4 are expressed in the retinal ganglion cell layer when retinal axons are crossing the midline. Knocking down CRMP2 causes reduced elongation and premature termination of the retinal axons, while knocking down CRMP4 results in ipsilateral misprojections of retinal axons that would normally project to the contralateral brain. Furthermore, CRMP4 synchronizes with neuropilin 1 in retinal axon guidance, suggesting that CRMP4 might mediate the semaphorin/neuropilin signaling pathway. These results demonstrate that CRMP2 and CRMP4 function differentially in axon development in vivo.

Chd7 Is Critical for Early T-Cell Development and Thymus Organogenesis in Zebrafish.

Coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, ear anomalies/deafness (CHARGE) syndrome is a congenital disorder affecting multiple organs and mainly caused by mutations in CHD7, a gene encoding a chromatin-remodeling protein. Immunodeficiency and reduced T cells have been noted in CHARGE syndrome. However, the mechanisms underlying T lymphopenia are largely unexplored. Herein, we observed dramatic decrease of T cells in both chd7knockdown and knockout zebrafish embryos. Unexpectedly, hematopoietic stem and progenitor cells and, particularly, lymphoid progenitor cells were increased peripherally in nonthymic areas in chd7-deficient embryos, unlikely to contribute to the T-cell decrease. Further analysis demonstrated that both the organogenesis and homing function of the thymus were seriously impaired. Chd7 might regulate thymus organogenesis through modulating the development of both neural crest cell-derived mesenchyme and pharyngeal endoderm-derived thymic epithelial cells. The expression of foxn1, a central regulator of thymic epithelium, was remarkably down-regulated in the pharyngeal region in chd7-deficient embryos. Moreover, the T-cell reduction in chd7-deficient embryos was partially rescued by overexpressing foxn1, suggesting that restoring thymic epithelium may be a potential therapeutic strategy for treating immunodeficiency in CHARGE syndrome. Collectively, the results indicated that chd7 was critical for thymic development and T-lymphopenia in CHARGE syndrome may be mainly attributed to the defects of thymic organogenesis. The current finding may benefit the diagnosis and therapy of T lymphopenia and immunodeficiency in CHARGE syndrome.

Molecular systematics and phylogenetic analysis of the Asian endemic freshwater sleepers (Gobiiformes: Odontobutidae).

The Odontobutidae is a group of freshwater sleepers endemic to East and Southeast Asia. The composition of the Odontobutidae is controversial and the systematics position of some species (e.g. Philypnus chalmersi) remains unknown. Phylogenetic relationship among the odontobutids has never been really tested due to the lack of informative morphological characters, and that molecular data have not been collected in many species. Here, we sampled 41 specimens, representing all known genera of the Odontobutidae except the Laotian genus Terateleotris, in addition to a disputable odontobutid species, Philypnus chalmersi and 14 outgroups (six families). We collected sequence data of 4434 single-copy nuclear coding loci using gene capture and Illumina sequencing. A robust phylogeny of the odontobutids and outgroups was built, confirming that the Odontobutidae is monophyletic and sister to the Rhyacichthyidae. We verified that Neodontobutis, Sineleotris and Philypnus chalmersi are members of the Odontobutidae based on the resulting phylogeny as well as patterns of pectoral girdle examined by X-ray microtomography. We proposed a new genus Microdous for Philypnus chalmersi based on the new morphological and molecular evidences. The family of the Odontobutidae can be divided into two clades: Microdous (=Philypnus) sister to a group consisting of Micropercops and Sineleotris, and Odontobutis sister to a group unifying Perccottus and Neodontobutis. Divergence time among the odontobutids was estimated based on 100 most clock-like loci and three fossil calibration points using BEAST. Ancestral range of the family was reconstructed using Reconstruct Ancestral States in Phylogenies (RASP) and BioGeoBEARS. The results suggest that the common ancestor of the odontobutids originated around 30.8 Ma (20.7-42.0 Ma, 95% HPDs) in South China. Orogeny, climatic change and river capture might account for diversification and current distribution of the odontobutids.

The population history of Garra orientalis (Teleostei: Cyprinidae) using mitochondrial DNA and microsatellite data with approximate Bayesian computation.

BACKGROUND: The South China landmass has been characterized by a complex geological history, including mountain lifting, climate changes, and river capture/reversal events. To determine how this complexity has influenced the landmass's phylogeography, our study examined the phylogeography of Garra orientalis, a cyprinid widely distributed in South China, using sequences from the mitochondrial DNA control region and cytochrome b gene (1887 bp) and polymorphisms of thirteen microsatellite loci. RESULTS: In total, 157 specimens were collected from eight populations. All 88 mtDNA haplotypes were identified as belonging to three major lineages, and these lineages were almost allopatric in their distributions. The results of a statistical dispersal-vicariance analysis suggested that the ancestral populations of G. orientalis were distributed south of the Yunkai Mountains, including on Hainan Island. The mtDNA data revealed a strong relationship between phylogeny and geography. In the microsatellite analysis, a total of 339 alleles with an average of 26 alleles per locus were observed across thirteen microsatellite loci. A clustering algorithm for microsatellite data revealed an admixture-like genetic structure. Although the mtDNA and microsatellite data sets displayed a discordant population structure, the results of an approximate Bayesian computation approach showed that these two markers revealed congruent historical signals. The population history of G. orientalis reflects vicariance events and dispersal related to the complex geological history of South China. CONCLUSION: Our results (i) found that the discordances between mtDNA and microsatellite markers were accounted for by admixtures; (ii) showed that the Wuzhishan and Yinggeling mountain ranges and Qiongzhou Strait were important barriers limiting gene exchange between populations on both sides; (iii) indicated that during glaciation and inter-glacial periods, the strait and continental shelves were exposed and sank, which contributed with the dispersion and differentiation of populations; and (iv) displayed that the admixtures between lineages took place in coastal populations and then colonized the tributaries of the Pearl River.

Variations existing in protein-coding genes of the complete mitochondrial genomes of two highly morphological similarity fish: Periophthalmus magnuspinnatus and P. modestus (Perciformes, Gobiidae).

Periophthalmus magnuspinnatus is a new record species of Gobiidae in China. It had been misidentified as P. modestus for long time early before 2006. Here, the complete mitochondrial genome (mtDNA) sequences of the two species were first reported and analyzed comparably. The two genomes were both consisted of 13 protein-coding genes, 22 tRNA genes, two rRNA genes and a control region. Except for eight tRNA and ND6 genes, all other mitochondrial genes were encoded on the heavy strand. It was 16,496 bp and 16,803 bp, respectively, and P. modestus had a 238 bp-gap between tRNAleu and ND5. There were high variations (10-19%) in the protein-coding genes. In the initiation condon and stop codons, the two fish also had tiny difference. Phylogenetic analysis showed that P. magnuspinnatus and P. modestus first clustered together and then they constituted Gobiidae clade with other 12 fish. Whereas Odontobutidae and Rhyacichthyidae formed the sister group, then clustered with Eleotridae, and finally joined with Gobiidae, which is consist with previous phenotypic report. The study will contribute to the phylogenetic analysis of the Gobiidae and natural resources conservation of P. magnuspinnatus and P. modestus.

New complete mitochondrial genome of the Odontobutis potamophila (Perciformes, Odontobutidae): genome description and phylogenetic performance.

A new complete sequence of mitochondrial genome of Odontobutis potamophila has been reported. It was 16,843 bp and consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. Except for 8 tRNA and ND6 genes, all other mitochondrial genes were encoded on the heavy strand. Phylogenetic comparison proved that ND4, ND5 and ND6 genes might be dependable "leading" indicators of lineage divergence in Odontobutis obscurus. Also, COII, ND5 and cytb genes are useful in examining intraspecific population genetic diversity.

The complete mitogenome of the oriental sucking barb, Garra orientalis (Cypriniformes, Cyprinidae): repetitive sequences in the control region.

The oriental sucking barb, Garra orientalis, is a small to moderate-sized freshwater fish. In this study, the complete mitochondrial genome of G. oriental was successfully sequenced for the first time with total length of 17,288 bp. The genome structure consists of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes and a control region. Moreover, tandem repeat unit ranged from 259 to 260 bp (repeated 3-4 times) was identified in the control region among G. orientalis individuals.

Complete mitochondrial genome of Osteochilus salsburyi (Cypriniformes, Cyprinidae).

Osteochilus salsburyi (Cypriniformes, Cyprinidae) is a small-sized fish of significant economic value. In this paper, the complete mitogenome sequence of O. salsburyi was first determined. It is 16,599 bp in length and consists of a typical vertebrate genome structure including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes, and a control region. Except for eight tRNA and ND6 genes, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand in descending order is A (33.0%), T (26.5%), C (25.4%), and G (15.0%), with a slight AT bias of 59.5%. This information of O. salsburyi mitogenome could contribute not only to the development of efficient conservation strategy on vulnerable genetic diversity but also to the identification of useful genetic markers for distinction across species boundary.

Genetic incorporation of D-lysine into diketoreductase in Escherichia coli cells.

Pyrococcus horikoshii lysyl-tRNA synthetase/tRNA orthogonal pair exhibited high selectivity towards D-lysine in the presence of excess amount of D-lysine. Based on the observation, this orthogonal pair was employed to encode D-lysine, and D-lysine was site-specifically incorporated into the diketoreductase in E. coli cells.

[Analysis of genetic diversity in wild populations of Trachidermus fasciatus by RAPD and the transformation of two SCAR markers].

Firstly, RAPD was conducted to analyze genetic diversity of Trachidermus fasciatus in the Fuchun River population (FR), Yellow River population (YR), Luan River population (LR), and Yalu River population (YL), with 32 polymorphic 10-bp random primers selected from 294 ones. Thirty wild individuals were detected in each population. The results indicated that the genetic diversity of T . fasciatus was relatively rich. The major results were as the following: 1) Altogether, 591 bands were detected and 515 of them were polymorphic, accounted for 87.14%. The range of proportion of polymorphic loci (P) was: FR(89.17%)>YR(87.99%)>YL(86.63%)>LR(83.25%). 2) The Shannon's information index(I(T)) and Nei's genetic diversity(H(T)) among populations were 0.3393-0.3566 and 0.2157-0.2279, respectively. Compare to other three populations, LR population had relative lower values. If took the populations as a whole, the total Nei's genetic diversity(H(T)) and Shannon's information index(I(T)) was 0.2336±0.1643 and 0.3710±0.2153, respectively. 3) The value of gene flow (N(m) ) (5.76103-19.84497) were high, indicating certain gene exchange existed among the four populations. But the AMOVA results exhibited significantly differentiation (P<0.05 or P<0.01) among the populations. 4) In the UPGMA tree constructed according to genetic distance, YL and YR populations clustered firstly, then with FR population, and finally they joined to LR population. Obviously, the YL, YR and FR populations had relatively close relationship according to their geographic distance, whereas LR population showed clear divergence to the other three populations. Secondly, out of the five special RAPD bands (S(1225)(525 bp), S(1225)(605 bp), S(1225)(841 bp), S(1345)(695 bp) and S(1345)(825 bp)), SCAR maker SCAR01(560 bp) and SCAR02(443 bp) were successfully transformed from S(1255)(605 bp) and S(1255)(841 bp), respectively. After large samples examination of the two markers, we found the highest frequency (96.67% and 93.33%) in the YL population, higher frequency (83.33% and 90%) in the FR population, high frequency (56.67% and 66.67%) in the YR population, and the lowest frequency (13.33% and 20 %) in the LR population. Therefore, SCAR01(560 bp) and SCAR02(443 bp) can be used as special molecular markers for the population identification between LR and other three populations.