Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>)

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(?21) used as a tail on each locus-specific forward primer and a single universal primer M13(?21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.     

Characterization of genome-wide microsatellites of <Emphasis Type="Italic">Saccharina japonica</Emphasis> based on a preliminary assembly of Illumina sequencing reads

Microsatellites or simple sequence repeats (SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp (Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N₅₀ of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.     

Location of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> resistance-associated trait loci in half-smooth tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis> at its microsatellite linkage map

A cultured female half-smooth tongue sole (Cynoglossus semilaevis) was crossed with a wild male, yielding the first filial generation of pseudo-testcrossing from which 200 fish were randomly selected to locate the Vibrio anguillarum resistance trait in half-smooth tongue sole at its microsatellite linkage map. In total, 129 microsatellites were arrayed into 18 linkage groups, ≥4 each. The map reconstructed was 852.85 cM in length with an average spacing of 7.68 cM, covering 72.07% of that expected (1 183.35 cM). The V. anguillarum resistance trait was a composite rather than a unit trait, which was tentatively partitioned into Survival time in Hours After V. anguillarum Infection (SHAVI) and Immunity of V. Anguillarum Infection (IVAI). Above a logarithm of the odds (LOD) threshold of 2.5, 18 loci relative to SHAVI and 3 relative to IVAI were identified. The 3 loci relative to IVAI explained 18.78%, 5.87% and 6.50% of the total phenotypic variation in immunity. The microsatellites bounding the 3 quantitative trait loci (QTLs) of IVAI may in future aid to the selection of V. anguillarum-immune half-smooth tongue sole varieties, and facilitate cloning the gene(s) controlling such immunity.     

Genetic transformation of <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> with a bacterial phleomycin resistance gene as dominant selective marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×10⁷ cells mL^?1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL^?1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.     

Analysis of phenotypic and genetic parameters for growthrelated traits in the half smooth tongue sole, <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Phenotypic and genetic parameters for growth-related traits in the half-smooth tongue sole, Cynoglossus semilaevis, were estimated in 22 full-sib families produced by normal and neo-male breeding stocks. As phenotypic males with female genotypes, neo-males are harmful in C. semilaevis aquaculture because they reduce overall production. The present study evaluated the difference in the growth-related traits: total length (TL), body weight (BW) and square root of body weight (SQ_BW) at the age of 570 days between normal and neo-male offspring (neo-males used as male parents). The difference in the proportion of females between normal and neo-male offspring was also assessed. Based on the linear mixed model, restricted maximum likelihood (REML) and best linear unbiased prediction (BLUP) were used to estimate various (co)variance components and estimated breeding values (EBVs) of growth-related traits. As a result, all the mean values of the three studied traits were significantly larger in normal offspring than in neo-male offspring. Additionally, the female proportion was significantly larger in normal offspring than in neo-male offspring. Heritability was 0.128±0.066 2 for TL, 0.128±0.065 5 for BW and 0.132±0.062 9 for SQ_BW, all of which were low level heritabilities. The correlation coefficients of EBVs and phenotypic values of the target traits were 0.516 for TL, 0.524 for BW and 0.506 for SQ_BW, all of which were highly significant (P <0.01). Genetic correlations among TL, BW and SQ_BW were positive high (0.921–0.969) and higher than those of phenotype (0.711–0.748), both of which had low standard errors (0.063–0.123 for genotype, and 0.010–0.018 for phenotype). Compared with normal offspring, neo-male offspring have lower breeding values for each studied trait through EBVs comparison. Therefore, neo-male offspring should not be used as broodstock in a C. semilaevis breeding programs.     

A comparative study of spatially clustered distribution of jumbo flying squid (<Emphasis Type="Italic">Dosidicus gigas</Emphasis>) offshore Peru

We examined spatially clustered distribution of jumbo flying squid (Dosidicus gigas) in the offshore waters of Peru bounded by 78°–86°W and 8°–20°S under 0.5°×0.5° fishing grid. The study is based on the catch-per-unit-effort (CPUE) and fishing effort from Chinese mainland squid jigging fleet in 2003–2004 and 2006–2013. The data for all years as well as the eight years (excluding El Niño events) were studied to examine the effect of climate variation on the spatial distribution of D. gigas. Five spatial clusters reflecting the spatial distribution were computed using K-means and Getis-Ord Gi* for a detailed comparative study. Our results showed that clusters identified by the two methods were quite different in terms of their spatial patterns, and K-means was not as accurate as Getis-Ord Gi*, as inferred from the agreement degree and receiver operating characteristic. There were more areas of hot and cold spots in years without the impact of El Niño, suggesting that such large-scale climate variations could reduce the clustering level of D. gigas. The catches also showed that warm El Niño conditions and high water temperature were less favorable for D. gigas offshore Peru. The results suggested that the use of K-means is preferable if the aim is to discover the spatial distribution of each sub-region (cluster) of the study area, while Getis-Ord Gi* is preferable if the aim is to identify statistically significant hot spots that may indicate the central fishing ground.     

Significant population genetic structure detected in the rock bream <Emphasis Type="Italic">Oplegnathus fasciatus</Emphasis> (Temminck &amp; Schlegel, 1844) inferred from fluorescent-AFLP analysis

Oplegnathus fasciatus (rock bream) is a commercial rocky reef fish species in East Asia that has been considered for aquaculture. We estimated the population genetic diversity and population structure of the species along the coastal waters of China using fluorescent-amplified fragment length polymorphisms technology. Using 53 individuals from three populations and four pairs of selective primers, we amplified 1 264 bands, 98.73% of which were polymorphic. The Zhoushan population showed the highest Nei’s genetic diversity and Shannon genetic diversity. The results of analysis of molecular variance (AMOVA) showed that 59.55% of genetic variation existed among populations and 40.45% occurred within populations, which indicated that a significant population genetic structure existed in the species. The pairwise fixation index F _st ranged from 0.20 to 0.63 and were significant after sequential Bonferroni correction. The topology of an unweighted pair group method with arithmetic mean tree showed two significant genealogical branches corresponding to the sampling locations of North and South China. The AMOVA and STRUCTURE analyses suggested that the O. fasciatus populations examined should comprise two stocks.     

Edible peanut worm (<Emphasis Type="Italic">Sipunculus nudus</Emphasis>) in the Beibu Gulf: Resource,aquaculture, ecological impact and counterplan

Sipunculus nudus is an important economic species because of its high nutritional and medicinal values. The exploitation and utilization of S. nudus primarily occur in the coastal regions of the Beibu Gulf. However, wild resource of S. nudus is rapidly decreasing because of the overexploitation, which has led to considerable developments of artificial breeding techniques. The cultivation scale of S. nudus has increased in response to successful artificial breeding; however, methods for culturing S. nudus in tidal flats or ponds require further study. Most studies have focused on the breeding, nutrition, medical value and ecological impact of these worms. Studies on the distribution, sediment requirements, nutrition characteristics, breeding techniques and aquaculture ecology of this species are summarized in this paper to promote the development of the aquaculture industry for S. nudus. The high biomass of S. nudus in the Beibu Gulf is positively correlated with the sediment characteristics and water quality of the region. The production of peanut worm has improved to some extent through culturing; however, the nutrient value and ecological environment problems have been observed, which reflect the over exploitation of trace elements and the sediment. These problems will worsen unless they are resolved, and the release of organic materials, nitrogen and phosphorus during harvesting impacts the coastal environment. Moreover, genetic erosion is a potential risk for larvae in artificial breeding programs in tidal flats. Therefore, culturing and collecting methods should be improved and the wild resource conservation should be implemented to promote the sustainable development of the peanut worm.     

Genome-wide mining,characterization, and development of microsatellite markers in <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis> by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats (SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class (44.08%), followed by mononucleotides (29.67%), trinucleotides (18.96%), tetranucleotides (5.66%), hexanucleotides (1.07%), and pentanucleotides (0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci (90.0%) were successfully amplified with specific products and 24 (80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17 (with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.     

Development of three multiplex PCR primer sets for ark shell (<Emphasis Type="Italic">Scapharca broughtonii</Emphasis>) and their validation in parentage assignment

Scapharca broughtonii is a commercially important and over-exploited species. In order to investigate its genetic diversity and population structure, 43 novel polymorphic microsatellites were isolated and characterized. The number of alleles per locus ranged from 3 to 22 with an average of 6.93, and the observed and expected heterozygosities varied between 0.233 and 1.000, and 0.250 and 0.953, with an average of 0.614 and 0.707, respectively. Three highly informative multiplex PCRs were developed from nine of those microsatellites for S. broughtonii. We evaluated and validated these multiplex PCRs in 8 full-sib families. The average polymorphism information content (PIC) was 0.539. The frequency of null alleles was estimated as 3.13% of all the alleles segregation based on a within-family analysis of Mendelian segregation patterns. Parentage analysis of real offspring demonstrated that 100% of all offspring were unambiguously allocated to a pair of parents based on 3 multiplex sets. Those 43 microsatellite loci with high variability will be helpful for the analysis of population genetics and conservation of wild stock of S. broughtonii. The 3 sets of multiplex PCRs could be an important tool of pedigree reconstruction, population genetic analysis and brood stock management.     

Development and validation of 89 novel expressed sequence tag-derived microsatellite markers in blood clam, <Emphasis Type="Italic">Tegillarca granosa</Emphasis>

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

Biodegradation of <Emphasis Type="Italic">Enteromorpha</Emphasis> polysaccharides by intestinal micro-community from <Emphasis Type="Italic">Siganus oramin</Emphasis>

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL^?1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

Variations in leaf functional traits and physiological characteristics of <Emphasis Type="Italic">Abies georgei</Emphasis> var. <Emphasis Type="Italic">smithii</Emphasis> along the altitude gradient in the Southeastern Tibetan Plateau

Variations in leaf functional traits of Abies georgei var. smithii at 3700, 3900, 4100, 4300, and 4390 m altitude were investigated in 15 typical plots in the Southeastern Tibetan Plateau. In each plot, three seedlings were selected, of which functional leaves in current-year sunny branches were chosen for the measurement of morphological, photosynthetic, and physiological and biochemical characteristics, and their variations were analyzed. Results showed that significant variations existed among the leaf functional traits of A. georgei var. smithii along the altitudinal gradient, as well as their physiological adaption indicators. Leaf area decreased, while the mass per area and thickness of leaf increased at an altitude above 4,100 m. The maxima of pigment, total nitrogen concentration, net photosynthesis rate during light-saturated, and when water use efficiency appeared at 4100 m altitude. In addition, A. georgei var. smithii seedlings regulated the activities of superoxide dismutase and ascorbate peroxidase to resist abiotic stress under 4100 m altitude. Meanwhile, malondialdehyde concentration and the dark respiration rate rapidly increased, which indicates that A. georgei var. smithii seedlings suffered from heavy abiotic stress from 4100 m to 4390 m altitude. Basing on variations in leaf functional traits along the altitude gradient, we inferred that 4100 m altitude was the suitable region for A. georgei var. smithii growth in the Sygera Mountain. Moreover, the harsh environment was the main limiting factor for A. georgei var. smithii population expansion to high altitude.     

Biochemical Composition and Nutritional Value of Different Shell Color Strains of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The shell color of Pacific oyster (Crassostrea gigas) is a desirable trait; but the nutritional studies on C. gigas with different shell colors have not been conducted. Through successive selective breeding, five shell color strains of black (B), purple (P), orange (O), golden (G) and white (W) C. gigas have been developed. The aim of this study was to evaluate the chemical composition and nutritional value of five shell color strains and one commercial population with a common color. The biochemical composition including moisture, total protein, glycogen, ash, total fat, fatty acids (FA), amino acids and minerals was detected. The results indicated that the protein (50.76%–56.57%) was the major component. The content of glycogen showed a significant difference between orange shell and golden shell strains, as well as between commercial population and golden shell strain. In addition, all shell color strains contained a large amount of essential amino acids (12.20–14.15 g (100 g)^?1), of them leucine (2.81–3.29 g (100 g)^?1) and lysine (2.79–3.28 g (100 g)^?1) were predominant. The oysters were rich in polyunsaturated fatty acids (42.26%–45.24% of total fatty acid) with high levels of DHA (18.53%–21.16% of total fatty acid) and EPA (17.23%–18.68% of total fatty acid). Significant differences of mineral contents (Mg, Zn, Fe and Cu) were identified among the six populations. These results indicated that C. gigas with different shell colors presented rich nutritional value with high protein, glycogen, essential amino acids and polyunsaturated fatty acids. The biochemical composition obtained in this study is useful for selective breeding of C. gigas with different shell colors.     

Identification and bioinformatics analysis of microRNAs from the sporophyte and gametophyte of <Emphasis Type="Italic">Pyropia haitanensis</Emphasis>

Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata (Porphyra haitanensis) is an economically important genus that is cultured widely in China. P. haitanensis is cultured on a larger scale than Pyropia yezoensis, making up an important part of the total production of cultivated Pyropia in China. However, the majority of molecular mechanisms underlying the physiological processes of P. haitanensis remain unknown. P. haitanensis could utilize inorganic carbon and the sporophytes of P. haitanensis might possess a PCK-type C₄-like carbon-fixation pathway. To identify microRNAs and their probable roles in sporophyte and gametophyte development, we constructed and sequenced small RNA libraries from sporophytes and gametophytes of P. haitanensis. Five microRNAs were identified that shared no sequence homology with known microRNAs. Our results indicated that P. haitanensis might posses a complex sRNA processing system in which the novel microRNAs act as important regulators of the development of different generations of P. haitanensis.     

Significance of different microalgal species for growth of moon jellyfish ephyrae, <Emphasis Type="Italic">Aurelia</Emphasis> sp.1

The scyphozoan Aurelia aurita (Linnaeus) sp. l., is a cosmopolitan species-complex which blooms seasonally in a variety of coastal and shelf sea environments around the world. The effects of different microalgal species on the growth of newly-released Aurelia sp.1 ephyrae were studied under laboratory conditions. We fed ephyrae with four different microalgal species (diatom, autotrophic dinoflagellate, heterotrophic dinoflagellate, and chlorophyta) plus Artemia nauplii for 12-24 d at 18°C. Results showed that the growth rate diverged significantly for Artemia nauplii compared to other food types. In addition, there was no significant variation between the growth rates for Skeletonema costatum and Prorocentrum donghaiense, and no significant variation was found in the growth rates for N. scintillans and P. subcordiformis. Artemia nauplii could support the energy requirement for the newly-released ephyrae to develop to meduase, and the ephyrae with Artemia nauplii showed a significant average growth rate of 25.85% d^?1. Newly-released ephyrae could grow slightly with some species of microalgae in the earliest development stage. Chain diatom Skeletonema costatum and autotrophic dinoflagellate Prorocentrum donghaiense, could not support the growth of the ephyrae, while heterotrophic dinoflagellate Noctiluca scintillans and chlorophyta Platymonas subcordiformis could support the growth of the ephyrae. However, none of the ephyrae fed with the tested phytoplankton could mature to medusae.     

Quantitative trait loci detection of <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> resistance in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese flounder (Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. olivaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis (BSA) and quantitative trait loci (QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333 (♀: F0768 ×♂: F0915) (Nomenclature rule: F+year+family number) were used to detect simple sequence repeats (SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers (Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group (LG)-1 exhibited a significant difference between DNA, pooled/bulked from the resistant and susceptible groups (P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of significance in LG1, where 17 and 18 SSR markers were identified, respectively. Each model found three resistance-related QTLs by composite interval mapping (CIM). These six QTLs, designated qE1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, qE-2 and qE-4, were located at the 66.7 cM region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese flounder in the future.     

Distribution and quantitative zonation of unloading cracks at a proposed large hydropower station dam Site

Rock mass unloading is an important rock engineering problem because unloading may impact the stability of a rock mass slope. Based on hydroelectric engineering principles, this study focuses on the classification of unloading zones to reflect the rock mass structure characteristics. Geological background and slope structure of the study region were considered to investigate the distribution and deformation of the unloading process. Quantitative indices were classified according to the formation mechanisms and the geological exhibition of unloading zones. The P-wave velocity (V _P ), the ratio of the wave velocity (K _V , the ratio of the test P-wave velocity along the adit depth to the P-wave velocity of intact rock), the sum of joint openings every 2 meters (S _t ), and the density of open joints (D _t ) were calculated as quantitative indices for the rock mass unloading zone. The characteristics of the unloading zone of rock mass slopes at the dam site were successfully determined. The method of combining qualitative data with quantitative indices was found to be effective for the classification of slope unloading zones.