The Study Of Biological Characteristic And Genetic Diversity On Popualtion Of The Endangered Brachymystax Lenok Tsinlingensis

Brachymystax lenok tsinlingensis (Qinling lenok) is only found in cold-water mountainstreams and rivers of Qinling Mountains，Shaanxi, China. The fish has been listed as a secondclass state protected wild animal in China Red Data Book of Endangered Animal in1988.Althougth some research on the taxonomic status and genetic resources of Qinling lenok havebeen done, the study of population structure and genetic diversity is very seldom, andclassification of the fish is still controversial. In this study, with Youshui River, Heihe River,Qianhe River and Taibai River populatins as objects, we anlaysed the biologicalcharacteristics, the microstructure and ultrastructure of the digestive system, the geneticdiversity and genetic variation using the biological methods, microsatellite marker andanalysis technics of mtDAN sequence. All the results as follows:1. Biological characteristics: the population age range, which collected from YoushuiRiver in Huayang town (Shaanxi), is between1and4. The whole body length was rangedfrom136mm to280mm, the range of body mass was22.9g to214g, and the age of thepopulation is decreasing. The proportion of female and male was1:1.38(χ2=2.362, P>0.05).The fish’s head and body side have the blossom-shaped round and irregular erythematous.Upper and lower jaw, vomer, the tongue and palate bone all have small teeth with inward tilt.There is a jaw wrapped in septum between upper and lower jaw, the jaw can move90degrees.The relationship expression between body length and body weight was W=1.97×10-5L2.8686(r2=0.9194, n=100). The relationship of power exponent “b” and “3” is no significantdifference. The Von Bertalanffy growth model was fitted to constant speed of estimated agesof individual fish and estimated growth parameters, Von Bertalanffy growth model: Lt=759.2[1-e-0.143(t+0.51)] and Wt=4408.31[1--0.143(t+0.51)].2. The microstructure and ultrastructure of the digestive system: The histologicalstructure of the alimentary canal consists of four layers: mucosa, submucosa, muscularis andserosa. The esophageal mucosa consists of undifferentiated mucous cells and surfaceepithelial cells. The U-shaped stomach was divided into cardiac, fundic and pyloric region.There were numerous gastric glands in the submucosa layer of the cardiac and fundic stomach,but none were present in the pyloric region. The convoluted tube-shape intestine is lined by simple columnar epithelial cells with microvilli at the apical surface. There were numerousgoblet cells in the intestine. Finger-like pyloric caeca were found in the front of intestine lube,with average number67. In ultrastructural level, mucous and glandular cells in the stomachwere found, the glandular cell with well-developed tubulovesicular system, a great amount ofpepsinogen granules, mitochondria and Golgi apparatus. The enterocytes with abundantmicrovilli contained mitochondria and lysosome, and mucous granules of goblet cell wereapparent. High density of lipid droplets of pyloric caeca might be concerned withfat-absorption。3. Isolation of the microsatellite marker: the study used the method of FIASCO (Fastisolation by AFLP sequences containing repeats) to constructed microsatellite enrichmentlibrary of Qinling lenok. One hundred and seventy-six clones contained microsatellites. Basedon these sequences, fifty-eight microsatellite primers was designed and synthesized. Twenty-five primers was validated polymorphism in the mix wild population. The observed numbersof alleles ranged from3to9. The expected and observed heterozygosities ranged from0.4183to0.8831and from0.3483to0.8992, respectively. These polymorphic microsatellitemarkers can be used to investigate the genetic diversity and population structure of wild B.lenok tsinlingensis.4. Genetic diversity based on microsatellite: eleven microsatellite loci were first used toevaluate the genetic characterization at the northern (Shitou River and Xianyi Riverpopulations) and southern population (Youshui River and Taibai River populations) of QinlingMountains. A total of59alleles were found and the number of alleles in each locus rangedfrom3to8，with an average of5.4. The average expected heterozygosity (He) and observed(Ho) heterozygosity in four population ranged from0.6421-0.6980and from0.4841-0.6999.Significant deviations from Hardy–Weinberg were found in the four populations at some ofthe loci. Analysis of molecular variance (AMOVA) revealed that relatively little (11.02%) ofgenetic diversity came from individual between a population. In contrast, the majority ofdiversity (88.98%) occurred among individual within a population. Result from Nei’s geneticdistance indicated that the Youshui River and Taibai River population were grouped in onecluster, which was clustered with the Shitou River population. The Xianyi River populationwas grouped in a separated cluster.5. MtDNA sequence analysis: the mitochondrial genomes organization is similar that ofthe other fish in salmonidae. The A content is4700bp(28.2%), the T content is4449bp(26.7%), the C content is4783bp (28.7%), the G content is2733bp (16.4%). The total lengthof thirteen protein-coding genes is11431bp in length, and share start codon ATG, stopcondons include TAA(ND5、ND4、ND4L、COX1、COX3、ATPase8and ATPase); TAA (Cytb and ND6gene); TAG (ND gene); and incomplete stop condon T (COX2and ND3gene).Several other conservative sequence were identified in the control region, which containingtermination associated sequence(ETAS), conserved sequence blocks (CSB-D, CSB-F andCSB-1, CSB-2, CSB-3), a T-type microsatellite and82bp tandem repeat sequences.Evolutionary developmental tree was constructed based on PCG and SrRNA sequence.Brachymystax and Hucho have closest relative, and located the root of developmental tree.The Kinmura-parameter distance of PCG between Qinling lenok and Brachymystax lenok is0.21, and sequence differences are2.1%, the results support the subspecies differentiation ofB. lenok tsinlingensis.6. Genetic variation based on control region (D-loop): the mtDNA control region(D-loop) of Qinling lenok was used to analyze its natural population structure and the geneticvariation of53individuals collected from four locations (Dajiangou in Taibai, Youshui Riverin Yangxian, Qianhe in Longxian and Heihe in Zhouzhi). Length heterogeneity was foundamong and between populations. The estimated haplotype and nucleotide diversity were9and0.0023, respectively. Genetic structure analysis showed a high level genetic diversity of B.lenok tisnlingensis (h=0.6060±0.1499). The AMOVA analysis indicated that26.02%of totalvariation came from individual populations, and73.98%from variation within the4geographic populations, which showed low genetic differentiation between the4geographicgroups. Test of neutral evolution and mismatch distribution indicated that no historicalexpansion occurred in these populations. The moderate genetic diversity and low geneticdifferentiation would provide new information for conservation and exploitation of thisspecies.Unique ecosystems of Qinling Mountains force the fish to form local subspecies. Wildpopulations have miniaturization trend. There have some genetic differenriation between thesourther and northern populations of Qinling Mountain. The genetic diversity of thesepopulations is moderate. These results are used to conservation of genetic resources preventdecling of genetic diversity of this endangered B. lenok tsilingensis species.