| Chinese Bahaba(Bahaba flavolabiata),is an endemic fish species in China,and distributing in the east China sea and the northern south China sea.Due to the environment deteriorated sharply and overfishing,its resources are rapidly reduced and the species are endangered.It is difficult to carry out the relevant investigation and research;there have been very few studies on the biology of B.flavolabiata in China.Based on the development of SSR markers and the analysis of genetic diversity,this paper established the theoretical basis for the identification of germplasm resources and breeding of B.flavolabiata.On the other hand,through the study of morphology,histology and mucous cells in the digestive tract,understanding its digestion and absorption mechanism is of great significance to its artificial culture research.1.Development of SSR markers in B.flavolabiata by RNA-SeqIn this study,it generated about 13.43 Gb data in total after Illumina Hiseq sequencing.Then assemble all samples together,we got 65,047 Unigenes,and a total of 29,797 SSR loci were found,which distributed in 19,664 unigenes.Among these SSR loci,the main repeat types were di-nucleotide repeats(39.8%),followed by mono-nucleotide repeats(32.5%)and finally tri-nucleotide(22.4%).The AC/GT motif was the most abundant(28%)in di-nucleotide repeats.In total 26,949 pairs of primers were designed by Primer3 software.186 pairs of SSR primers were randomly selected and used to amplify the SSR loci in 6 B.flavolabiata samples,and 121 SSR primer pairs could amplify the target fragments with expected size,of which 47 pairs of primers produced polymorphic bands.The results indicated that it is feasible to develop SSR markers on a large scale by RNA-Seq,and the developed SSR markers can lay a foundation for the analysis of genetic diversity in B.flavolabiata.2.The genetic diversity of the natural population of B.flavolabiata was evaluated by SSR markersIn this paper,29 pairs of high polymorphic SSR primers are screened by the dual fluorescent PCR reaction.19 samples genes of B.flavolabiata are amplified by PCR and the genetic diversity of their products is analyzed.The results showed that: The number of alleles(Na)about 29 microsatellite loci was 2-7,the total number of alleles was 121,and the average number of alleles was 4.17.The effective allele(Ne)is between 1.0606 and 6.0561 and the sum is 85.2050.Respectively,the average values of the apparent heterozygosity(Ho)and the expected heterozygosity(He)are 0.6593 and 0.5973.The polymorphism information content PIC value was between 0.0555-0.8137 with an average of 0.5423.There were still 5 loci that deviated significantly from "Hardie Weinberg’s Law"(corrected P ≤ 0.05)with "Hardie Weinberger balance" significantly detecting P-values.The gene richness(Rs)is between 2 and 6.976 and the sum is 119.638.The gene diversity(GD)is between 0.059 and0.856.These data reflect that most of the SSR loci developed in this study are highly polymorphic,and the genetic diversity of the B.flavolabiata population is at a higher level.3.Histology and the distribution of mucous cells in digestive system of B.flavolabiataThis work was done to investigate histological structure and distribution features of mucous cell in digestive system of B.flavolabiata,and paraffin section,HE and AB-PAS(Alician blue and periodic acid Schiff reagent(AB,p H2.5))staining were used.The oesophagus was crude and short,and the Bu-type stomach was divided into three portions: cardiac stomach,fundic stomach and pyloric stomach.There were eight pyloric caeca,and intestine was detoured two inflections in the body cavity.Based on the histological analysis,digestive tract wall composed of serosa,muscularis submucosa and mucosa from outer to inner.The muscularis of esophagus was thick but mucosa was thin without mucous in the epithelium.Muscularis and mucosa composed of simple columnar epithelium in stomach were thick,there was gastric gland found in lamina propria.Features of pyloric could be characterized as large cavity diameter and nemours mucosa folds,but muscularis were thin hardly.Muscularis in intestine were thicker than in pyloric caeca with nemours mucosa folds and mucous cells were found in epithelium.AB-PAS staining reviewed that there only type Ⅰ and type Ⅱ mucous cells were detested out in digestive tract except esophagus of B.flavolabiata.Numorous type Ⅰ mucous cells were found in the gastric gland and epithelium of stomach.Only type Ⅱ mucous cells distributed in epithelium of pyloric caeca mucosa.A mounts of type Ⅱ and little bit type Ⅰ mucous cells were found in intestine,the former distributed mostly in the surface of epithelium and the latter in basal layer of epithelium.The structure of the digestive system and the distribution of mucous cells reflect the characteristic of the coordination between feeding habits and structure. |
