The Analysis Of Genetic Diversity And The Development Of Microsatellite Markers On Glyptosternum Maculatum (Regan) In The Yarlung Zangbo River, China

Glyptosternum maculatum(Regan)(Sisoridae,Siluriformes) is one of the GlyptosternoM catfish species distributed only in the middle-up reach of the Yarlung Zangbo River,Tibet,China.Nevertheless,three spawning grounds have been proposed, one each in the Nyang river,Lhasa river(two branches of the Yarlung Zangbo River) and Shetongmon reach.The individuals collected from these three populations are obviously fifferent in both body weight and body length.It is very important to make sure whether the geographical populations evolved independent or not for the conservation and proper utilization of Glyptosternum maculatum resources.In the present study,AFLP and SSR molecular methods were utilized to study the genetic diversity of the different Glyptosternum maculatum populations,the important results as bellows,1.The genetic diversity of Glyptosternum maculatum revealed by AFLP and SSR on species level(1) 81 AFLP primer combinations in total had been used to select the polymorphic primer combinations in Glyptosternum maculatum samples,then five ones selected as polymorphic primers,detected 332 products,51 of them(15.4%) were polymorphic in at least 1 population,no band was found to be specific for a population.The number of polymorphic bands ranged from 8(E-AAC/T-AAG,E-AAG/T-AAT and E-AAT/T-AGA) to 17(E-AAC/T-AAT) with an average of 10.2.The genetic distance between the three populations ranged from 0.0015 to 0.0042.(2) 28 SSR primer pairs had been used to select the polymorphic ones,at the end,6 polymorphic primer pairs amplified 20 alleles in all 96 Glyptosternum maculatum samples,on the average of 3.2,the information content(PIC) ranged from 0.0394 to 0.4159,on the average of 0.2837,indicating that these markers could be used to analyzed the genetic diversity of Glyptosternum maculatum.The genetic distance between the three populations ranged from 0.0022 to 0.0778.2.The comparison between SSR and AFLP:The AFLP markers detected 332 products,among which there were 51 polymorphic bands;The SSR markers amplified 20 alleles in total,which suggested that AFLP marker could provide more genetic information than SSR marker.Nevertheless,the results obtained by the two marker systems were very similar:they both revealed that the genetic diversity of Glyptosternum maculatum was very low,especially at the area of Lhasa,in addition to,there was no genetic variance existed among the populations.Both AFLP and SSR markers were effective to evaluate the genetic diversity of Glyptosternum maculatum.3.The development of microsatellite for Glyptosternum maculatumAll of the enriched library clones constructed by biotin-(CA)₁₅,there were 720 clones containing mierosatellite sequence(two or three bands in PCR results),145 clones were randomly selected to sequence,among which,139 sequences had the microsatellte sequence repeats above 7,15 sequences were the same.The repeats ranged from 8 to 165, on the average of 52.Among 124 microsatellite sequences,there were 80 of perfect,40 of imperfect and 14 of compound categories,part of sequences had been registered in Genebank.4.The screening of SSR markers for Glyptosternum maculatumThirty-three microsatellite primers were designed with their amplification conditions optimized,there were 29 primers amplified products and 25 one were polymorphism.The number of alleles per locus(A) was 2-10 with an average of 4.6,the observed heterozygosity(H_o) ranged from 0 to 1.The ranges of PIC varied from 0.2846 to 0.8173 with an average of 0.5793,which indicated their applicability for genetic analysis.5.The cross-amplification in other related taxa of SSR markers for Glyptosternum maculatumAll of the 14 primer pairs were revealed polymorphism for cross-species amplification in P.sulcatus(McClelland) using eighteen individuals.The number of alleles per loci ranged from 2 to 7(3.85 on the average) with the expected heterozygosity (H_E) ranging from 0.3476 to 0.8571(0.6054) and the observed heterozygosity(H_o) varying from 0 to 1(0.5992).The results suggested that there were 14 SSR marker developed from Glyptosternum maculatum successfully amplifying in other taxa Pseudechermeis sulcatus(McClelland).