| The genus Megalobrama(M.pellegrini,M hoffmanni,M.skolkovii,M.amblycephala)and the genus Parabramis(Parabramis pekinensis)are China's economic fish.They belongs to subfamily Culterinae,family Cyprinidae,Order Cypriniformes and distribute widely in Yangtze River,Pearl River,Heilongjiang River,Qiangtangjiang River,Yellow River,Minjiang River and lakes of Hainan island.These species were very similar in morphology.Here,we study the species of the genus Megalobrama and Parabramis in morphology and molecular genetics.1.Interspecific morphological comparison analysis of the genus Megalobrama and ParabramisTo investigate the difference among 5 species,41 morphometric characters and 23 meristic characters were measured from 446 wild individuals and analyzed by multivariate statistical.The result showed that significant differences(P<0.05)in the morphological characters(the number of caudal vertebrae)and meristic characters(length of spine in ventral fin/head length,body height/body length,caudal peduncle height/body length,4-5/body length,4-7/body length,caudal peduncle length/caudal peduncle height)among the 5 species which can be used to distinguish the species of the genus Megalobrama and P pekinensis.According to the principal component analysis(PCA)of the morphometric characters,three principal components were extracted and the related contribution ratios were 31.90%?17.12%?10.28%respectively,with the cumulative contribution ratio of 59.30%.As to the meristic characters,three principal components were extracted and the related contribution ratios were 23.83%?12.18%?9.86%respectively,with the cumulative contribution ratio of 45.87%.PCA scatterplots of meristic characters showed:P.pekinensis formed 1 cluster separated from the genus Megalobrama;the genus Megalobrama formed 4 clusters,M.skolkovii was clearly separated from the other 3 species in the genus Megalobrama;M.hoffmanni was clearly separated from M.skolkovii,rarely overlapped with M.pellegrini,partly overlapped with M amblycephala;M.pellegrini and M.amblycephala were with much overlap and hard to distinguish.PCA scatterplots of morphometric characters showed:P.pekinensis formed 1 cluster separated from the genus Megalobrama,which rarely overlapped with the genus Megalobrama;M.hoffmanni was separated from the other 3 species of the genus Megalobrama;thecluster of M skolkovii was clearly separated from M.hoffmanni,partly overlapped with M.amblycephala,mixed with M.pellegrini;M.pellegrini overlapped with M.amblycephala and M.skolkovii,and was hard to distinguish.Clustering analysis of meristic characters showed:M.hoffmanni got together with M.pellegrini firstly,finally got together with P.pekinensis,indicating that meristic characters of P.pekinensis were most different with the genus Megalobrama,and in the genus Megalobrama,M.skolkovii was the most different one.Clustering analysis of morphometric characters showed:M.skolkovii was the most different one,the second was P.pekinensis,M amblycephala and M.pellegrini were the closest species.5 discrimination functions of 41 morphometric characters from P.pekinensis and the genus Megalobrama were established,indicating the comprehensive discriminant rate of the 5 groups was 98.21%.2.Morphological comparison analysis of different populations of the genus Megalobrama and ParabramisIn the present study,based on 35 morphological proportional characters of 414 specimens of P.pekinensis which collected from Liangzi Lake,Futou Lake,Yangtze River Reservoir,Heilongjiang River,and based on 37 morphological proportional characters of 472 specimens of M.skolkovii which collected from Beijiang River,Qiantang River,Jinsha River Reservoir,Heilongjiang River,4 multi variation analysis methods(one-way analysis of variance,cluster analysis,principle component analysis,discriminant analysis)were used to comparably investigate the morphological variation and population biology among the 4 population of each species using traditional morphological and truss network analysis.The result of one-way analysis of variance revealed that 11 morphological proportional characters(TL/SL,MGL/HL,ED/HL,LSD/HL,LSP/HL,LSV/HL,LSA/HL,1-2/SL,1-3/SL,2-3/SL,3-5/SL)were significant different(P<0.05)among the partial 4 populations of P.pekinensis;17 morphological proportional characters(TL/SL,HL/SL,CPL/SL,CPH/SL,MGL/HL,PD/HL,MW/HL,1-2/SL,1-3/SL,3-5/SL,4-5/SL,4-6/SL,5-6/SL,6-8/SL,6-9/SL,7-8/SL,7-9/SL)were significant different among the 4 populations of M.skolkovii.The result of cluster analysis indicated that P.pekinensis in Liangzi Lake were most similar to P.pekinensis in Futou Lake with morphological character,while P.pekinensis in Heilongjiang River were most different from other 3 populations;M.skolkovii in Beijiang River were most similar to M.skolkovii in Jinsha River Reservoir with morphological characters,while M.skolkovii in Heilongjiang River were most different from other 3 populations.In the result of principal component analysis,7 principal were constructed by factor loading of P.pekinensis,the cumulative contribution ratio was 60.97%;10 principal were constructed by factor loading of M.skolkovii,the cumulative contribution ratio was 60.26%.PCA scatterplots of meristic characters showed:P.pekinensis of Heilongjiang River formed 1 cluster separated from the other populations;M.skolkovii of Heilongjiang River and Qiantang River formed 2 clusters independently separated from the other populations.In discriminant analysis,these four populations of each species could be divided.The discriminant functions of 4 populations of P.pekinensis were established based on 15 morphological proportional characters which were significant different among the populations.The total accuracy of discrimination of P.pekinensis from 4 populations all was 89.13%.The discriminant functions of 4 populations of M.skolkovii were established based on 18 morphological proportional characters which were significant different among the populations.The total accuracy of discrimination of M.skolkovii from three populations all was 99.58%.The results showed that the attribution of 4 populations for each species can be determined by differences in morphological proportional characters.3.Development of molecular genetic markers and genetic analysis of the genus Megalobrama and ParabramisAccording to the genome data,we got 132,637,162135,164571 and 152997 sequences contained microsatellite loci of M.skolkovii,M.hoffmanni,M.Pellegrini and P.pekinensis,which could be used to design primer pairs.Total of 1047 primer pairs(380 pairs of M skolkovii,285 pairs of M hoffmanni,192 pairs of M.pellegrini,190 pairs of P.pekinensis)were sent to synthesized.According to the genome data,we got 132,637,162135,164571 and 152997 sequences contained microsatellite loci of M skolkovii,M.hoffmanni,M.Pellegrini and P.pekinensis,which could be used to design primer pairs.Total of 1047 primer pairs(380 pairs of M.skolkovii,285 pairs of M.hoffmanni,192 pairs of M.pellegrini,190 pairs of P.pekinensis)were sent to synthesized.We successfully developed 26 polymorphic microsatellite loci of M.skolkovii,37 polymorphic microsatellite loci of M.hoffmanni,29 polymorphic microsatellite loci of M.Pellegrini,30 polymorphic microsatellite loci of P.pekinensis.AMOVA analysis revealed that most of the total variation occured main within populations of M.skolkovii and P.pekinensis,while the percentage of variation within populations were 86.62%and 86.62%,respectively.Among the 4 populations of M.the Heilongjiang River population showed the highest genetic diversity and the Beijiang River populatin showed the lowest.Among the 4 populations of P.Pekinensis,the Heilongjiang River population showed the highest genetic diversity and the Liangzihu Lake populatin showed the lowest.Nei's genetic distance clustering of 4 M.skolkovii geographical population showed that the Beijiang River population was most close to the Jinshahe Reservoir population,and farthest from the Heilongjiang River population.Structure analysis showed the genetic structure of the 4 population was different.Nei's genetic distance clustering of 4 P.pekinensis geographical population showed that the Yangtze River population was most close to the Futou River population,and farthest from the Heilongjiang River population.Structure analysis showed the genetic structure of the 4 population was different.We evaluated the transferability of the polymorphic microsatellite loci of 4 species by cross-amplification in the genus genus Megalobrama and P.pekinensis.The transferability rate of M skolkovii loci in M amblycephala,M.hoffmanni,M.Pellegrini and P.pekinensis were 88.46%,80.77%,73.08%,and 92.31%.The transferability rate of M.hoffmanni loci in M.amblycephala,M.Pellegrini,M.skolkovii and P.pekinensis were 81.08%,81.08%,91.89%and 89.19%.The transferability rate of M.Pellegrini loci in M.amblycephala,M hoffmanni,M skolkovii and P.pekinensis were 96.55%,86.21%,86.21%and 75.86%.The transferability rate of P.pekinensis loci in M.amblycephala,M.hoffmanni,M.Pellegrini and M.skolkovii were 83.33%,83.33%,70.00%,96.97%.These microsatellite loci can be used to identify the genus Megalobrama and P.pekinensis according to the bands(with or without and the size)of PCR(for example:HHF-63,HHF-104,HHF-113,HHF-148 and HHF-163).4.Genome sequence analysis based on resequencing of the genus Megalobrama and ParabramisWe built sequencing library of M.pellegrini,M.hoffmanni,M.skolkovii and P.pekinensis,the sequence numbers of Q20 were all over 95%,Q30 were all over 90%,sequencing depth was more than 35×.It showed no contaminant by other species after Blast in TN bank.Compared with mitochondria data,it showed the mitochondrial contents of the 4 species were low.The genome size of M.skolkovii,M.hoffmanni,M.pellegrini and P.pekinensis were 0.98 Gb,0.99 Gb,1.00Gb and 0.99 Gb,the hybrid rate were 0.49%,0.49%,0.37%,0.47%,the repetitive sequence content were 46.17%,41.85%,44.05%,40.80%.The low hybrid rate and repetitive rate indicated the genomes of the 4 species were relatively simple.When M.amblycephala genome was the reference genome,the compare rates of P.pekinensis,M.skolkovii,M.hoffmanni and M.Pellegrini were 96.48%,98.32%,99.54%and 99.64%.Within the scope of the M.amblycephala genome,we got 7,624,328,13,978,887,8,971,456 and 16,790,911 single nucleotide polymorphisms(SNPs)in M.skolkovii,M.hoffmanni,M.pellegrini and P.pekinensis,and annotated them.Functional annotation distribution of short insertionsand deletions(Short InDel)showed 17107 Short InDel mutated in 4 species,76 Short InDel only mutated in M.skolkovii,254 Short InDel only mutated in M.hoffmanni,127 Short InDel only mutated in M.Pellegrini,and 464 Short InDel only mutated in P.pekinensis.The number of structural variation(SV)of M skolkovii,M.hoffmanni,M.Pellegrini and P.pekinensis were 92782,21575,15063,140293.The number of copy number variation(CNV)of M.skolkovii,M.hoffmanni,M.Pellegrini and P.pekinensis were 11375,11359,11957 and 9419.The neighbor-joining(NJ)constructed according to SNPs showed M.hofffmanni clustered together with P.pekinensis first,M.Pellegrini second,M.skolkovii last,which indicated M.hoffmanni genome was most close to P.pekinensis genomeand most different with M.skolkovii genome. |
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