Multilocus Genetic Diversity And Population Structure For The Odontobutis Distributed In Yangtze River Basin And Huai River

In the paper, we collected 163 fish of 17 populations to the Odontobutis from Yangtze river basin and Huai River. They were Yi Dou(YD), Dang Yang(DY), Jing Zhou(JZ), Hong Hu(HH) and Yingtan population, belonged to the Odontobutis sinensis. Others were Lu An(LA), Chao Hu(CH), Xuan Cheng(XC), Huang Shang(HS), Huai An(HA), Yang Zhou(YZ), Gao You(GY), Jing Jiang(JJ), Hu Zhou Jiuliqiao(HZJ), Hu Zhou Taihu(HZT), Su Zhou(SZ) and Shang Hai population(SH), attached to O. potamophila. By the mitochondrial markers(complete mitochondrial DNA and control region) and nuclear markers(three EPICs and ten microsatellites), we analyzed the genetic diversity and population structure of these 17 populations, and discussed the possible reason why it had such a different spatial distribution pattern of the genetic diversity.The main results were as follows:1 Mitochondrial gene rearrangement and molecular marker selection for the Odontobutis distributed in Yangtze riverThe river sleeper, Odontobutis potamophila(Perciformes, Odontobutidae), is a pint-sized demersal freshwater goby and promising candidate for aquaculture during recent years in China. In fact, its wild populations have declined sharply due to overfishing. However, the molecular genetics information of O. potamophila is limited till now. The complete mitochondrial genome(mt DNA) of O. potamophila was obtained by primer-walking PCR amplification and its length was 16846 bp. Then, its structure, gene rearrangement mechanism and application in the phylogenetics were analyzed. The mt DNA of O. potamophila contained 37 genes(13 protein-coding genes, two r RNAs and 22 t RNAs) and non-coding control regions. In addition to ND6 and eight t RNAs(t RNAGln, t RNAAla, t RNAAsn, t RNACys, t RNATyr, t RNASer, t RNAGlu, t RNAPro), the remainders were encoded on the heavy strand(H strand). All protein-coding genes initiated with the orthodox ATG except for COI gene with GTG and ATP6 gene with ATA. These protein-coding genes had TAA, TA– and T–as termination codons. Because of shuffling rearrangement from different t RNA, the classic mt DNA arrangement HSL(t RNAHis- t RNAsert RNALeu) changed into SLH(t RNAser- t RNALeu- t RNAHis). As a result, the 320 bp-length and 42 bp-length anonymous regions were inserted into t RNALeu and t RNAHis, ND4 and t RNASer, respectively. However, the content of A+T(55.3%) was higher than that of G+C(46.7%), and it was similar to other fish mitochondrial genomes. Among the detected 112 fish of Perciformes, there were only 13 species(11.61%) existed mt DNA gene rearrangement. In particular,the gene rearrangement position of O. potamophila(KF874495) was consistent with O.platycephala(NC010199) and O.sinensis(KF154120), indicating an important molecular "tag" for the evolution of the Odontobutis. By selection pressure and genetic diversity analysis on the 13 protein coding genes, we proved that ND4 and ND5 might be the two suitable molecular candidate markers for reconstructing phylogenetic relationship in the family of Gobioidei. In the ML phylogenetic tree for ten species of Gobioidei based on ND4 or ND5 gene, the relationship of the three species in the Odontobutis was closer to Perccottus glenii and this was consistent with traditional morphological classification.2. Genetic diversity and population structure of the Odontobutis based on the mitochondrial control region(D-loop) from the Yangtze River basin and the Huai RiverThe Odontobutis sinensis and O. potamophila were collected from the middle and upper reaches, lower reaches of Yangtze River and the Huai River, respectively. For O. sinensis, a total of five populations, including 44 individuals, were selected from nine streams or lakes. The amplified length of D-loop was 1062 bp for every individual. Also, we selected the 12 populations, a total of 119 individuals, for the O. potamophila from eight streams or lakes. Amplified length of D-loop was 1041 bp for every individual. After alignment, we captured the 906 bp sequence to analyze population genetics.Results were listed as followed:(1) Genetic diversity. In O. sinensis, there were 73 polymorphic loci, accounting for 8.57% of the total sequence for the selected 906 bp. 22 haplotypes was defined from a total of 44 sequences, and the shared haplotype was H6. The ratio between the included number of H6 haplotype and all individuals accounted for 34.09%. Also, as for O. potamophila, there were 21 polymorphic loci, accounting for 2.32% of the selected 906 bp sequence. A total of 12 haplotypes is classfied for these 119 sequences, and the shared haplotypes were H11, H16 and H18. The ratio between the included number of three haplotypes and all individuals accounted for 63.86%, 3.36% and 10.08%, respectively.(2) Population genetic diversity. The average haplotype diversity and nucleotide diversity were 0.7197 and 0.0057, 0.1796 and 0.0007 for O. sinensis and O. potamophila, respectively. Three populations(YT population, DY population, CH population) had higher levels of haplotype diversity. However, other species had lower haplotype diversity. Especially HS population and LA population, the average haplotype diversity is 0. The average nucleotide diversity in each group are low. These results showed that genetic diversity was lower relied on D-loop, and genetic diversity for the O. sinensis was higer than for the O. sinensis.(3) Genetic differentiation and population genetic structure. Population genetic differentiation index Fst in five populations for O. sinensis and 12 populations for O. potamophila was 0.4822 and 0.9023, at a higher level genetic difference(Fst > 0.25). AMOVA results showed that two species had significant genetic structure. Based on double parameters Kimura genetic distances, NJ phylogenetic relationships showed that 17 populations were significantly divided into two branches. Namely, five populations from the O. sinensis(YT population, YD, HH population, DY, JZ population) consist of Cluster 1. 12 populations from the O. potamophila gathered for another big team(Cluster 3). At the same time, 12 populations can be subdivided into two big clustering, respectively, cluster 2 from the Yangtze river middle reaches(LA population, CH population, XC population, HS population) and the Cluster 3 distributed the lower reaches of Yangtze and Huai River(other eight populations). Three branches presented a obviously separate clustering phenomenon according to the different geographical regions. These results agreeed with result of the minimum span network.(4) Population dynamics. Neutrality test(Tajima’s D, Fu’ s FS, Rg and R2 and SSD test) and mismatch analysis showed the O. sinensis had experienced historical population expansion. In addition to GY population, SZ population, HZT population and CH population, the O. potamophila had also experienced historical population expansion. According to the clustering relations, Cluster1 and Cluster 2 groups have experienced the historical population expansion. However, Cluster3 did not occur the population expansion. Relied on evolution rate of 3%- 12% in one million for the D-loop gene, we estimated the population expansion time were 0.0237- 0.0946 million and 0.0048- 0.0193 million years ago for O. sinensis and O. potamophila, respectively, approximately in late pleistocene. The O. sinensis have a earlier differentiation time, and the result is consistent supported by phylogenetic tree using haplotypic data.3. Genetic diversity, population structure and phylogeography of the sleepers based on the nuclear EPICs from the Yangtze River basin and the Huai River17 population genetic structure was further analyzed using cloned the three EPICs(Exon-primed intron crossing markers).The results were listed as follows:(1) After cutting off the recombination sequences, the length of selected sequence 3733 bp based on three concatenated markers. As for O. sinensis, there were 279 polymorphic sites, accounting for 7.47% in the selected sequence. All 44 sequences could be defined to be 44 haplotypes. As for O. potamophila, there were 343 polymorphic sites, accounting for 9.19% in the selected sequence. All 119 sequences could be defined to be 115 haplotypes. The value of Tajima ’s D test is-0.9900, not significant. It indicated these two speices obeyed neutral evolution.(2) Population genetic diversity analysis. As for O. sinensis, haplotype diversity(h) and nucleotide diversity(π) were 1.0000 and 0.0118 for all populations, the average haplotype diversity and average nucleotide diversity were 1.0000 and 0.0077, nucleotide diversity varied from 0.0034 to 0.0121. As for O. potamophila, haplotype diversity and nucleotide diversity were 0.9994 and 0.0039 for all populations, the average haplotype diversity and average nucleotide diversity were 0.9875 and 0.0029, haplotype diversity and nucleotide diversity ranged from 0.9167 to 1.0000, 0.0007 to 0.0037, respectively. It suggested that genetic diversity was relatively high for the Odontobutis from Yangtze River and Huai River. It always showed the haplotype diversity was relatively high, and nucleotide diversity was relatively low. Genetic diversity in O. sinensis was higher than that’s of O. potamophila.(3) Population NJ clustering analysis. Genetic distance varied from 0.0086 to 0.0149, from 0.0026 to 0.0067 in O. sinensis and O. potamophila relied on two Kimura parameters. 17 populations were significantly divided into two branches(Cluster 1 and Cluster 3). Cluster 1 consisted of five populations from O. sinensis, and Cluster 3 consisted of 12 populations from O. potamophila. These two large clustering branches expressed obviously different geographical clustering. Namely, these individuals of Cluster 1 were distributed in the upper and middle of Yangtze River. However, these individuals of Cluster 3 were distributed in the middle and lower of Yangtze River and Huai River. Also, it could be seen that Cluster 2 was subdivided from the Cluster 3. There were less cross clustering from different geographical regions for Cluster 2 and Cluster 3.(4) Population genetic structure analysis. The index of genetic differentiation(FST) were relatively high between 17 populations(0.7325 to 0.7709). AMOVA showed that the difference among populations(0.7709) is greater than the difference between individuals within a population(0.1992)(**P<0.01). Haplotype minimum span network showed: 17 populations were obviously divided into two clustering, and these two species have experienced a multiple mutations to distinguish(177). All populations differentiated from H78 haplotype. Especially, the LA, CH, XC and HS, distributed in the middle of Yangtze River and Huai River, differentiated from H29 haplotype. These results showed that 17 populations could be divided into three large clustering.(5) Neutral evolution and nucleotide mismatch distribution analysis. The Tajima ’s D was-1.29130(P>0.05) 、-2.03645(P<0.05*) and-2.61750(P<0.01**) for Cluster 1, Cluster 2 and Cluster 3, orderly. The value of Fu ’s FS was-17.0490(P>0.05),-23.1810(P<0.01**) and-92.8640(P<0.01**) for Cluster 1, Cluster 2 and Cluster 3, orderly. The value of Rg and R2 was greater than zero(P > 0.05). These results showed that the three clustering group has experienced the historical population expansion.(6) Southern mountainous area in Anhui Province(Huang Shan and Dabie mountain) might be the geographical barriers between O. sinensis and O. potamophila. The test results of ‘Distance- isolation’ model showed that positive correlation was Y(x)= 0.0027*x + 0.4104 between the geographic distance(linear distance) and genetic distances [F’st/(1-F’st] for Cluster 2. Also, correlation was Y(x) = 0.0071 + 0.9465*x between the geographic distance and genetic distances for Cluster 3. Internal genetic distance between populations of Cluster 2 was smaller than that of Cluster 3, and the distribution was more centralized.(7) Phylogeography pattern for the 17 populations should belong to the third or fifth Avise geographical systems. Namely, phylogenetic relationships were continuous between different populations, and the geographic distribution sites was different or partial continuous. Some population occurred the transitory population replacement. Especially, the populations, distributed in the lower Yangtze River, presented a moderate level of gene flow, and only partial genotype has wide geographic distribution, sharing less haplotypes.4. Genetic diversity and population structure of the Odontobutis Based on microsatellite marker from the Yangtze River basin and the Huai River(1) Microsatellite markers were developed for O. sinensis and O. potamophila using biotinylated-magnetic enrichment beads methods. 13 and 14 pairs of SSR primers were successfully isolated for O. sinensis and O. potamophila and showed the highly polymorphic when they were examined using 27 individuals from JZ population and 45 individuals from SZ population, respectively. The results were listed as follows: as for O. sinensis, the value of NA, PIC, HO and HE varied from 6 to 21(average 14.2778), from 0.7447 to 0.9303(average 0.8816), from 0.3333 to 1.0000(average 0.5412) and from 0.7764 to 0.9518(average 0.8812). Three loci(O.sin7, O.sin13 and O.sin 17) were significantly deviated from the Hardy-weinberg equilibrium(HWE)(*P<0.05). Also, as for O. potamophila, the value of NA, PIC, HO and HE ranged from 8 to 30(average 20.9286), from 0.8020 to 0.9354(average 0.8962), from 0.7500 to 1.0000(average 0.9508) and from 0.8436 to 0.9628(average 0.9101). Six loci(O.pot1, O.pot2, O.pot4, O.pot5, O.pot10 and O.pot14) were significantly deviated from the Hardy-weinberg equilibrium(HWE)(*P<0.05). No locus have been detected linkage disequilibrium for these two sleepers after Bonferroni correction. Because of higher PIC(PIC>0.5), these 27 pairs SSR markers could provide a more abundant genetic information.(2) Then, the 27 primers were cross-species amplified. We screened out 10 primers, which could strongly amplified two species of sleepers in the meanwhile. These microsatellite loci could further be used to analyze genetic diversity and population genetic structure for the Odontobutis from Yangtze River and Huai River. The results were listed as follows: ① Genetic diversity. The average value of NA, AR, HE, HO, and PIC were 10.6000 and 10.8333, 8.2728 and 8.0890, 0.9678 and 0.9600, 0.9240 and 0.9143, 0.8559 and 0.8546 for O. sinensis and O. potamophila, orderly. Inbreeding coefficient varied from 0.0820 to 0.0690(average 0.0595), and it indicated these populations tended to random mating. All parameters suggested that genetic diversity of wild populations is rich for the Odontobutis distributed in the Yangtze River and Huai River. And genetic diversity of the O. sinensis is higher than that’s of the O. potamophila. ② Population genetic structure and clustering. The genetic distance between different populations ranged from 0.0179 to 0.0413, and from 0.0038 to 0.0943 for the O. sinensis and O. potamophila, respectively. AMOVA analysis showed the genetic differenciation was much bigger between the groups than that’s between populations. STRUCTURE clustering analysis found that the best clustering was three groups for the 17 populations. YD, DY, JZ, HH and YT belonged to Cluster 1. Cluster 2 consisted of LA, CH, XC and HS. Cluster 3 is included other eight populations. These results were consistent with that’s from principal coordinates analysis, but also it could be seen that LA and HS had a bigger population differentiation. ③ Population historical dynamic analysis. Five populations of the O. sinensis did not have experienced the bottleneck effect, and only four populations of the O. potamophila might have suffered the discontinuous bottleneck effect.