Li Ya-juan, Qi Hong-rui, Ma Hai-yan, Zhou He, Xu Wen, Sui Yi, and Li Jia-qi

Key Laboratory of Marine Bio-resources Restoration and Habitat Reparation in Liaoning Province, Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China

Introduction

Natural polyploidy is commonly found in aquatic animals, and in the reported karyotype of freshwater fishes, 30 are found to be polyploidy (Ma, 1996).Most of the polyploid fi shes are important commercial fishes or cultured species, therefore, researches on polyploidy fishes are very important for ascertaining and making use of the resources. Polyploidy exists in loach, Misgurnus anguillicaudatus. A large quantity of triploid are found in Japan, but no tetraploid(Morishima et al., 2002; Arai et al., 1991; Zhang and Arai, 1999). As it was reported before, the diploid(2n=50)and tetraploid (4n=100)population existed in China (Li et al., 1983; Li et al., 1987; Yin et al.,2005); however, after ploidy investigation from 29 sites around China, a few natural triploid (3n=75)were found (Li et al., 2008). Former studies on loach mainly focused on karyptype of tetraploid(Li et al., 1983; Li et al., 1987; Yin et al., 2005),ploidy identification (Gao et al., 2007; Guo et al.,2009), cyt b sequence (Yang et al., 2009), population genetic structure (Zhou et al., 2011)and gene polymorphism of mitochondria (Zheng et al., 2011).In the last 10 years, we had systematically studied the distribution of natural polyploidy in China (Li et al., 2008), chromosome karyotype of somatic cells(Li et al., 2009), chromosome band and FISH (Li et al., 2010), chromosome behavior of meiosis (Li et al.,2010), artificially induced gynogenesis (Li et al.,2013)and reproductive characteristics (Li et al., 2012).However, genetic diversity researches on loaches of different ploidy have not been reported yet. Microsatellite DNA is a newly developed marker with lots of kinds, high polymorphism, high heterozygosity and low recombination rate. It also has lager genetic variation grope and makes up rich polymorphism,highly individual specific and follows the rules of Mendel's Laws. It has many highly provided heritable information of polymorphic locus. Therefore, the microsatellite molecular marker is the most widely used markers for constructing genetic map, and population genetic structure analysis (Norris et al.,1999; Du et al., 2000; Zhou et al., 2001; Sun et al.,2001; Liang et al., 2002; Dong et al., 2007; Zhang et al.,2010; Liu et al., 2012). Seven microsatellite markers were used to analyze its genctic diversity, in order to provide genetic theory basis for protecting and making use of the loach genetic resources, reveal the origin and evolution mechanism of the polyploid loaches, and to discuss the genetic relationship between loaches of different ploidy for clarifying the formation mechanism of polyploid loaches.

Materials and Methods

Experimental materials

A total of 54 loaches were collected from Honghu City, Hubei Province. Ploidy was detected by measuring the volume of erythrocyte nuclei using a flow cytometer. Of the 54 individuals, 23 were diploid,three were triploid and 28 were tetraploid. Genomic DNA was extracted by using the phenol-chloroform method and diluted to 100 ng · μL-1in 100 μL of TE buffer (pH 8.0)and stored at -20℃.

Microsatellite markers

Microsatellite loci were identified from the simple sequence repeats (SSRs)registered with GenBank.PCR primers were designed on either side of the core sequence. In this study, seven loach microsatellite loci were selected for the analysis (Table 1). The primers were synthesized by Sangon Biotech.

Table 1 Primer sequence of microsatellite DNA

PCR

PCR reagent conditions were as follows: 2.5 μL of 10×buffer (Mg2+), 2.5 μL of MgCl2(2.5 mmol · L-1),0.5 μL of each dNTP (2.5 mmol · L-1), 1.0 μL of each of the forward and reverse primers (10 μmol · L-1), 0.3 μL of Sangon Taq DNA polymerase (5 U · μL-1), 2.0 μL of DNA and 17.2 μL of double distilled water, to give a total volume of 27 μL. The thermocycling conditions were as follows: initial denaturation for 1 min at 94℃,followed by 30 cycles of denaturation for 15 s at 94℃,annealing for 15 s at 56℃ and extension for 30 s at 72℃. This was followed by a fi nal extension for 60 s at 72℃. PCR products were examined using an 8%non-denatured polyacrylamide gel. The gel photograph was taken after coloration by sliver staining.

Statistics

Gel electrophoresis results were analyzed using Gel-Pro (version 4.5)and PopGen (version 1.32). The number of alleles (A), observed mean heterozygosity value (Ho), expected mean heterozygosity value (He),genetic distance (Ds)and genetic similarity index (I)were calculated. Polymorphism information content(PIC)was calculated using Bostein's formula:

piand pjindicated the rate of alleles ith and jth in species respectively, and m indicated the number of alleles.

Linkage disequilibrium amongst loci was analyzed using GenePop (version 3.4). The Hardy-Weinberg equilibrium was assessed using a χ2test. Neil's genetic distance was calculated using the gendist program of PHYLIP (Phylogeny Inference Package)v3.6. A clustering analysis was performed using UPGMA (Unweighted Pair Group Method with Arithmetic means).

Results

Result of PCR amplif i cation

Genetic variations among diploid, triploid and tetraploid individuals were analyzed using seven pairs of microsatellite primers that were able to amplify corresponding sequences in loaches with the three types of ploidy (Fig. 1).

Polymorphism of genetic

Polyacrylamide gel electrophoresis banding patterns of diploid (2n), triploid (3n)and tetraploid loaches(4n)were analyzed with PopGen software. A total of 68 alleles were obtained from seven microsatellite loci, which were divided across the loci as follows:11 Mac3 alleles, 8 Mac36 alleles, 6 Mac37 alleles, 12 Mac45 alleles, 9 Mac49 alleles, 10 Mac60 alleles and 12 Mac63 alleles, and the polymorphism information content (PIC)indices were all above 0.5. The average expected mean heterozygosity values (He)were 0.8420, 0.7186 and 0.8521; the average observed mean heterozygosity values (Ho)were 0.9674, 0.9785 and 0.8928; the Hardy-Weinberg P values were 0.3078,0.3151 and 0.3762, for diploid, triploid and tetraploid individuals, respectively. The results indicated that the three populations were highly polymorphic, with no deviations from Hardy-Weinberg equilibrium observed at the seven microsatellite loci (Table 2).

Fig. 1 Electrophoretic patterns of polyacrylamide by Primer Mac 49

Table 2 Heterozygosity and P values of seven loci of Misgurnus anguillicaudatus with three levels of ploidy

Genetic similarith, genetic distance and clustering analysis

Genetic distance and similarity between the loaches were calculated using the Neil's method (Neil, 1972).Results showed that the genetic distance was the lowest (0.2065)and the similarity was the highest(0.7998)(Table 3)between diploid and tetraploid individuals. The clustering analysis, using UPGMA,also indicated a close genetic relationship between diploid and tetraploid individuals (Fig. 2).

Table 3 Genetic identity and genetic distance in three types of ploidy observed in loach

Fig. 2 Dendrogram of three types of ploidy observed in loach

Discussion

Genetic diversity

Reports on the genetic diversity of the loaches in China using microsatellite markers are still rare, especially on loaches of different ploidy from different waters.Genetic diversity is the basis for creatures to acclimatize and the force for evolutionary. For a spices,higher genetic diversity means stronger ability to adapt and survive, and abudant possibility for breeding and genetic improvment. On the contratry, lower genetic diversity may cause a reduction for the ability to adapt and survive, and eventually degeneration of species. Polymorphism information content (PIC)and heterozygosity (H)are important parameters to measure genetic variation and population genetic diversity, as the bigger the value, the higher the gene richness, and the stronger the ability to adapt envioronment (Nei, 1978).

Statistics of 40 000 individuals of 78 species by 542 microsatellites, Dewoody and Avise (2000)obtained an average He of freshwater fi shes as 0.46. Zeng et al.(2008)showed that the average He of three different kinds of spot loaches (Misgurnus anguillicaudatus)was respectively 0.1554, 0.1799 and 0.0857, and the average PIC was respectively 0.2737, 0.3626 and 0.1989, indicating a low genetic diversity of three different kinds of spot loaches. Shan et al. (2009)used 12 pairs of microsatellites to analyze the genetic diversity of four diploid loach populations, and the results showed that the average He was 0.360-0.515,and the average PIC was 0.332-0.465, indicating a medium genetic diversity. In this research, the average Ho of diploid, triploid and tetraploid loaches was respectively 0.9647, 0.9785 and 0.8928, and the average He was respectively 0.8420, 0.7186 and 0.8521, which were higher than the results above,indicating an abudant genetic diversity of loaches of the three ploidy and a certain genetic variation potential.

Polymorphism information content (PIC)is the main parameter to show the population genetic variation. According to the standards of Botstein et al.(1980), a site was considered high polymorphism when PIC>0.5, medium polymorphism when 0.25

Genetic similarity and genetic distance

Genetic distance was a dependable parameter to measure genetic diversity between populations, as the closer the relationship between populations, the lower the genetic variation, the bigger the similarity, and the smaller the genetic distance (Shao et al., 2009). In this research, genetic diversity analysis using seven microsatellites showed that the genetic distance between diploid and tetraploid was the smallest (0.2065),the similarity was the highest (0.7998), the genetic distance between triploid and tetraploid was farther(0.5882), and the similarity was the lowest (0.4358).UPGMA clustering tree based on genetic distance showed that the diploid and the tetraploid were clustered as one branch, while the triploid was another. This result was consistent with the result by morphological analysis of Li et al (2009). Therefore,loaches of the three ploidy showed an abundant genetic diversity, and could be used as excellent materials for breeding.

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