**2. Development and characterization of microsatellite loci**

### **2.1. Development of microsatellite loci**

Genetic properties of microsatellite loci are briefed as follows (Fig. 1). These loci are repeat‐ ing sequences of 2 to 6 base pairs of DNA, for instance CACA…, CTCTCT… and CAT‐ CAT… Microsatellites that are typically neutral and co-dominant are used as molecular markers in genetics for kinship, population and other studies, because of often presenting high levels of inter- and intra-specific polymorphism [33-35]. Especially, CA nucleotide re‐ peats appear to be very frequent in human and other genomes and present every few 10,000 to 100,000 base pairs. A repeat size in a locus is treated as an allele and a pair of repeat sizes which are inherited from both of parents is used as genotypes at a locus for a diploid organ‐ ism. Heterozygous describes a genotype consisting of two different sizes (alleles), while ho‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

mozygous does it consisting of two identical ones (Fig. 1).

Applications

352

**Figure 1.** Scheme of microsatellite loci in nuclear genome DNA (unpublished figure)

In this chapter, to detect the existence of the above serious genetic issues, we carried out a series of analysis for genetic diversity and population structure in population of the Hotoke loach (Fig. 2). Novel microsatellite loci applied in this loach were developed and character‐ ized in Section 2. Using these developed loci, genetic diversity and population structure were investigated for populations in the upper Kokai River along with adjacent rivers, the southeast part of Tochigi Prefecture as a case study in Section 3. Technical terms related to population and conservation genetics are often used in the sections; thus, details of mean‐

ings of these terms are able to be known by references cited in the end of this chapter.

In Section 2, a total of 19 novel microsatellite loci for the Hotoke loach were isolated with an individual obtained in the Shitada R., Chiba Pref. and characterized using 32 individuals collected from the Koise R., Ibaraki Pref. The following development procedure [36] is parti‐ ally improved based on the latest studies [39, 40].

A sample of this loach was collected from an agricultural canal in the Shitada R., Chiba Pref. in 2005 and preserved in 99% EtOH, and then stored at -30 °C. Genomic DNA was extracted from single caudal fin clip, approximately 5 mm × 5 mm, using a standard phenol-chloro‐ form procedure [41]. Microsatellite enriched libraries were developed following the previ‐ ous study [42] with some modifications. Briefly extracted DNA was digested with RsaI (New England Biolabs) and then ligated to SuperSNX linkers (SuperSNX24 Forward: 5'-GTT TAA GGC CTA GCT AGC AGA ATC-3' and SuperSNX24+4P Reverse: 5'-phosphate-GAT TCT GCT AGC TAG GCC TTA AAC AAA A-3'). Linker-ligated DNA was enriched for mi‐ crosatellites using streptavidin-coated magnetic beads (Dynal) treated with a blocking step [43] and using the pooled biotinylated probes (CA)12 and (CT)12.

Recovered DNA was amplified by the polymerase chain reaction (PCR) and PCR products were cloned using a TOPO-TA Cloning Kit (Invitrogen) following the manufacture's proto‐ col. A total of 192 positive clones were sequenced on a 3130*xl* Genetic Analyzer (Applied Bi‐ osystems; ABI) using BigDye Terminator kit version 3.1 (ABI) and resultant sequences were

proofread for repeat regions using the software DNA BASER version 3.2 (Heracle BioSoft). Oligonucleotide primers (Table 1) were designed in flanking regions of the 19 targeted mi‐ crosatellite loci using the software DNASIS PRO version 3.0 (Hitachi Software Engineering).

**2.2. Characterization of microsatellite loci**

GENEMAPPER version 4.0 (ABI).

populations of the Hotoke loach.

**3.1. Study sites**

Each microsatellite locus was characterized for polymorphisms among 32 individuals ob‐ tained the Koise R., Ibaraki Pref. in 2006. DNA of the individuals was extracted using an au‐ tomated DNA isolation system (GENE PREP STAR PI-80X, KURABO) following the manufacturer's instructions. PCR amplifications were performed on the 32 DNA extracts across all loci using 10 μl reaction volumes containing approximately 10 ng DNA template, 0.5 U Taq DNA polymerase (BIOTAQ, Bioline), 1×NH4 buffer (BIOTAQ), 2.5 mM MgCl2, 0.25 mM each dNTP, 0.03 μM M13-tailed forward primer, 0.25 μM reverse primer and 0.25 μM labeled M13 primer (5'-GCC AGT CAC GAC GTT GTA-3') [44]. The M13 primer was

Genetic Diversity and Population Structure of the Hotoke Loach, *Lefua echigonia*, a Japanese Endangered Loach

http://dx.doi.org/10.5772/53022

355

labeled at the 5' end with 6-FAM, VIC, NED or PET fluorescent dyes (ABI, Table 1).

Thermal profiles on iCycler and C1000 (both of Bio-Rad) of thermal cyclers were as follows. Initial denaturation at 94°C for 2 min was followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 56°C for 15 s and extension at 72°C for 30 s. A single final extension at 72°C was done for 30 min. PCR products were resolved on a 3130*xl* Genetic Analyser with GeneScan 500 LIZ size standard (ABI). Electropherograms were analyzed with the software

Measures of genetic diversity, tests for deviations from Hardy-Weinberg equilibrium (HWE) and estimates of linkage disequilibrium (LD) between loci were calculated using the soft‐ ware GENEPOP on the web version 4.0.10 [45]. The possible presence of null alleles was as‐

All the 19 loci were polymorphic (Table 1). The number of observed alleles per locus ranged from 2 to 9. The observed heterozygosity ranged from 0.125 to 0.844, while the expected het‐ erozygosity varied from 0.148 to 0.876. No significant deviations from HWE or signs of LD were observed after sequential Bonferroni correction with the significant level at 0.05 [47] and there was no evidence of null alleles in any of the tested loci. Consequently, the high level of polymorphisms observed in these microsatellite loci may have to support future in‐ vestigations to improve our knowledge of the genetic differentiation and genetic structure of

In Section 3, genetic diversity and population structure of populations of the Hotoke loach in the upper Kokai R. including 4 adjacent rivers, the southeast part of Tochigi Pref. (Fig. 3) was detailed using the microsatellite loci developed in Section 2 (Table 1). As mentioned in Section 1, populations of the Hotoke loach have been often diminished and isolated by land consolidation projects in rural areas. Therefore it appears difficult to find populations dis‐ tributed with a certain area. However, rich biota still continues to exist in the upper Kokai R. due to delay of land consolidation. This area sounds attractive for field scientists, and then their some activities were carried out to conserve and recover such a sound rural ecosystem

sessed with the software MICRO-CHECKER version 2.2.3 [46].

**3. Analysis of genetic diversity and population structure**


a Sequence of the M13 tails on forward primers: GCC AGT CAC GAC GTT GTA

**Table 1.** Characterization of 19 polymorphic microsatellite loci for 32 individuals of the Hotoke loach. Loci with gray color were used in analysis of genetic diversity and population structure in Section 3. (Modified from one of previous study [36])

### **2.2. Characterization of microsatellite loci**

proofread for repeat regions using the software DNA BASER version 3.2 (Heracle BioSoft). Oligonucleotide primers (Table 1) were designed in flanking regions of the 19 targeted mi‐ crosatellite loci using the software DNASIS PRO version 3.0 (Hitachi Software Engineering).

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

GenBank accession no.

(CA)13 6-FAM AB286032

(CA)30AA(CA)5 VIC AB286033

(CA)14CG(CA)3 6-FAM AB286034

(GA)29 6-FAM AB286035

(GT)13 NED AB286036

(CT)10 PET AB286037

(CT)7(GT)11 NED AB286038

(GA)14 PET AB286040

(GT)7 6-FAM AB286041

(GT)11 NED AB286042

(CT)21 NED AB286044

(GT)7 NED AB286045

(GT)11 PET AB286046

(CA)10 6-FAM AB286047

(GT)9 NED AB439725

(GT)6 PET AB439726

6-FAM AB286039

VIC AB286043

VIC AB286048

(GA)5AA(GA)8 AA(GA)17

(CT)14CA(CT)2 (CA)6

(GT)7GCGTGG (GT)5

**Locus Primer sequence (5'-3')a Repeat motif Dye**

*Lec01*

Applications

354

*Lec02*

*Lec03*

*Lec04*

*Lec05*

*Lec06*

*Lec07*

*Lec08*

*Lec09*

*Lec10*

*Lec11*

*Lec12*

*Lec13*

*Lec14*

*Lec15*

*Lec16*

*Lec17*

*Lec18*

*Lec19*

study [36])

a

F: M13-ATC CCT CCC TTC ACC GTC TG R: TCC GAA ACC AGC AGC ACC AC

F: M13-TGT GCT GTA GGA TTG CTT GAG C R: ATG TCA GAG GCT GAT GG GAT AC

F: M13-CGT CCA CCA GCC TTA CGA AC R: TGA CGC TCA GTA GTC GGA CC

F: M13-GCA CTG CTG ATG ACA ATC ATT G R: GCT TTG GGT TAG AAC ATC AGT G

F: M13-TGT CTG CTG TGA TGA TGA CAT C R: CTC ACA GCA CTA TTC ACT GAT G

F: M13-CCG TGT CTG TTT TGC TTT CTC R: CTC CCT TCA CAA AGT AAC TGG

F: M13-TGT GAA GAA ACC TGA ACA CGC R: ATT CTG TGT CCC TGA ACA CAC

F: M13-GAC GCA ACA ATC TCA GGG TC R: ACA GGA CCA AGT GGA CTC TC

F: M13-GGG GAT AGT GGA GAT GGG TG R: TTC ATC CCT CTT CCG CCC AC

F: M13-GGT TGG CAA TGC CAG CAA TG R: TGC TTT ACC AAG GTG ACG GC

F: M13-CTG ACA CTG TGT GTG TAG CAG R: GGT TTC ACC TGG TCC ATA CAC

F: M13-GGC ACC AAA GGC AGA TTT TAC R: AGA GTG TGA GAT TAT GGC AGC

F: M13-GAC GCC ACG ACA AGA CGA AC R: TAT GTG TGG AGG GGG GTG AG

F: M13-ATT AGG AGC ATT ACC CAA CAG C R: CAA AGG AAG CAA AAA CAA GGG C

F: M13-GAG CAA GAG GTG TGT GCT TC R: TGC TGG TTC ACG CTC TAC AC

F: M13-CAC ACT AAC ACT TCT CCA GCG R: CAC AGT GAC CAA AGT CAC CAG

F: M13-GTC CCC ATA AAA CAG GAA ACC C R: GAC TAT TGA GTG AGT GCC ACA C

F: M13-CGA CCA TCT TCT GGG GTT ACG R: CCT CGG ATG GGC TAA ATG ACC

F: M13-CTG TGT GTG GGT GTA TCT GAA C R: AAA GTG GCT CTT CTT CTG CTG G

Sequence of the M13 tails on forward primers: GCC AGT CAC GAC GTT GTA

**Table 1.** Characterization of 19 polymorphic microsatellite loci for 32 individuals of the Hotoke loach. Loci with gray color were used in analysis of genetic diversity and population structure in Section 3. (Modified from one of previous Each microsatellite locus was characterized for polymorphisms among 32 individuals ob‐ tained the Koise R., Ibaraki Pref. in 2006. DNA of the individuals was extracted using an au‐ tomated DNA isolation system (GENE PREP STAR PI-80X, KURABO) following the manufacturer's instructions. PCR amplifications were performed on the 32 DNA extracts across all loci using 10 μl reaction volumes containing approximately 10 ng DNA template, 0.5 U Taq DNA polymerase (BIOTAQ, Bioline), 1×NH4 buffer (BIOTAQ), 2.5 mM MgCl2, 0.25 mM each dNTP, 0.03 μM M13-tailed forward primer, 0.25 μM reverse primer and 0.25 μM labeled M13 primer (5'-GCC AGT CAC GAC GTT GTA-3') [44]. The M13 primer was labeled at the 5' end with 6-FAM, VIC, NED or PET fluorescent dyes (ABI, Table 1).

Thermal profiles on iCycler and C1000 (both of Bio-Rad) of thermal cyclers were as follows. Initial denaturation at 94°C for 2 min was followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 56°C for 15 s and extension at 72°C for 30 s. A single final extension at 72°C was done for 30 min. PCR products were resolved on a 3130*xl* Genetic Analyser with GeneScan 500 LIZ size standard (ABI). Electropherograms were analyzed with the software GENEMAPPER version 4.0 (ABI).

Measures of genetic diversity, tests for deviations from Hardy-Weinberg equilibrium (HWE) and estimates of linkage disequilibrium (LD) between loci were calculated using the soft‐ ware GENEPOP on the web version 4.0.10 [45]. The possible presence of null alleles was as‐ sessed with the software MICRO-CHECKER version 2.2.3 [46].

All the 19 loci were polymorphic (Table 1). The number of observed alleles per locus ranged from 2 to 9. The observed heterozygosity ranged from 0.125 to 0.844, while the expected het‐ erozygosity varied from 0.148 to 0.876. No significant deviations from HWE or signs of LD were observed after sequential Bonferroni correction with the significant level at 0.05 [47] and there was no evidence of null alleles in any of the tested loci. Consequently, the high level of polymorphisms observed in these microsatellite loci may have to support future in‐ vestigations to improve our knowledge of the genetic differentiation and genetic structure of populations of the Hotoke loach.
