26. Determination of DNA sequence as Genetic diversity indicator of the Japanese
endangered species: the Japanese loach with eight-barbel (Lefua echigonia)
[Abstract] In elucidating the ecosystem of rice paddy fields the nuclear DNA sequences that are
possibly genetic diversity indicators were determined for Japanese endangered species, the
Japanese eight-barbel loach. The genetic identities of individuals in a population and genetic
differentiation between populations corresponding to genetic diversity indicators can be evaluated
using the differences in sequences of sample populations.
[Keywords] Japanese eight-barbel loach (Lefua echigonia), genetic diversity, DNA sequence, rice
paddy field, Japanese endangered species
[Charge] Laboratory of Ecological Engineering, Department of Rural Environment, National
Institute for Rural Engineering
[Telephone number] TEL: 029-838-7686
[Category] Rural Engineering
[Class] Technology, dissemination
[Background and objectives]
Conservation and restoration of biodiversity in rice paddy fields are major issues in agricultural
and rural development projects. Genetic diversity (existence of various genes within a specific
population), species diversity (existence of various species) and ecosystem diversity can all be
defined as part of biodiversity. Although a lot research has been conducted in which biodiversity
is represented by species diversity, due to the clarity of its definition, research on genetic diversity
and ecosystem diversity has made little progress. In this research the DNA sequences, which are
potential genetic indicators, were determined for Japanese endangered species, the Japanese
eight-barbel loach (Lefua echigonia) that is often used as an environmental indicator in rice paddy
field and/or a targeted conservation species in development projects.
[Content and characteristics of the results]
1. Nuclear DNA sequences with repeat units of 2-5 bases such as CA and CTA (microsatellites;
hereinafter referred to as ‘index sequences’) were determined as genetic diversity indicators.
2. Nineteen sequences were determined from a sampled individual from the Shitada River in
Chiba pref. by cloning the enriched library of the index indicators (Table 1). Each sequence
was identified as Lec01-Lec19 and registered in GenBank (AB286032-AB286048,
AB439725 and AB439726), thus allowing the primer sequences necessary for PCR
amplification to be determined (Table 1).
3. To validate the index sequences six sample populations (10-24 specimens in each population,
Figure 1) from different river systems in the southeast part of Tochigi pref. were analyzed for
genetic diversity. In the analysis PCR amplification was conducted using the primers in Table
1 and the reaction conditions in Table 2, with genotype data where variation in the array of
each individual was sequenced being used.
4. Of the nineteen index sequences the sample population had twelve polymorphic sequences
with two or more genotypes. For the genetic identity of an individual in a population, being
the genetic diversity indicator of the population, it was estimated that the mean of the number
of alleles per locus was 4.92-6.25 while the means of the observed values and expected values
of heterozygosity per locus were 0.45-0.62 and 0.54-0.61, respectively (Figure 2), thus
revealing that no population had the trend of identification of the genes of different
5. Genetic differentiation between populations, which corresponds to another indicator of
genetic diversity, was estimated using the coefficient of gene differentiation, FST, the
difference of which can be visualized using the phylogenetic tree. The FST was 0.06-0.21,
revealing that genetic diversity between populations is related to the geographical location of
the river system, as revealed by the phylogenetic tree (Figure 2, below). The index sequences
(Table 1) proved useful in analyzing genetic diversity.
[Utilization of the results and points to be considered]
1. When using the index sequences first conducting preliminary analysis on a small number of
individuals is desirable since the polymorphic sequence can vary depending on the population
2. The index sequence is also applicable to analysis of assignment (e.g. place of origin) and
population structure and to individual identification. New index sequences will need to be
determined for species other than the Japanese eight-barbel loach.
3. Experiment and research institutions involved in conservation genetics, ecology and biology
are assumed to be the users of the results given here.
Table 1 Nineteen sequences of nuclear DNA, potentially being genetic diversity indicators of the Japanese eight-barbel loach
Repeat motif Repeat motif
Locus Primer sequences (5' - 3') Locus Primer sequences (5' - 3')
Lec01* (CA)13 Lec11 (GT)11
F:TGTGCTGTAGGATTGCTTGAGC (CA)30AA F:GGCACCAAAGGCAGATTTTAC (CT)14CA
R:ATGTCAGAGGCTGATGGGATAC (CA)5 R:AGAGTGTGAGATTATGGCAGC (CT)2(CA)6
F:CGTCCACCAGCCTTACGAAC (CA)14CG F:GACGCCACGACAAGACGAAC
Lec03 Lec13 (CT)21
R:TGACGCTCAGTAGTCGGACC (CA)3 R:TATGTGTGGAGGGGGGTGAG
Lec04 (GA)29 Lec14* (GT)7
Lec05* (GT)13 Lec15* (GT)11
Lec06* (CT)10 Lec16* (CA)10
F:TGTGAAGAAACCTGAACACGC F:GTCCCCATAAAACAGGAAACCC (GT)7GCG
Lec07 (CT)7(GT)11 Lec17*
R:ATTCTGTGTCCCTGAACACAC R:GACTATTGAGTGAGTGCCACAC TGG(GT)5
F:GACGCAACAATCTCAGGGTC (GA)5AA(GA)8 F:CGACCATCTTCTGGGGTTACG
Lec08* Lec18* (GT)9
R:ACAGGACCAAGTGGACTCTC AA(GA)17 R:CCTCGGATGGGCTAAATGACC
Lec09 (GA)14 Lec19* (GT)6
* Polymorphic sequences (two or more genotypes) in sample populations collected from the southeast part of Tochigi pref. (Figure 1)
Table 2 Reaction conditions for use in PCR amplification
of index sequences
Step Cycle Temperature and reaction time
1 1 94 celsius degrees, 2 min.
94 celsius degrees, 15 sec.
2 50 56 celsius degrees, 15 sec.
72 celsius degrees, 30 sec.
3 1 72 celsius degrees, 30 min.
Fig. 1 Sampling sites (●) of the six sample Fig. 2 Genetic identity [above; number of alleles
populations in the southeast part of and heterozygosity per locus (error bars
Tochigi pref. and their specimen sizes (N) show standard error)] and differentiation
(below; phylogenetic tree of FST by
neighbor-joining method) in the sample