Molecular Analysis for Genetic Diversity and Distance of Introduced Grus antigone sharpii L. to Thailand
The genetic relationship was examined in a population
of Grus antigone sharpii L. using DNA markers from the ISSR technique
for applying towards breeding purposes for conservation of species. Since
their extinction from Thailand, sixteen eastern sarus cranes: Grus
antigone sharpii L. provided from Cambodia were fed and bred to sixty
individuals at Nakhonratchasima Zoo, Northeastern Thailand to re-exist
in Thai natural sites. Their genetic diversity and distance were examined
to test their possibility to adapt to environmental variation. Blood samples
from 27 individuals of Grus antigone sharpii L. were collected
and DNA was extracted. These DNA samples were amplified using the successful
fifteen from twenty four primers inter simple sequences repeat markers.
A dendrogram was constructed and shows distance values of the species
between 12.1 and 53.5. The samples produced 63.96% polymorphic banding
profiles. The genetic diversity (Hβ²) in this population was estimated
using Shannonβ²s index. The high Hβ² value of 0.501 reflected
the somewhat wide range of distribution sites, which would adapt to environmental
variations. Genetic evenness is 0.152. This value supports that all the
studied samples have a small equal genetic abundance.
The sarus crane: Grus antigone L. 1758 is in the crane family, Gruidae,
which comprises 15 extant species of large, graceful birds distributed across
five continents. Unfortunately, cranes worldwide are threatened by habitat loss,
excessive harvest and disturbance (Meine and Archibald, 1996),
making Gruidae one of the most threatened groups of birds in the world. The
sarus crane, the worlds tallest flying bird, has a broad distribution
that spans two continents and is the only species of crane that breeds both
in India and in Southeast Asia (Meine and Archibald, 1996).
There are four recognized subspecies of sarus crane, namely Indian (Grus
a. antigone), eastern (Grus a. sharpii), Australian (Grus
a. gillae) and the extinct Philippine sarus (Grus a. luzonica;
Blanford, 1896; Schodde et al.,
1988; Meine and Archibald, 1996).
The eastern sarus crane, Grus antigone sharpii L., became extinct from
Thailand because of habitat loss with forest destruction which occurred violently
throughout the country. In 1988, Thailand obtained sixteen individuals of eastern
sarus cranes originating in Cambodia. They were fed and the population enlarged
to sixty individuals at Nakhonratchasima Zoo for releasing them as natural resources
in Thailand. There genetic diversity and distance should therefore be examined
to test their possibility of adapting to the environmental variations. Genetic
diversity allows species to adjust to a changing world, which is caused by both
natural and human factors. Understanding genetic diversity within the genus
or species is essential for establishing effective and efficient conservation
and breeding practices (Chaveerach et al., 2006).
Traditionally, morphological characters have been used to characterize levels
and patterns of diversity or similarity. Since, these traits represent only
a small portion of the plant genome and are influenced by environmental factors,
they have limited utility for describing the potentially complex genetic structure
which may exist within and between taxa (Avise, 1993).
Various molecular approaches have been devised to overcome these constraints
(Soltis and Soltis, 1990). A number of PCR-based DNA markers,
including Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeat (SSR),
Inter-Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism
(AFLP) techniques have been used widely to investigate population genetics.
Each of these methods has many advantages and limitations. ISSR markers are
extremely variable and have proven to be sensitive enough to differentiate cultivars
and natural populations (Wolfe et al., 1998; Chaveerach
et al., 2006). These markers of genetic variation are generally independent
of environmental factors and more numerous than phenotypic characters, thereby
providing a clearer indication of the underlying variation in the genome (Avise,
Genetic distance is a measure of the dissimilarity of genetic material between
different species or individuals of the same species. It can be used to measure
the relatedness of samples by cluster analysis (Nybom and
Hall, 1991; Welsh et al., 1991). The result
is a branching diagram that connects all of Operational Taxonomic Units (OTUs)
and OTU clusters at levels corresponding to their degree of distance (Weier
et al., 1982).
The present study aims to examine genetic relationships consisting of
genetic distance and diversity in a population of Grus antigone sharpii
L. using DNA markers from the ISSR technique.
MATERIALS AND METHODS
Sample collection and DNA extraction: Blood samples from 27 individuals
of Grus antigone sharpii L. identified by microchip coding (Table
1) and an outgroup of West African crown crane were collected from Nakhonratchasima
Zoo, Northeastern Thailand in 2006. Genomic DNA was isolated from blood samples
using proteinase K digestion and treatment with phenol/chloroform (Sambrook
et al., 2001). The quality and quantity of extracted DNA was checked
by 0.8% agarose gel electrophoresis stained with ethidium bromide.
ISSR analysis: Amplification reactions were performed using a
PCR machine (Gene Amp PCR system 9700, Applied Biosystems). Amplifications
were carried out in 20 μL using PCR master mix (Promega) with 1 μM
primer and 2 ng of DNA template. Twenty-four ISSR primers were screened
and the 15 ISSR primers (Table 2) were successfully
amplified clear bands for Grus antigone sharpii L. and the outgroup,
West African Crown Crane. The reaction mixture was incubated at 94°C
for 3 min and the amplification was performed with the following thermal
cycles: 35 cycles of denaturation for 1 min at 94°C, 1 min annealing
temperature Tm-5, extension for 2 min at 72°C and 7 min final extension
at 72°C. Amplification products were detected by agarose gel electrophoresis
in TAE buffer (0.4 M Tris, 0.114% acetic acid 1 mM EDTA pH 8.0) and visualized
by ethidium bromide staining.
||Twenty-seven studied individuals and microchip coding
of Grus antigone sharpii L. from Nakhonratchasima Zoo, Northeastern
||Summary of ISSR primers, number of bands scored, number
of polymorphic bands and percentage of polymorphisms for amplification
profile of 27 individuals of Grus antigone sharpii L.
ISSR data analysis: The total number of ISSR bands discerned from the
agarose gel was documented as diallelic characters: present = 1, absent = 0;
as the ISSR markers are considered the dominant markers. The resulting bands
were used to construct a dendrogram following the UPGMA with the Fingerprinting
II program version 3.0 (Bio Rad). The distance values were obtained from the
dendrogram. The percentage of polymorphic loci was calculated with the Shannons
diversity index (H) for each population, H= -Σpi ln pi, where
pi is the frequency of a given ISSR band. Genetic evenness (E) is calculated
from genetic diversity/ln individual richness (ln 27 = 3.296) (Nei,
Twenty-four primers were screened and fifteen informative ISSR primers
produced a total of 146 band locations with sizes ranging from 100-2,500
and an average of 9.733 bands per primer. Of these bands, 63.96% (95 bands)
were polymorphic. Percentages of Polymorphic Bands (PPB) for each primer
ranged from 25 to 87.50%. Primer (AC)8CG generated the highest
number of bands (18 bands). The minimum number of bands (7) was produced
by primer (GT)8C (Table 2). Figure 1 shows
the banding pattern using the primer (CAC)3GC, one of the 15
informative primers which demonstrate the differences of banding pattern
between individuals in a population of Grus antigone sharpii L.
|| ISSR profile of Grus antigone sharpii L. and
an outgroup, West African Crown Crane with primer (CAC) 3GC
||A dendrogram constructed from 15 ISSR primers determined
by UPGMA (Fingerprinting II, Bio Rad) to clarify the genetic relationships
of Grus antigone sharpii L. and West African Crown Crane. ♀
indicates female and ♂ indicates male individuals. Individuals
with * are parents of natural reproduction and individuals with **
are of insemination
The dendrogram (Fig. 2) clearly distinguishes the studied
samples into three groups without consideration of male and female or
artificial breeding (insemination) or natural reproduction. The first
group comprises 13 individuals, namely 114567283A♀, 115311146A♀,
114822755A♀, 116412280A♂, 116427254A♂, 115224744A♂,
114562111A♂, 122913516A♀, 115311352A♀, 126811095A♀,
123179520A♀, 114673192A♂ and 114752571A♂. The second
includes 115232651A♀, 114754651A♂, 123211537A♀, 113556597A♂,
122911611A♂, 122652573A♂, 122914754A♀, 122917266A♂,
115229467A♀, 122638531A♂, 114833192A♂, 122762133A♀,
115224744A♀ and 115229154A♂. The third is an outgroup, West
African Crown Crane.
||Genetic distance (D) values of Grus antigone sharpii
L. population and West African Crown Crane
||Genetic diversity (H) and evenness (E) values
of Grus antigone sharpii L. population and an outgroup
The genetic distance (D) values of a population range display from 12.1
between 115311146A♀ and 114567283A♀ to 53.1 between 114833192A♀
and 115311352A♀ (Fig. 3).
The genetic diversity of the population of the 27 studied samples in
60 individuals enlarged from 16 original individuals is 0.501 (Table
3). The value reflects the fairly good range of distribution sites
of the species. Genetic richness is 27, ln genetic richness (ln 27) is
3.296, so the genetic evenness is 0.152.
The high genetic distance values of 12.1-53.3 in a species indicate that
they possess several different genetic variations and the population can
be further enlarged toward the goal of existing naturally in Thailand.
Genetic diversity is an important factor for genetic variation measurement.
The genetic diversity in the population of the introduced eastern sarus crane
is 0.501. It is not at satisfactory level. However, it reveals a fairly good
value to support the broad range of distribution sites. This shows that the
genetic diversity of Grus antigone sharpii L. may be able to fit environmental
variation (Fu et al., 2003). High genetic diversity
is important, allowing Grus antigone sharpii L. to adjust to the ever-changing
environment in Thailand, whether the changes are due to the natural or human
factors (Chamberlain and Hubert, 2001).
The genetic evenness value is very low, 0.152, showing that the studied samples
have little equal genetic abundance. The genetic evenness is the measure of
whether the number of alleles at a locus occurs equally frequently. For example,
if three alleles are available at a locus, do they occur equally frequently
(1:1:1) or not, such as one prominently and the other two rarely (100:2:1).
When using genetic diversity data, genetic richness is defined as the number
of alleles potentially found at a locus and genetic evenness is a measure of
whether the number of alleles at a locus occurs equally frequently (Brown
and Weir, 1983).
The dendrogram cannot show the result position of natural reproduction
of recent parents, 122638531A♂* x 115311352A♀* and offsprings,
122917266A♂*, 115232651A♀* and 115229467A♀* and similarly
cannot show the recent position of artificial insemination, namely microchip
coding 114673192A♂** x 114567283A♀** and offspring, 115229154A♀**.
Since for a small population in a limited area, human factors for enlarging
the population have varied methods including natural and artificial reproduction.
The Grus antigone sharpii L. in this area may change their behavior
from living in lone male/female pairs. As shown in the dendrogram, the
sixteen original parents from Cambodia, males and females parents were
The measurements of richness, evenness, genetic distance and diversity
show that the population of Grus antigone sharpii L. introduced
to Thailand are rather suitable to be maintained in a Thai natural habitat
in the form of traditional variety.
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