Genetic Diversity in Mazandaranian Native Cattle: A Comparison with Holstein Cattle, using ISSR Marker
This study was carried out to investigate genetic diversity in Mazandaranian native cattle population comparised to the Holstein breed, using Inter Simple Sequence Repeats (ISSR) marker. A total of 175 animals, including 71 native and 104 cattle of Holstein breed were screened. The extraction of DNA samples were carried out, using modified salting out method. A 19-mer oligonucleotide, (GA)9C, was used as primer in PCR reactions. The PCR products showed 15 different fragments with length ranged from 120 to1600 bp in the two breeds.. Genetic variation indexes, including effective number of alleles, Shannon index, Neis gene diversity and standard genetic distance were estimated, using POPGene software. Generally, the estimated genetic variation indexes showed low levels of diversity in the two breeds. However, Nei's gene diversity and Shannon index estimation was observed almost two folds in native cattle compared to Holstein breed. Less levels of diversity in Holstein cattle may be because of applying intensive selection programs. Conversely, native cattle have been less affected by selection. Therefore, it seems that Mazandaranian native cattle probably are better for breeding programs than Holstein cattle. Results showed that ISSR Markers are reliable and can be used in genetic diversity investigations.
Breeds are commonly classified as indigenous and exotic, where indigenous breeds
are mainly kept in low input low output production systems while exotic breeds
are usually adapted to intensive, high-output systems and do not flourish in
unimproved local production environments (Hoffman and Scherf,
2005). A period of traditional selection over the last few centuries has
resulted in the establishment of numerous breeds. Then, in the quit recent past,
more and more effective breeding programs have been implemented and led to an
emphasis on a few specialized stocks. Consequently, breeds that are less suited
to current needs tend to see their numbers reduced and to be eventually lost
(Ollivier and Foulley, 2005). Today, Holstein cattle
have become the pre-dominant dairy breed worldwide. There are about 7.5 million
cattle in Iran and Holstein cattle in more than 90% of industrial dairy farms
are used. Extensive use of artificial insemination has reduced the number of
breeding sires and effective population size. Likewise, it has resulted the
high level of inbreeding or homozygosity in herds. Replacement of local breeds
with more productive ones (Holstein) has increased the number of endangered
breeds globally. Many traditional European breeds have disappeared because of
farmers focus on the new cattle breeds. Around 16% of them have become extinct
and 15% - are rare or endangered. Losing genetic diversity is considered as
the main reason of high level of breeds uniformity and will increase frequency
of genetical defects and negatively affects fertility (FAO,
2000). The most famous Iranian cattle breeds are Sarabi, Golpayegani, Mazandarani
and Sistani.The Mazandarani is a zebu type breed found in Northern Iran. They
are kept for meat and milk production and are see in all colors (Mason,
1996). However, these native cattle breeds are being interbred or replaced
by improved Western breeds, so seems all are endangered. The conservation of
genetic variation is an essential component of many species management programs.
Ultimately, genetic variation allows species to adapt the change of environmental
conditions and respond to selection/breeding programs. To manage any biological
resource effectively, researchers must identify the level of genetic variation
within and among livestock populations. Since, the mid 1980s, genome identification
and selection has progressed rapidly with the help of PCR technology. The large
numbers of marker protocols that are rapid and require only small quantities
of DNA have been developed. Each marker technique has its own advantages and
disadvantages (Bornet and Branchard, 2001). Since 1994,
new molecular marker techniques called Inter Simple Sequence Repeats (ISSR)
has been available. ISSRs are semi arbitrary markers amplified by PCR in the
present of one primer complementary to a target microsatellite. In such amplification
period genome sequence information isnt required and leads to multi locus
and highly polymorphous patterns (Bornet and Branchard,
2001). ISSR primers could be successfully used for the detection of new
genomic loci and applied in a new way for genomic mapping, fingerprinting, gene
tagging (Ye et al., 2005) and genetic diversity
studies. Different studies have been performed on animals, using these markers
(Triapitsyna and Glazko, 2005; Bannikova,
2004; Vaulin and Zakharov, 2008; Glazko
et al., 1999; Lovenko, 2002; Gorodnaya
and Glazko, 2006; Chatterjee and Mohandas, 2003).
Furthermore, many studies have shown that, the approach can be used as a useful
tool for the genetic diversity monitoring in different populations (or breeds)
of animals (Ahani Azari et al., 2007; Kol
and Lazebny, 2006) . In current study, GA-ISSR marker based on the Polymerase
Chain Reaction (PCR), have been developed to establish the genetic diversity
within and between Mazandaranian native and Holstein cattle.
MATERIALS AND METHODS
The blood samples from two different cattle breeds, Mazandaranian native cattle
(n = 71) and Holstein (n = 104) were collected randomly. The used Holstein cattle
were sampled from an industrial dairy cattle farm (Kabiri) on the north of Gorgan
(Golestan Provience) and the native cattle belonged to Sabegh Mahaleh Village
at the same region. The experiments was performed in Molecular Genetics Laboratory
of Animal Science, Department of Gorgan University of Agricultural Sciences
and Natural Resources from July 2008 to 20 January 2009. DNA was extracted,
using modified salting out as described by Miller et al.
(1988). Extracted DNA was dissolved in Tris-EDTA (TE) buffer. Extracted
DNA was quantified by gel electrophoresis and its quality was verified by spectrophotometer.
DNA samples stored at -20°C. In PCR reaction, a 19-mer oligonucleotide GAGAGAGAGAGAGAGAGAC
(Glazko et al., 1999), was used. PCR was performed
in a final volume of 25 μL, using PCR Master Mix kit (SinageneTM.
Iran). Amplification was performed in a DNA thermal cycler ( Biometra Thermo-Cycler
(PersonalTM ) with the following parameters: 94°C for 2 min;
35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for
30 sec and extension at 72°C for 2 min followed by final extension at 72°C
for 10 min. PCR products were electrophoresed at 120 V in 2% agarose gel containing
1X Tris-Boric acid-EDTA (TBE) buffer. Twelve microliter of PCR products were
loaded with 2 μL of tracking dye and run until the dye was scatted 10 cm
from the wells. The profiles were stained with ethidium bromide (0.5 μg
mL-1) and documented by the gel documentation under UV and then were
photographed. The amplicons later were used for constructing of binary file.
Allele sizing for different DNA fragments was carried out by Dscan-ONE
(1994-1997) software package (V1.31). Molecular data analyses were carried
out using POPGene V1.31 software (Yeh et al., 1997).
The following parameters were computed at the reference population level: expected
heterozygosity (Nei, 1978) as:
where, pi is the frequency of the ith allele.
Effective alleles number (Hartl and Clark, 2007) as:
Shannon index (Shannon and Weaver, 1949) as:
Standard genetic distance (Nei, 1978) as:
where, xij and yij are frequency of the ith allele in jth locus in x and y populations, mj refers to number of alleles in j locus and r is the number of loci.
All the locus-population combinations were tested for deviations from Hardy-Weinberg equilibrium (HWE), using Chi-square (χ2) goodness of fit test.
An ISSR-PCR pattern obtained by GA-ISSR marker is shown in Fig.
1. The observed amplicon patterns were complete and clear by contrasting.
In the patterns of PCR products totally 15 different fragments were found, showing
different length between 120 to 1600 bp. The fragments size (bp) and frequency,
accompanied with Chi-square and G2 ( likelihood ratio) tests for
evaluating gene frequencies homogeneity between two breeds (Yeh
et al., 1997) are presented in Table 1.
The number of polymorphic loci in native and Holstein herds were 6 and 3, respectively.
The monomorphic fragments (frequency= 1) were 9 fragments in native cattle,
while it was 11 fragments in Holstein breed. The monomorphic fragments between
the two breeds were the same in 9 fragments and differ in 5 fragments. Gene
frequency differences between two breeds were significant (p<0.05) for fragments
number 2, 4, 6, 11 and 14 (Table 1). Fragment number 9 (525-590
bp) was observed only in native cattle. The proportion of polymorphic fragments
in Mazandaranian and Holstein breeds were 0.46 and 0.20, respectively. To analysis
inter population diversity four parameters, including number of observed alleles,
effective number of the alleles, Neis gene diversity and Shannon index
were estimated using POPGene software (Table 2).
|| ISSR-PCR pattern, obtained by GA-ISSR marker, M: GeneRulerTM
bp DNA Ladder standard marker
|| Fragment length, gene frequencies, G2 and chi-square test
results for evaluating gene frequencies homogeneity in two cattle breeds
|Values in the brackets are the probabilities. Values less
than 0.05 are significant.
|| The mean of genetic diversity indexes in two breeds
|N: Number of animals, Na: Effective allele number, Ne: Observed
allele number, H: Nei's gene diversity, I: Shannon index. Data are expressed
as Mean ± SD
Based on Table 2, all of the mentioned parameters in native
cattle are more than Holstein breed. High standard deviation estimates are mainly
related to low number of detected fragments and number of the used animals (Nei,
This is the first attempt to specifically quantify the genetic diversity of
the Mazandaranian native cattle with ISSR markers. Based on the results Mazandaranian
native and Holstein were different in the fragment size and frequency. Some
fragments were found in both breeds but didn't show the same frequency.
The fragment number 9 was not detected in Holstein but since, it's frequency
was very low (0.029) can't be used as an important breed-specific marker. Based
on allelic frequencies (Table 1) the two investigated breeds
did not show high polymorphic bands and lower polymorphism rate in Holstein
than Mazandaranian native cattle could be due to higher genome uniformity. Low
levels of polymorphic fragments in other breeds investigated by the same marker,
was reported by Ahani Azari et al. (2007). In
the present study, most of the loci-population combinations deviated from HWE
were in agreement with some prior studies (Rachagani et
al., 2006; Elbeltagy et al., 2008; Santos-silva
et al., 2008). Means of heterozygosity and effective number of alleles
in indigenous cattle (Table 2) were more than Holstein breed.
Furthermore, Shannon genetic diversity in Mazandaranian and Holstein cattle
were 0.21 and 0.11, respectively (Table 2). On the other hand,
genetic diversity estimation was high in Mazandaranian cattle compared to Holstein
breed ( 0.21 and 0.11, respectively (Table 2)). As well as,
Neis gene diversity estimation in Mazandaranian cattle was almost two
folds of Holstein cattle. Consequently, as expected, all genetic diversity indexes
in native cattle were higher than Holstein. Findings by Ahani
Azari et al. (2007) reported more genetic diversity indexes in the
breeds of Bos taurus and Bos indicus than Holstein. Furthermore,
similarity indices of animals in these breeds were high. Present results indicated
that because of intensive selection inbreeding programs in Holstein, it's genetic
diversity has been lost. Generally, reduction of the number and genetic diversity
of other breeds have been happened in the past decades. In the year 2000, over
6300 breeds of domesticated livestock were identified. Of these, over 1300 are
now extinct or considered to be in danger of extinction. Many others have not
been formally identified and may disappear, before they are recorded or widely
known. Europe records the highest percentage of extinct breeds or breeds at
risk (55% for mammalian and 69% for avian breeds). Asia and Africa record only
14 and 18%, respectively, but the data for developing countries are much less
fully documented in the World Watch List for Domestic Animal Diversity than
those of developed countries (Hoffman and Scherf, 2005).
The biological unit for conservation in domesticated animals is usually the
breed. Obtaining information from molecular markers in different breeds made
it possible to create a hypothetical scenario for assessing different methods
of analyzing diversity for conservation (Solis et al.,
2005). Genetic distance of the two breeds was estimated 0.105 and no considerable.
Although, estimated genetic diversity indexes in Mazandaranian cattle were lower
than expected values, but were higher than Holstein breed. These results may
be because of sampling of the native animals in a limited area and undesirable
inbreeding. With the relatively small total population size and small individual
flock sizes, genetic drift is an important factor affecting within-breed genetic
diversity, so it is expected that random gene frequency changes would be cumulative
over generations (Maiwashe and Blackburn, 2004). The
balance between drift, natural and artificial selection and mutation needs further
evaluation for the indigenous breed. Since, designing any selection program
at the first step needs knowledge of the genetic diversity of the herds, specially
pedigree-less ones, conducting of such researches seems very essential. Meanwhile,
it should be emphasized that genetic distances between breeds provide an initial
guide for conservation decisions, which need to be completed by more detailed
characterization. In conclusion, the marker (ISSR-GA) indicated genetic diversity
indexes successfully and will be useful in other researches that, screening
more number of animals. Since, the use of ISSR-PCR markers on biodiversity of
the animals are rare and usually other markers are used, it is suggested to
increase the number of ISSR primers and investigated animals and perform comparative
analysis with other molecular markers to obtain reliable results. In this way,
may test the potential of this marker in monitoring genetic variability of animals.
Results of this study clearly showed that genetic diversity in both investigated
cattle breeds, especially in Holestein breed, has been lost. Although, sampling
of the studied native cattle was conducted in a limited region and animals undergo
close matings and inbreeding at some rate, but all the genetic diversity indexes
in native cattle were better than Hostein breed. Furthermore, earlier studies
were in agreement with our findings. Consequently, for avoiding negative effects
of genome uniformity, extinction of breeds and inbreeding defects, it is suggested
permanent investigating genetic variability in all domestic species with more
reliable molecular markers. Conducting such researches certainly helps specialists
to design selection breeding programs more carefully in the future and prevents
or limits the trend of diminishing genetic variability in breeds of livestock.
1: Ahani Azari, M., O.E. Lazebny and G.E. Sulimova, 2007. Determination of heterozygosity level in fifteen various cattle breeds using ISSR-PCR method. Proceedings of the 5th National Biotechnology Congress of Iran. Summit Metting Conference Hall, November 24-26, 2007, Tehran, Iran -
2: Bannikova, A.A., 2004. Molecular markers and modern phylogenetics of mammals. Zh Obshch Biol., 65: 278-305.
3: Bornet, B. and M. Branchard, 2001. Nonanchored Inter Simple Sequence Repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting. Plant Mol. Biol. Rep., 19: 209-215.
CrossRef | Direct Link |
4: Chatterjee, S.N. and T.P. Mohandas, 2003. Identification of ISSR markers associated with productivity traits in silkworm, Bombyxmoril. Genome, 46: 438-447.
5: Elbeltagy, A.R., S. Galal, A.Z. Abdelsalam, F.E. El Keraby, M. Blasi and M.M. Mohamed, 2008. Biodiversity in Mediterranean buffalo using two microsatellite multiplexes. Lives. Sci., 114: 341-346.
6: FAO, 2000. Global project for the maintenance of domestic animal genetic diversity (MoDAD). http://www.fao.org/dad-is/.
7: Glazko, V.I., T.N. Dyman, S.I. Tarasiuk and A.V. Dubin, 1999. The polymorphism of proteins, RAPD-PCR and ISSR-PCR markers in European and American bison and cattle. Tsitol Genet., 33: 30-39.
PubMed | Direct Link |
8: Gorodnaya, A.V. and V.I. Glazko, 2006. Population-genetic study of the polymorphism of structural genes and ISSR-PCR markers in some cattle breeds. Cytol. Genet., 40: 49-57.
Direct Link |
9: Hartl, D.L. and A.G. Clark, 2007. Principles of Population Genetics. 4th Edn., Sinauer Associates, Sunderland, MA
10: Hoffman, L. and B. Scherf, 2005. Animal genetic resources- time to worry? Lives. Rep., 1: 57-74.
Direct Link |
11: Kol, N.V. and O.E. Lazebny, 2006. Polymorphism of ISSR-PCR markers in Tuvinian population of Reindeer Rangifer tarandusl. Rus. J. Genet., 42: 1469-1476.
12: Iovenko, V.N., 2002. Genetic diversity of protein markers in sheep population from Ukraine. Genetika, 38: 1669-1676.
13: Maiwashe, A.N. and H.D. Blackburn, 2004. Genetic diversity in and conservation strategy considerations for Navajo Churro sheep. Anim. Sci., 82: 2900-2905.
Direct Link |
14: Miller, S.A., D.D. Dykes and H.F. Polesky, 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16: 1215-1215.
PubMed | Direct Link |
15: Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89: 583-590.
PubMed | Direct Link |
16: Ollivier, L. and J.L. Foulley, 2005. Aggregate diversity new approach combining within-and between-breed genetic diversity. Lives. Prod. Sci., 95: 247-254.
17: ONE-Dscan, 1994-1997. One-Dimensional gel analysis Scanalytics divition of CSP. Scanalyticas Attach processor System.
18: Rachagani, S., I.D. Gupta, N. Gupta and S.C. Gupta, 2006. Genotyping of β-lactoglobulin gene by PCR-RFLP in Sahiwal and Tharparkar cattle breeds. BMC Genet.
CrossRef | Direct Link |
19: Santos-silva, F., F.S. Ivo, M.C.O. Sousa, M.I. Carolino, C. Ginja and L.T. Gama, 2008. Assessing genetic diversity and differentiation in Portuguese coarse-wool sheep breeds with microsatellite markers. Small Rum. Res., 78: 32-40.
20: Solis, A., B.M. Jugo, J.C. Meriaux, M. Iriando and L.I. Mazon et al., 2005. Genetic diversity within and among four South European native horse breeds based on microsatellite DNA analysis: Implications for conservation. Heredity, 96: 670-678.
21: Triapitsyna, N.V.I. and V. Glazko, 2005. Polymorphism of DNA fragments flanked by microsatellite loci(ISSR-PCR) in cattle reproduced under low-dose irradiation conditions. Tsitol Genet., 39: 41-50.
22: Vaulin, O.V. and I.K. Zakharov, 2008. Temporal dynamics and variation of multilocus ISSR-PCR DNA markers in the Uman population of Drosophila melanogaster over two decades. Genetika, 44: 359-365.
23: Ye, C., Z. Yu, F. Kong, S. Wu and B. Wang, 2005. R-ISSR as a new tool for genomic fingerprinting, mapping and gene tagging. Plant Mol. Biol. Rep., 23: 167-177.
24: Yeh, F.C., R.C. Yang, T.B.J. Boyle, Z.H. Ye and J.X. Mao, 1997. POPGENE, the User-Friendly Shareware for Population Genetic Analysis. Molecular Biology and Biotechnology Center, Alberta
Direct Link |
25: Mason, I.L., 1996. A World Dictionary of Livestock Breeds, Types and Varieties. 4th Edn., CAB International, Wallingford, UK., Pages: 273
Direct Link |
26: Shannon, C.E. and W. Weaver, 1949. The Mathematical Theory of Communication. 1st Edn., University of Illinois Press, Urbana, IL., ISBN-10: 0252725484