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ISSN No. (Print): 0975-1130
ISSN No. (Online): 2249-3239
Genetic Diversity of Gastrointestinal tract Fungi in Buffalo by
Molecular methods on the basis of Polymerase Chain Reaction
Abbas Mo'azami Goudarzi*, Mohammad Chamani**, Naser Maheri-Sis*, Mehdi Amin Afshar**
and Ramin Salamatdoost-Nobar*
*Department of Animal Science, Shabestar Branch, Islamic Azad University, Shabestar, IRAN
**Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, IRAN
(Corresponding author: Mohammad Chamani)
(Received 14 December, 2014, Accepted 04 January, 2015)
(Published by Research Trend, Website: www.researchtrend.net)
ABSTRACT: Buffalo are able to utilize feed more efficiently than beef cattle where the feed supply is of low
quantity and or quality. Therefore, the present study was conducted to establish the community structure of
anaerobic rumen fungi in buffalo using molecular approaches. Polymerase chain reaction approach was used
in this study to determine the population of major anaerobic rumen fungi in buffalo in digesta and rumen
fluid. Total community DNA was extracted, and the ribosomal internal transcribed spacer (ITS) 1 region was
amplified, cloned, and sequenced. The resulting nucleotide sequences were used to construct a phylogenetic
tree. A total of 12 clones were analyzed. Sequence analysis of ITS1 spacer seems a promising tool for
comparing a variety of rumen fungal isolates.
Key words: Anaerobic rumen fungi, buffalo, ITS1, rRNA, PCR
INTRODUCTION
The anaerobic gut fungi are the only known obligately
anaerobic fungi. For the majority of their life cycles,
they are found tightly associated with solid digesta in
the rumen and or hindgut. They produce potent
fibrolytic enzymes and grow invasively on and into the
plant material they are digesting making them
important contributors to fibre digestion. This close
association with intestinal digesta has made it difficult
to accurately determine the amount of fungal biomass
present in the rumen, Orpin (1984) suggesting fungal
8% contribution to the total microbial biomass. It is
clear that the rumen microbial complement is affected
by dietary changes, and that the fungi are more
important in digestion in the rumens of animals fed
with high-fibre diets (Bauchop, 1979 and Lee et al.,
2000). It seems likely that the gut fungi play an
important role within the rumen as primary colonizers
of plant fibre (Akin et al., 1983).
Present knowledge of anaerobic gut fungal population
diversity within the gastrointestinal tract is based upon
isolation, cultivation and observations in vivo (Davies
et al., 1993). It is likely that there are many species yet
to be described, some of which may be nonculturable.
The development of molecular techniques has greatly
broadened our view of microbial diversity and enabled
a more complete detection and description of microbial
communities (Bauchop, 1979 and Ranjard et al., 2001).
The development of molecular biological techniques in
the last decade has enabled the new approach to the
characterization of fungi. Li and Heath (1992) studied
relationship of gut fungi based on ITS1 sequences and
found that Anaeromyces isolates were more distant
from other rumen fungi. The whole DNA sequences
showed above 80% similarity among Piromyces,
Neocallimastix and Orpinomyces, whereas the
similarities between Anaeromyces and these three
genera were only 70% (Fliegerova et al., 2002).
Molecular analyses have therefore been used in
attempts to clarify the classification of anaerobic fungi.
Ribosomal sequences of internal transcribed spacer
regions ITS1 and ITS2 (Brookman et al., 2000;
Fliegerova et al., 2004) have been applied successfully
for discrimination between Orpinomyces and
Anaeromyces. Sequencing and subsequent comparison
of acquired results with gene-bank data is time
consuming. In this research, we try to determine the
genetic diversity of the gastrointestinal tract anaerobic
fungi in Azarbayejan Iranian buffalos.
MATERIAL AND METHODS
This research was done in the Department of Animal
Science, Shabestar Branch Islamic Azad University in
Iran. For the sampling of buffalo rumen, the necessary
coordination was carried out by the industrial
slaughterhouse of Uromia. Buffalo were slaughtered
and samples of rumen contents were taken.
Biological Forum – An International Journal 7(1): 20-25(2015)
Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 21
Samples of rumen content were collected randomly
from rumen in the slaughter house. Finally, after 24-48
hours, the samples were transferred to the laboratory for
DNA extractions.
Total genomic DNA was extracted by using RBB+C
method that described at follow (Yu and Morrison
2004).
A. Cell lysis:
(a) Transfer 0.25 g of sample into a fresh 2-mL screw-
cap tube. Add 1 mL of lysis buffer [500 mM NaCl, 50
mM Tris-HCl, pH 8.0, 50 mM EDTA, and 4% sodium
dodecyl sulfate (SDS)] and 0.4 g of sterile zirconia
beads (0.3 g of 0.1 mm and 0.1 g of 0.5 mm).
(b) Homogenize for 3 min at maximum speed on a
Mini-Beadbeater™ (BioSpec Products, Bartlesville,
OK, USA).
(c) Incubate at 70°C for 15 min, with gentle shaking by
hand every 5 min.
(d) Centrifuge at 4°C for 5 min at 16,000× g. Transfer
the supernatant to a fresh 2-mL Eppendorf® tube.
(e) Add 300 μL of fresh lysis buffer to the lysis tube
and repeat steps 2–4, and then pool the supernatant.
B. Precipitation of nucleic acids:
(a) Add 260 μL of 10 M ammonium acetate to each
lysate tube, mix well, and incubate on ice for 5 min.
(b) Centrifuge at 4°C for 10 min at 16,000× g.
(c) Transfer the supernatant to two 1.5-mL Eppendorf
tubes, add one volume of isopropanol and mix well, and
incubate on ice for 30 min.
(d) Centrifuge at 4°C for 15 min at 16,000× g, remove
the supernatant using aspiration, wash the nucleic acids
pellet with 70% ethanol, and dry the pellet under
vacuum for 3 min.
(e) Dissolve the nucleic acid pellet in 100 μL of TE
(Tris-EDTA) buffer and pool the two aliquots.
C. Removal of RNA, protein, and purification:
(a) Add 2 μL of DNase-free RNase (10 mg/mL) and
incubate at 37°C for 15 min.
(a) Add 15 μL of proteinase K and 200 μL of Buffer
AL (from the QIAamp DNA Stool Mini Kit), mix well,
and incubate at 70°C for 10 min.
(a) Add 200 μL of ethanol and mix well. Transfer to a
QIAamp column and centrifuge at 16,000× g for 1 min.
(b) Discard the flow through, add 500 μL of Buffer
AW1 (Qiagen), and centrifuge for 1 min at room
temperature.
(c) Discard the flow through, add 500 μL of Buffer
AW2 (Qiagen), and centrifuge for 1 min at room
temperature.
(d) Dry the column by centrifugation at room
temperature for 1 min.
(e) Add 200 μL of Buffer AE (Qiagen) and incubate at
room temperature for 2 min.
(f) Centrifuge at room temperature for 1 min to elute
the DNA.
(g) Aliquot the DNA solution into four tubes. Run 2 μL
on a 0.8% gel to check the DNA quality.
(h) Store the DNA solutions at -20°C.
The quality of the community DNA was assessed by
1% agarose gel electrophoresis. The ribosomal ITS1
region defined by primers Good92F GM1 (5΄-
TGTACACACCGCCCGTC-3΄) and GM2 (5΄-
CTGCGTTCTTCATCGAT-3΄) as described by Li and
Heath (1992). The PCR reaction was performed in 100
μl reactions containing (final concentration): forward
and reverse primers, 0.2 μM; dNTPs mixture, 200 μM;
MgCl2, 1.5 mM; KCl, 50 mM; Tris/HCl pH 8.4, 10
mM; and Taq polymerase, 0.25 Units. Approximately
50 ng genomic DNA were used as template for each
amplification. The temperature conditions were as
follows: initial denaturation at 94°C for 5 min, followed
by 30 cycles of denaturation at 94°C for 1 min,
annealing at 48 °C for 1 min and extension at 72°C for
1.5 min. Final step was carried out at 72°C for 10
min.The PCR products quality was assessed by 0.8%
agarose gel electrophoresis and the amplified DNA was
purified with a QIAquick PCR purification kit
(QIAGEN) according to the manufacturer’s
instructions. The DNA was then ligated into the
pTG19-T PCR cloning vector system and transformed
into competent Escherichia coli (DH5α) cells, before
plasmid isolation using a GF-1 Plasmid DNA
Extraction Kit. After the plasmid extraction, 15μl of the
extracted plasmid was sent to the ShineGene Company
of China for sequencing with Universal M13 primers.
Sequences from the current study were trimmed
manually and analysed by the CHECK_CHIMERA
program (Maidak et al., 2001). The similarity searches
for sequences were carried out by BLAST (Madden et
al., 1996) and alignment was done using CLUSTAL W
(Thompson et al., 1997). The phylogenetic analysis was
carried out using MEGA software version4 (Tamura et
al., 2007) and the phylogenetic relatedness was
estimated using the neighbour-joining method and by
using the MEGA4 program (Saitou and Nei, 1987).
RESULTS AND DISCUSSION
Detection of microbes by their DNA requires a
sensitive methodology and PCR is commonly used. The
ribosomal genes are often chosen as a suitable amplicon
for environmental studies as they are multicopy in
eukaryotic genomes providing enhanced sensitivity, and
can be used for phylogenetic analyses and delimitation
of diverse groups of organisms across and between the
kingdoms.
Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 22
The rDNA genes are particularly well suited for this
purpose as they are present in all forms of life and
consist of alternating variable and conserved regions.
The small subunit rRNA gene sequences, the ITS1 gene
in eukaryotes, are conventionally used for phylogenetic
comparisons. However, as this gene is more than 97%
identical between genera of the anaerobic fungi (Dore
and Stahl, 1991) the less conserved internal transcribed
spacer (ITS) regions have proven to be more useful
(Brookman et.al, 2000). Molecular data has been used
to clarify the classification of the anaerobic rumen
fungi. Favored indicators of genetic diversity are the
rRNA encoding gene sequences, particularly the
internal transcribed spacers ITS1, this can be used to
identify micro-organisms and to determine pylogenetic
relationship within communities, including the rumen
fungi (Hausner et. al, 2000 and Vainio and Hantula,
2000). In this research, our purpose is to determine the
genetic diversity of the rumen anaerobic fungi in
buffalos of the Azerbayjan in Iran. PCR products
quality was assessed by 0.8% agarose gel
electrophoresis (Fig. 1).
Fig. 1. Analysis of PCR products by agarose gel (0.8 %) electrophoresis.
The GenBank accession numbers for the sequences
determined are: AIB01-1, KJ130471; AIB01-2,
KJ130472; AIB01-3, KJ130473; AIB01-4, KJ130474;
AIB01-5, KJ130475; AIB01-6, KJ130476; AIB01-7,
KJ130477; AIB01-8, KJ130478; AIB01-9, KJ130479;
AIB01-10, KJ130480, AIB01-11, KJ130481, AIB01-
12, KJ130482. Table 1 showed Phylotypes of ITS1
gene sequences of anaerobic rumen fungi retrieved
from the rumen samples of buffalo. The phylogenetic
tree was drawn using the Neighbor-joining method and
the MEGA4 software (Fig. 2). The results show that the
ITSI sequence is less conserved in one genera and it can
have a little differences in a genera or between the
different genera. In this study the observed changes
were in 1% of the number of ITS1 region nucleotides.
Novel groups identified in the fungal ITS data may be
assigned to newly defined genera as characterization of
isolated strains progresses (Tuckwell et al., 2005).
Table 2: Phylotypes of ITS1 gene sequences of anaerobic rumen fungi retrieved from the rumen samples of
buffalo.
% sequence similarityNearest valid taxonSize (bp) GenBankAccession no.Phylotype
99Caecomyce sp.364KJ130471AIB01-1
100Caecomyces sp.375KJ130472AIB01-2
99Orpinomyces sp.483KJ130473AIB01-3
99Orpinomyces sp.435KJ130474AIB01-4
98Piromyces sp.414KJ130475AIB01-5
98Neocallimastix sp.401KJ130476AIB01-6
97Neocallimastix sp.485KJ130477AIB01-7
96Neocallimastix sp.445KJ130478AIB01-8
97Piromyces sp.426KJ130479AIB01-9
99Orpinomyces sp.432KJ130480AIB01-10
98Piromyces sp.460KJ130481AIB01-11
99Anaeromyces sp.416KJ130482AIB01-12
Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 23
Fig. 2. Neighbor-joining phylogenetic tree of aligned ITS1 sequences of anaerobic rumen fungi.
The anaerobic gut fungi are eukaryotic organisms and
therefore require a different genetic marker to the
rumen bacteria for identification and differentiation.
The use of 18S rDNA has not been found to be useful
in differentiating between the members of the gut
fungal family, Neocallimastigaceae, as they are too
similar. We have used the more variable, ITS region 1
between the structural genes of the ribosomal repeat as
sequences with suitable levels of variability for
phylogenetic studies. The ITS regions from the
anaerobic gut fungi show length polymorphisms, and
there is an approximate relationship between the length
of the ITS1 and genera. The paucity of morphological
features presents a problem regarding the taxonomy of
anaerobic fungi. While examining plant material from
the digestive tract, fungi often appear as the complex
cluster and this makes the classification even up to
genus level difficult. At a time when there is little
disagreement as to the status of the six genera,
subgeneric classification is problematic since
difficulties associated with exchange and long-term
maintenance of cultures impeded direct morphological
and physiological comparisons among isolates. With
the advent of molecular taxonomy, it is hoped that
DNA sequence comparisons and phylogenetic
reconstruction will elucidate the relatedness of the
various taxa.
Majority of the sequences deposited relate to the
ribosomal RNA genes widely used in phylogenetic
reconstruction. The small ribosomal (18S) subunit is
highly conserved in different taxa and thus contains
little phylogenetically useful information for subgeneric
classification (Li and Heath, 1992). In contrast, the
internal transcribed spacer (ITS) regions, widely used
for study of closely related fungal taxa, show a high
level of variability (Li and Heath, 1992; Brookman et
al., 2000; Fliegerova et al., 2004), and has been used to
differentiate the morphologically similar monocentric
(Neocallimastix, Piromyces) and polycentric
(Anaeromyces, Orpinomyces) genera. Brookman et al.,
(2000) also reported that the two multi-flagellated taxa
(Neocallimastix, Orpinomyces) were closely related
based on the ultrastructure of the zoospores.
Neocallimastixsp.KJ130477
Neocallimastixsp.KJ130478
Neocallimastix sp. KJ130476
Caecomyces sp. KJ130472
Piromyces sp. KJ130475
Caecomycessp.KJ130471
Piromycessp.KJ130479
Orpinomycessp.KJ130474
Orpinomyces sp. KJ130480
Orpinomyces sp. KJ130473
Piromyces sp. KJ130481
Anaeromycessp.KJ130482
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 24
Unfortunately, various problems including the presence
of divergent ITS sequences within individual isolates
has hampered widespread use of this locus for
taxonomic studies (Ozkose, 2001), though PCR
amplification of DNA from environmental samples
(rumen fluid, digesta etc.) using ITS primers may prove
valuable for ecological studies (Tuckwell et al., 2005).
In conclusion, it was well shown that the applicability
of PCR techniques for the quantification of rumen
anaerobic fungi in the digesta and rumen fluid of
buffalo have provided additionally useful data. The
most reliable method to detect genetic variation
between fungal species is analysis of rDNA that
contains highly conserved DNA sequences as well as
more variable regions. Sequence analysis of ITS1
spacer seems a promising tool for comparing a variety
of rumen fungal isolates. However, molecular
techniques will become useful techniques for rumen
ecology research to manipulate rumen fermentation to
improve ruminant feeding efficiency especially under
conditions of low-quality roughage.
ACKNOWLEDGEMENT
This article is part of Ph.D. thesis (supervisors; Dr.
Mohammad Chamani and Dr. Naser Maheri-Sis and
Advisors; Dr. Mahdi Amin Afshar and Dr. Ramin
Salamat Doust-Nobar) in animal nutrition, Islamic
Azad University, Shabestar Branch. Authors thanks
from Animal Research Centre and Animal Nutrition
Laboratories of Islamic Azad University, Shabestar
Branch, Shabestar, Iran.
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5 mohammad chamani

  • 1. ISSN No. (Print): 0975-1130 ISSN No. (Online): 2249-3239 Genetic Diversity of Gastrointestinal tract Fungi in Buffalo by Molecular methods on the basis of Polymerase Chain Reaction Abbas Mo'azami Goudarzi*, Mohammad Chamani**, Naser Maheri-Sis*, Mehdi Amin Afshar** and Ramin Salamatdoost-Nobar* *Department of Animal Science, Shabestar Branch, Islamic Azad University, Shabestar, IRAN **Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, IRAN (Corresponding author: Mohammad Chamani) (Received 14 December, 2014, Accepted 04 January, 2015) (Published by Research Trend, Website: www.researchtrend.net) ABSTRACT: Buffalo are able to utilize feed more efficiently than beef cattle where the feed supply is of low quantity and or quality. Therefore, the present study was conducted to establish the community structure of anaerobic rumen fungi in buffalo using molecular approaches. Polymerase chain reaction approach was used in this study to determine the population of major anaerobic rumen fungi in buffalo in digesta and rumen fluid. Total community DNA was extracted, and the ribosomal internal transcribed spacer (ITS) 1 region was amplified, cloned, and sequenced. The resulting nucleotide sequences were used to construct a phylogenetic tree. A total of 12 clones were analyzed. Sequence analysis of ITS1 spacer seems a promising tool for comparing a variety of rumen fungal isolates. Key words: Anaerobic rumen fungi, buffalo, ITS1, rRNA, PCR INTRODUCTION The anaerobic gut fungi are the only known obligately anaerobic fungi. For the majority of their life cycles, they are found tightly associated with solid digesta in the rumen and or hindgut. They produce potent fibrolytic enzymes and grow invasively on and into the plant material they are digesting making them important contributors to fibre digestion. This close association with intestinal digesta has made it difficult to accurately determine the amount of fungal biomass present in the rumen, Orpin (1984) suggesting fungal 8% contribution to the total microbial biomass. It is clear that the rumen microbial complement is affected by dietary changes, and that the fungi are more important in digestion in the rumens of animals fed with high-fibre diets (Bauchop, 1979 and Lee et al., 2000). It seems likely that the gut fungi play an important role within the rumen as primary colonizers of plant fibre (Akin et al., 1983). Present knowledge of anaerobic gut fungal population diversity within the gastrointestinal tract is based upon isolation, cultivation and observations in vivo (Davies et al., 1993). It is likely that there are many species yet to be described, some of which may be nonculturable. The development of molecular techniques has greatly broadened our view of microbial diversity and enabled a more complete detection and description of microbial communities (Bauchop, 1979 and Ranjard et al., 2001). The development of molecular biological techniques in the last decade has enabled the new approach to the characterization of fungi. Li and Heath (1992) studied relationship of gut fungi based on ITS1 sequences and found that Anaeromyces isolates were more distant from other rumen fungi. The whole DNA sequences showed above 80% similarity among Piromyces, Neocallimastix and Orpinomyces, whereas the similarities between Anaeromyces and these three genera were only 70% (Fliegerova et al., 2002). Molecular analyses have therefore been used in attempts to clarify the classification of anaerobic fungi. Ribosomal sequences of internal transcribed spacer regions ITS1 and ITS2 (Brookman et al., 2000; Fliegerova et al., 2004) have been applied successfully for discrimination between Orpinomyces and Anaeromyces. Sequencing and subsequent comparison of acquired results with gene-bank data is time consuming. In this research, we try to determine the genetic diversity of the gastrointestinal tract anaerobic fungi in Azarbayejan Iranian buffalos. MATERIAL AND METHODS This research was done in the Department of Animal Science, Shabestar Branch Islamic Azad University in Iran. For the sampling of buffalo rumen, the necessary coordination was carried out by the industrial slaughterhouse of Uromia. Buffalo were slaughtered and samples of rumen contents were taken. Biological Forum – An International Journal 7(1): 20-25(2015)
  • 2. Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 21 Samples of rumen content were collected randomly from rumen in the slaughter house. Finally, after 24-48 hours, the samples were transferred to the laboratory for DNA extractions. Total genomic DNA was extracted by using RBB+C method that described at follow (Yu and Morrison 2004). A. Cell lysis: (a) Transfer 0.25 g of sample into a fresh 2-mL screw- cap tube. Add 1 mL of lysis buffer [500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 4% sodium dodecyl sulfate (SDS)] and 0.4 g of sterile zirconia beads (0.3 g of 0.1 mm and 0.1 g of 0.5 mm). (b) Homogenize for 3 min at maximum speed on a Mini-Beadbeater™ (BioSpec Products, Bartlesville, OK, USA). (c) Incubate at 70°C for 15 min, with gentle shaking by hand every 5 min. (d) Centrifuge at 4°C for 5 min at 16,000× g. Transfer the supernatant to a fresh 2-mL Eppendorf® tube. (e) Add 300 μL of fresh lysis buffer to the lysis tube and repeat steps 2–4, and then pool the supernatant. B. Precipitation of nucleic acids: (a) Add 260 μL of 10 M ammonium acetate to each lysate tube, mix well, and incubate on ice for 5 min. (b) Centrifuge at 4°C for 10 min at 16,000× g. (c) Transfer the supernatant to two 1.5-mL Eppendorf tubes, add one volume of isopropanol and mix well, and incubate on ice for 30 min. (d) Centrifuge at 4°C for 15 min at 16,000× g, remove the supernatant using aspiration, wash the nucleic acids pellet with 70% ethanol, and dry the pellet under vacuum for 3 min. (e) Dissolve the nucleic acid pellet in 100 μL of TE (Tris-EDTA) buffer and pool the two aliquots. C. Removal of RNA, protein, and purification: (a) Add 2 μL of DNase-free RNase (10 mg/mL) and incubate at 37°C for 15 min. (a) Add 15 μL of proteinase K and 200 μL of Buffer AL (from the QIAamp DNA Stool Mini Kit), mix well, and incubate at 70°C for 10 min. (a) Add 200 μL of ethanol and mix well. Transfer to a QIAamp column and centrifuge at 16,000× g for 1 min. (b) Discard the flow through, add 500 μL of Buffer AW1 (Qiagen), and centrifuge for 1 min at room temperature. (c) Discard the flow through, add 500 μL of Buffer AW2 (Qiagen), and centrifuge for 1 min at room temperature. (d) Dry the column by centrifugation at room temperature for 1 min. (e) Add 200 μL of Buffer AE (Qiagen) and incubate at room temperature for 2 min. (f) Centrifuge at room temperature for 1 min to elute the DNA. (g) Aliquot the DNA solution into four tubes. Run 2 μL on a 0.8% gel to check the DNA quality. (h) Store the DNA solutions at -20°C. The quality of the community DNA was assessed by 1% agarose gel electrophoresis. The ribosomal ITS1 region defined by primers Good92F GM1 (5΄- TGTACACACCGCCCGTC-3΄) and GM2 (5΄- CTGCGTTCTTCATCGAT-3΄) as described by Li and Heath (1992). The PCR reaction was performed in 100 μl reactions containing (final concentration): forward and reverse primers, 0.2 μM; dNTPs mixture, 200 μM; MgCl2, 1.5 mM; KCl, 50 mM; Tris/HCl pH 8.4, 10 mM; and Taq polymerase, 0.25 Units. Approximately 50 ng genomic DNA were used as template for each amplification. The temperature conditions were as follows: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 48 °C for 1 min and extension at 72°C for 1.5 min. Final step was carried out at 72°C for 10 min.The PCR products quality was assessed by 0.8% agarose gel electrophoresis and the amplified DNA was purified with a QIAquick PCR purification kit (QIAGEN) according to the manufacturer’s instructions. The DNA was then ligated into the pTG19-T PCR cloning vector system and transformed into competent Escherichia coli (DH5α) cells, before plasmid isolation using a GF-1 Plasmid DNA Extraction Kit. After the plasmid extraction, 15μl of the extracted plasmid was sent to the ShineGene Company of China for sequencing with Universal M13 primers. Sequences from the current study were trimmed manually and analysed by the CHECK_CHIMERA program (Maidak et al., 2001). The similarity searches for sequences were carried out by BLAST (Madden et al., 1996) and alignment was done using CLUSTAL W (Thompson et al., 1997). The phylogenetic analysis was carried out using MEGA software version4 (Tamura et al., 2007) and the phylogenetic relatedness was estimated using the neighbour-joining method and by using the MEGA4 program (Saitou and Nei, 1987). RESULTS AND DISCUSSION Detection of microbes by their DNA requires a sensitive methodology and PCR is commonly used. The ribosomal genes are often chosen as a suitable amplicon for environmental studies as they are multicopy in eukaryotic genomes providing enhanced sensitivity, and can be used for phylogenetic analyses and delimitation of diverse groups of organisms across and between the kingdoms.
  • 3. Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 22 The rDNA genes are particularly well suited for this purpose as they are present in all forms of life and consist of alternating variable and conserved regions. The small subunit rRNA gene sequences, the ITS1 gene in eukaryotes, are conventionally used for phylogenetic comparisons. However, as this gene is more than 97% identical between genera of the anaerobic fungi (Dore and Stahl, 1991) the less conserved internal transcribed spacer (ITS) regions have proven to be more useful (Brookman et.al, 2000). Molecular data has been used to clarify the classification of the anaerobic rumen fungi. Favored indicators of genetic diversity are the rRNA encoding gene sequences, particularly the internal transcribed spacers ITS1, this can be used to identify micro-organisms and to determine pylogenetic relationship within communities, including the rumen fungi (Hausner et. al, 2000 and Vainio and Hantula, 2000). In this research, our purpose is to determine the genetic diversity of the rumen anaerobic fungi in buffalos of the Azerbayjan in Iran. PCR products quality was assessed by 0.8% agarose gel electrophoresis (Fig. 1). Fig. 1. Analysis of PCR products by agarose gel (0.8 %) electrophoresis. The GenBank accession numbers for the sequences determined are: AIB01-1, KJ130471; AIB01-2, KJ130472; AIB01-3, KJ130473; AIB01-4, KJ130474; AIB01-5, KJ130475; AIB01-6, KJ130476; AIB01-7, KJ130477; AIB01-8, KJ130478; AIB01-9, KJ130479; AIB01-10, KJ130480, AIB01-11, KJ130481, AIB01- 12, KJ130482. Table 1 showed Phylotypes of ITS1 gene sequences of anaerobic rumen fungi retrieved from the rumen samples of buffalo. The phylogenetic tree was drawn using the Neighbor-joining method and the MEGA4 software (Fig. 2). The results show that the ITSI sequence is less conserved in one genera and it can have a little differences in a genera or between the different genera. In this study the observed changes were in 1% of the number of ITS1 region nucleotides. Novel groups identified in the fungal ITS data may be assigned to newly defined genera as characterization of isolated strains progresses (Tuckwell et al., 2005). Table 2: Phylotypes of ITS1 gene sequences of anaerobic rumen fungi retrieved from the rumen samples of buffalo. % sequence similarityNearest valid taxonSize (bp) GenBankAccession no.Phylotype 99Caecomyce sp.364KJ130471AIB01-1 100Caecomyces sp.375KJ130472AIB01-2 99Orpinomyces sp.483KJ130473AIB01-3 99Orpinomyces sp.435KJ130474AIB01-4 98Piromyces sp.414KJ130475AIB01-5 98Neocallimastix sp.401KJ130476AIB01-6 97Neocallimastix sp.485KJ130477AIB01-7 96Neocallimastix sp.445KJ130478AIB01-8 97Piromyces sp.426KJ130479AIB01-9 99Orpinomyces sp.432KJ130480AIB01-10 98Piromyces sp.460KJ130481AIB01-11 99Anaeromyces sp.416KJ130482AIB01-12
  • 4. Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 23 Fig. 2. Neighbor-joining phylogenetic tree of aligned ITS1 sequences of anaerobic rumen fungi. The anaerobic gut fungi are eukaryotic organisms and therefore require a different genetic marker to the rumen bacteria for identification and differentiation. The use of 18S rDNA has not been found to be useful in differentiating between the members of the gut fungal family, Neocallimastigaceae, as they are too similar. We have used the more variable, ITS region 1 between the structural genes of the ribosomal repeat as sequences with suitable levels of variability for phylogenetic studies. The ITS regions from the anaerobic gut fungi show length polymorphisms, and there is an approximate relationship between the length of the ITS1 and genera. The paucity of morphological features presents a problem regarding the taxonomy of anaerobic fungi. While examining plant material from the digestive tract, fungi often appear as the complex cluster and this makes the classification even up to genus level difficult. At a time when there is little disagreement as to the status of the six genera, subgeneric classification is problematic since difficulties associated with exchange and long-term maintenance of cultures impeded direct morphological and physiological comparisons among isolates. With the advent of molecular taxonomy, it is hoped that DNA sequence comparisons and phylogenetic reconstruction will elucidate the relatedness of the various taxa. Majority of the sequences deposited relate to the ribosomal RNA genes widely used in phylogenetic reconstruction. The small ribosomal (18S) subunit is highly conserved in different taxa and thus contains little phylogenetically useful information for subgeneric classification (Li and Heath, 1992). In contrast, the internal transcribed spacer (ITS) regions, widely used for study of closely related fungal taxa, show a high level of variability (Li and Heath, 1992; Brookman et al., 2000; Fliegerova et al., 2004), and has been used to differentiate the morphologically similar monocentric (Neocallimastix, Piromyces) and polycentric (Anaeromyces, Orpinomyces) genera. Brookman et al., (2000) also reported that the two multi-flagellated taxa (Neocallimastix, Orpinomyces) were closely related based on the ultrastructure of the zoospores. Neocallimastixsp.KJ130477 Neocallimastixsp.KJ130478 Neocallimastix sp. KJ130476 Caecomyces sp. KJ130472 Piromyces sp. KJ130475 Caecomycessp.KJ130471 Piromycessp.KJ130479 Orpinomycessp.KJ130474 Orpinomyces sp. KJ130480 Orpinomyces sp. KJ130473 Piromyces sp. KJ130481 Anaeromycessp.KJ130482 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
  • 5. Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 24 Unfortunately, various problems including the presence of divergent ITS sequences within individual isolates has hampered widespread use of this locus for taxonomic studies (Ozkose, 2001), though PCR amplification of DNA from environmental samples (rumen fluid, digesta etc.) using ITS primers may prove valuable for ecological studies (Tuckwell et al., 2005). In conclusion, it was well shown that the applicability of PCR techniques for the quantification of rumen anaerobic fungi in the digesta and rumen fluid of buffalo have provided additionally useful data. The most reliable method to detect genetic variation between fungal species is analysis of rDNA that contains highly conserved DNA sequences as well as more variable regions. Sequence analysis of ITS1 spacer seems a promising tool for comparing a variety of rumen fungal isolates. However, molecular techniques will become useful techniques for rumen ecology research to manipulate rumen fermentation to improve ruminant feeding efficiency especially under conditions of low-quality roughage. ACKNOWLEDGEMENT This article is part of Ph.D. thesis (supervisors; Dr. Mohammad Chamani and Dr. Naser Maheri-Sis and Advisors; Dr. Mahdi Amin Afshar and Dr. Ramin Salamat Doust-Nobar) in animal nutrition, Islamic Azad University, Shabestar Branch. Authors thanks from Animal Research Centre and Animal Nutrition Laboratories of Islamic Azad University, Shabestar Branch, Shabestar, Iran. REFERENCES Akin, D. E., Gordon, G. L. R., Hogan, J. P. (1983). Rumen bacterial and fungal degradation of Digitaria pentzii grown with or without sulfur. Applied and Environmental Microbiology, 46: 738–748. Bauchop, T. (1979). Rumen anaerobic fungi of cattle and sheep. Applied and Environmental Microbiology, 38: 148–158. Brookman, J.L., Mennim, G.A., Trinci, P.J., Theodoru, M.K., and Tukwell, D.S. (2000). Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 18S rRNA. Microbiology. 146: 393–403. Davies, D.R., Theodorou, M.K., Lawrence, M.I., and Trinci, A.P.J. (1993). Distribution of anaerobic fungi in the digestive tract of cattle and their survival in faeces. Journal of General Microbiology. 139: 1395–1400. Dore, J., and Stahl, D. A. (1991). Phylogeny of anaerobic rumen Chytridiomycetes inferred from small subunit ribosomal-RNA sequence comparisons. Canadian Journal of Botany. 69: 1964–1971. Fliegerova, k., Paioutova, S., Mrazeki, J., and Kopecny, J. (2002). Special Properties of Polycentric Anaerobic Fungus Anaeromyces mucronatus. Acta Veterinary Brno. 71: 441–444. Fliegerova K., Hodrova, B., and Voigt, K. (2004). Classical and molecular approaches as a powerful tool for the characterization of rumen polycentric fungi. Folia Microbiology. 49: 157–164. Hausner, G., Inglis, G., Yanke, L.M., Kawchuk, L.M., and McAllister, T.A. (2000). Analysis of restriction fragment length polymorphisms in the ribosomal DNA of a selection of anaerobic chytrids. Canadian Journal of Botany. 78: 917–927. Lee, S.S., Ha, J.K., and Cheng, K.J. (2000). Relative contributions of bacteria, protozoa, and fungi to in vitro degradation of orchard grass cell walls and their interactions. Applied and Environmental Microbiology. 66: 3807– 3813. Li, J., and Heath, J.B. (1992). The phylogenetic relationship of the anaerobic chytridiomycetous gut fungi (Neocallimasticacea) and the Chytridiomycota. I. Cladistic analysis of rRNA sequences. Canadian Journal of Botany. 70: 1738-1746. Madden, T.L., R.L. Tatusov, and J. Zhan (1996). Application of network BLAST server. Methods in Enzymology. 266: 131–141. B.L., Maidak, Cole, J.R., Lilburn, T.G., Parker, C.T.J.R., Saxman, P.R., Farris, R.J. Garrity, G.M., Olsen, G., Schmidt, T., and Mvand Tiedje, J.M. (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Research. 29: 173–174. Orpin, C.G. (1984). The role of ciliate protozoa and fungi in the rumen digestion of plant cell walls. Animal Feed Science and Technology. 10: 121–143. Ozkose, E. (2001). Morphology and molecular ecology of anaerobic fungi. Ph.D. dissertation, University of Wales Aberystwyth.
  • 6. Goudarzi, Chamani, Maheri-Sis, Afshar and Salamatdoost-Nobar 25 Ranjard, L., Poly, F., Lata, J.C., Mougel, C., Thioulouse, J., and Nazaret, S. (2001). Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: biological and methodological variability. Applied and Environmental Microbiology. 67: 4479–4487. Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4: 406–425. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution. 24: 1596– 1599. Thompson, J.D., Gibson, T.J. Plewniak, F., Jeanmougin, F., and Higgins, D.G. (1997). The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research. 25: 4876–4882. Tuckwell, D.S., Nicholson, M.J., McSweeney, C.S., Theodorou, M.K., and Brookman, J.L. (2005). The rapid assignment of ruminal fungi to presumptive genera using ITS1 and ITS2 RNA secondary structures to produce groupspecific fingerprints. Microbiology. 151: 1557–1567. Vainio, E.J., and Hantula, J. (2000). Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycological Research. 104: 927–936. Yu, Z.T., and Morrison, M. (2004). Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechnology. 36: 808–812.