| Research Article |
Open Access |
|
| Isolation of Nucleotide Binding Site (NBS)-Leucine Rich Repeat (LRR)
Resistant Gene Analogs (Rgas) In Arabica Coffee (Coffea Arabica L. Cv
S.288) |
| Deepak Kumar* and H.L. Sreenath |
| Plant Biotechnology Division, Coffee Board, Unit of Central Coffee Research Institute (CCRI), Dr. S. Radhakrishnan Road, Manasagangothri, Mysore-570 006, Karnataka,
India. |
| *Corresponding author: |
Deepak Kumar
Plant Biotechnology Division,Coffee
Board
Unit of Central Coffee Research Institute (CCRI)
Dr. S. Radhakrishnan Road,Manasagangothri
Mysore-570 006, Karnataka,India
Tel:+91-8971703863
E-mail: deepakkumardeo@gmail.com |
|
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| Received June 18, 2012; Accepted August 08, 2012; Published August 11, 2012 |
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| Citation: Deepak Kumar, Sreenath HL (2012) Isolation of Nucleotide Binding
Site (NBS)-Leucine Rich Repeat (LRR) Resistant Gene Analogs (Rgas) In
Arabica Coffee (Coffea arabica L. Cv S.288). J Biotechnol Biomater 2:146.
doi:10.4172/2155-952X.1000146 |
| |
| Copyright: © 2012 Deepak Kumar , et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. |
| |
| Abstract |
| |
| Cloning of resistance gene analogues against diverse pathogens from variety of plants in last decade has
revealed that many of them share high level of conserved sequence motifs. The conserved backbone of amino acid
motifs present in Nucleotide Binding Site (NBS) domain makes it possible to isolate resistance gene analogues
by Polymerase chain reaction (PCR) with degenerate primers. Oligo-nucleotide primers combinations that target
conserve motif of NBS domain as mentioned in earlier studies were used to amplify resistance gene analogues from
Coffea arabica (S.288). PCR product amplified from genomic DNA as well as cDNA were cloned and sequenced. In
the present study amplified resistance gene analogues from C.arabica genomic DNA and cDNA using Ploop-cof and
GLPL-cof primers were cloned, sequenced using T7 / SP6 primers, analyzed at NCBI/SGN Nucleotide Data Bank.
Analysis of these RGA leads to the understanding that difference in expression profile might be due to the difference
present at sequence level of Resistant Gene Analogs (RGA) isolated from DNA and cDNA. Analysis also revealed
presence of high level similarity at their sequences. Seven RGA isolated from genomic DNA of S.288 using nondegenerate
primers, eleven more RGA were isolated from S.288 genomic DNA with degenerate oligo-nucleotide
primers and thirty two RGA isolated from cDNA of S.288. Fifteen RGA clones isolated from cDNA prepared from rust
race I infected leaf sample for 24 hours. BLASTN result showed these C.arabica RGA has a high level of similarity
with C.canephora RGA. This confirm the integrity maintained among RGA even it was isolated from different coffee
variety which has difference at there genome (C.arabica 2n= 44 and C.canephora 2n= 22). RGA isolated which has
below 475 bp or above 530 bp in size along with both primer sequences in their end has either no match with any
RGA or they match with microsatellite. There is one independent sequence from genomic DNA and four independent
sequences from cDNA which has not given any BLASTN result, these sequences has primer sequence with them
that indicates these may be belong to new type of RGA. The RGA reported in current study are mainly from the class
A type of RGA. |
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| Keywords |
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| Nucleotide Binding Site (NBS); Leucine Rich Repeat
(LRR); Resistant Gene Analogs (Rgas); Coffea arabica. |
| |
| Introduction |
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| Coffee is one of the important beverage crops for Global point
of view. Coffee is a stimulating non-alcoholic beverage, which earns
a substantial amount of foreign exchange to India. Coffee belongs to
the genus Coffea, family Rubiaceae. The bean of the crop is used to
prepare drinking coffee. Commercially arabica and robusta are the two
important coffee species where arabica produces high quality beverage
and originated in southwestern Ethiopia (Kaffa region -centre of
diversity). Coffee is the second largest traded commodity in the world,
next only to petroleum. About 3,40,306 ha of area is covered with coffee
in India producing 4372000 bags and exported 3145000 bags in the year
of 2008/09 earnings of US $395.04 million [1]. The disease affecting
coffee are many, among them major disease of economical importance
are leaf rust caused by Hemieleia vastatrix. Hemieleia vastatrix is
a fungus belongs to the Phylum Basidiomycota, order Uredinales.
Parasitic in nature to plant causes serious damage to the crop. |
| |
| Disease resistance genes from various plant species have been
cloned and sequenced. Some of them are distinguished by the presence
of N-terminal nucleotide binding site (NBS) and C-terminal stretch
of leucine-rich repeats (LRR). Although these gene products are
structurally related, but DNA sequences are poorly conserved in the
case of Arabidopsis [2]. Nucleotide binding sites (NBS) domains related
to R-genes show a highly conserved backbone of amino-acid motifs,
which makes it possible to isolates resistance gene analogues (RGAs) by
PCR with low degeneracy primers. The amino-acid sequence of coffee RGAs were identified, that showed strong sequence similarity to almost
all known non-TIR (Toll/Interleukin1 receptor)-type R-genes. The
high degree of similarity between particular coffee RGAs and R-genes
isolated from other angiosperm species, such as Arabidopsis, tomato
and rice indicates an ancestral relationship and existence of common
ancestors [3]. |
| |
| Part of nucleotide binding site has enough DNA identity to design
primer for the polymerase chain reaction to amplify some of these
regions which is related to the DNA sequences. Such primers are
used to get certain resistance gene like (RGL) DNA fragments from
Arabidopsis thaliana. |
| |
| Most of these RGL DNA fragments were found in a clustered or
dispersed multi-copy sequence organization. R-genes that have been
molecularly characterized so far can be grouped into classes based on similarities in the function or amino acid sequence of the proteins they
encode. Examples are: RPS2 [4,5], RPM1 [6], RPP5 [7], L6 gene from
flax. |
| |
| These R-genes are found in the diverse plant species and the
conserved NBS-LRR features suggest a common function in the
defence response against pathogen attack, probably as part of the
signal transduction pathway. Knowledge about the genes involved
in disease resistance in plants speed up the works carried out against
plant diseases. The gene products of some of these can be distinguished
by the presence of an N- terminal nucleotide-binding site and a
C-terminal stretch of leucine rich repeats. The NBS sequences of R
genes are characterized by the presence of up to seven conserved
domains including the Ploop, Kinase2 and GLPL motifs [8] (Figure 1).
The presence of these conserved domains has facilitated the cloning
of resistance gene analogues from diverse species by PCR using
degenerate oligo nucleotide primers. Thus the identification of RGAs represents a potentially powerful strategy for the generation of markers
for map based cloning of resistance genes. |
| |
|
Figure 1: Schematic model of the structure of nucleotide binding site- leucine
rich repeat (NBS-LRR) type resistance genes. Relative positions of the
degenerate primers Ploop, Kinase 2, GLPL and MHD, designed to conserved
domains within the NBS. |
|
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| Nine distinct classes of RGAs of the NBS-like type, representing
a highly diverse sample, were isolated from Coffea arabica and Coffea
canephora species. Coffee RGA family suggests point mutation as
the primary source of diversity. Coffee RGA family appeared to be
closed related in sequence to cloned R-gene [3]. Coffee RGAs amino
acid sequence showed strong sequence similarity to almost all known
non-TIR (Toll/Interleukin I Receptor) type R-genes. High degree
of similarity between coffee RGAs and R-genes isolated from other
angiosperm species, such as arabidopsis, tomato and rice indicates an
ancestral relationship. The data obtained from coffee species suggests
that the evolution of NBS-encoding sequence involves the gradual
accumulation of mutations and slow rates of divergence within distinct
R-gene families. |
| |
| The majority of the studies that utilize the PCR-based method use
genomic DNA as the template. Although a large number of RGAs are
amplified in such studies, many of the identified RGAs are probably
pseudo genes [9] or non-functional genes. RGAs amplified from
cDNA are, on average, more likely to be functional R genes than
those amplified from genomic DNA, since the transcription of
non-functional genes would be associated with an overall fitness cost to
the plant. However, only very few reports of PCR-based amplification
of RGAs from cDNA template were found [10,11]. |
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| We have taken up a study for isolation of RGAs in Coffea arabica
Cv. S.288 with the objective of getting molecular markers linked to
Coffee Leaf Rust resistance. |
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| Materials and Methods |
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| Plant materials |
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| C. arabica S.288 plants were maintained in the nursery of Plant
Biotechnology Centre, Mysore. |
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| Bacterial strains |
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| Cloning of the RGA fragments obtained from genomic DNA was
carried out in E. coli strain DH 5α whereas the strain JM109 was used
to clone RGA fragments amplified from cDNA. |
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| Primers used for the study |
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| We used 16 pairs of degenerate primers combinations and two pair
of non degenerate primer on genomic DNA and cDNA sample (Table
1). |
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|
Table 1: Primers details. |
|
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| Genomic DNA Isolation |
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| Leaf sample of C. arabica S.288 was washed thoroughly with tap
water and, rinsed with sterile distilled water. The leaf was wiped dry with
tissue paper and ground to fine powder with a mortar and pestle using
liquid nitrogen. Around 100 mg of powdered leaf was used to isolate
genomic DNA using CTAB extraction buffer. RNA contamination was
removed by RNAase treatment. |
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| For RGA isolation from Ploop 4 and GLPL 4, genomic DNA
was isolated by DNeasy Plant Mini Kit (Qiagen). DNA isolation
protocol was followed as per the manufacturer instruction. RNAase A
(Pancreatic RNAase A Bangalore Genei), treatment was also performed
according to the instruction provided. |
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| DNA Quantification |
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| DNA was quantified using spectrophotometer (Beckman DU 640
B). |
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| RNA Isolation and Purification of mRNA |
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| For RNA isolation young green leaves of S.288 plant, was detached
from the plant immediately frozen in liquid nitrogen and ground to
fine powder using pre-chilled mortar and pestle (DEPC treated and
baked). RNA extraction was performed using Qiagen RNeasy kit
according to manufacturer’s protocol with modification required for
Coffee leaf tissue. |
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| RNA Quantification |
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| RNA was quantified by spectrophotometer (Beckman DU 640B).
Poly-A mRNA was isolated using mRNA purification kit (Bangalore
Genei) according to users manual. |
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| First Strand cDNA Synthesis |
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| Complementary DNA was prepared by using Bangalore Genei
cDNA synthesis kit as per the manufacturer’s protocol. The reaction
was stopped by incubation at 92oC for 2 minutes. The cDNA was
snapped chilled on ice and used as template for PCR reaction. |
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| PCR Amplification of RGAs |
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| Oligonucleotide primers which were used for the present study
designed based on the Ploop and GLPL motifs of existing RGA’s. The
non degenerate primers and degenerate primers were used for isolation
of RGA fragments from genomic DNA and cDNA of C. arabica S.288.
PCR amplification was performed in a 50 μl reaction volume on a
PTC 200 thermal cycler (MJ research). Either 100 ng of first strand leaf cDNA or 60 ng of genomic DNA was used as template for the
PCR amplification in a reaction mixture composing Taq Polymerase
assay buffer 1X final concentration (Bangalore Genei), 1 μM of each
primer, 400 μM dNTP (10 mM), 1.5 mM MgCl2 and 3 Units Taq DNA
polymerase (Bangalore Genei). The PCR program parameters include
initial denaturation at 94°C for 5 minutes followed by 35 cycles of
denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds,
and elongation at 72°C for 1 minute 30 seconds and finally extension at
72°C for 10 minutes. The PCR amplified products were size fractionated
on 1% of agarose gel in 1X TBE buffer under constant voltage of 80V
for 45 minutes. |
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| Cloning of RGA Fragments and Screening of Clones |
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| The DNA bands were cut from the gel using razor and extracted
by Gel Extraction Kit (Qiaquick) as instructed by the manufacturer
instruction. The TA based cloning system (pGEM-T Easy TA cloning
kit, Promega) was used to clone the RGA fragments according to the
user’s guidelines in 1:2 ratio. Ligation was performed in 20 μl reaction
volume overnight at 16°C using 3 units of T4 DNA ligase and 1X of
buffer provided with the kit. |
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| Blue white colonies appear which was counted for the efficiency
of transformation. High transformation efficiency 8 x 107 indicates the
successive ligation reaction and transformation. Single/independent
round white colonies were picked up from the transformation
petriplate using a sterile loop/tooth pick and re inoculated on selection
plate containing Ampicilline, IPTG and X-gal. Those clones retain the
white colour were used to inoculate 3 ml of LB medium supplemented
with antibiotic. Overnight grown cultures were pelleted down by
centrifuging at maximum speed for 1 minute. Plasmid DNA was
isolated from the cells using the Qiagen Mini-Prep Kit according to users manual. PCR was performed with RGA/M13 primers to amplify
them from DNA/cDNA were employed to screen positive clones. The
PCR was conducted in similar program conditions as described before.
The amplified product from the selected clone plasmid were analysed
on 1% agarose gel. Those clones which retain the desired fragment used
for cloning were picked and sent for sequencing. These clones were
properly numbered. Glycerol stock prepared with 15% glycerol and
preserved at -80°C. |
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| The positive clones carrying the RGA fragments were sequenced
using the T7/Sp6 Universal sequencing primer. RGA amplified
fragment size of 450bp to 550bp range were selected and sequenced.
Sequences were edited using DNA STAR/NCBI software for vector
contamination. |
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| Sequence submitted to NCBI nucleotide database |
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| All RGA sequences identified have more than 200bp sequence
length is submitted at NCBI database. The details are provided in Table
2. |
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|
Table 2: Coffea arabica RGA sequence (>200bp) submitted to NCBI Gene Bank database details. |
|
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| Results |
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| RGA cloning using degenerate primers |
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| Genomic DNA Isolation |
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| Genomic DNA isolated from S.288 using CTAB method has yielded
50 μg of genomic DNA from 100 mg of leaf tissue. CTAB method
has yielded DNA with RNA contamination. RNAase treatment has successfully removed RNA contamination. |
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| RGA amplified from genomic DNA using degenerate primers
has yielded desire fragment of 500 bp with only one set of primer
combination Ploop4 and GLPL4. Most of the other combination failed
to amplify, only combination of Ploop2 with GLPL1, Ploop2 with
GLPL2, Ploop2 with GLPL3, Ploop2 with GLPL4, Ploop4 with GLPL1,
Ploop4 with GLPL2, Ploop4 with GLPL3 and Ploop4 and GLPL4. Out
of these eight combinations only one set of primer Ploop4 and GLPL4
has yielded desire 500 bp fragments without any unspecific fragment
therefore used for cloning. Although Ploop2 with GLPL4 and Ploop4
with GLPL2 has yielded desire 500 bp amplified fragment with some
un-specific fragment, not used for cloning. Surprisingly as mentioned
by Noir et al. (2001) work says that Ploop4 and GLPL4 combination
has not yielded any RGA. Therefore it was interesting to clone and
study these fragments. |
|
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| Other RGA primer combination of Int-Cof primers and twelve
degenerate primers F1 to F6 forward and HDR1 to HDR6 reverse tried
with S.288 DNA has failed to amplify in any of its 36 combination tried.
This may be the primer sequence did not have best annealing with
coffee genomic DNA /cDNA sequence of S.288 at NBS region. Out of
16 degenerate primers along with Ploop 4 and GLPL 4 primers were
also used on genomic DNA of S.288 to isolate RGAs. Out of 16 pairs of
degenerate primer combinations used on genomic DNA, only one pair
(Ploop 4 and GLPL 4) resulted into expected size amplified products. |
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| RGA cloning using non-degenerate primers |
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| RGA amplified from genomic DNA |
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| RGA amplified from genomic DNA using non-degenerate primers has yielded desire fragment of 500 bp. Some unspecific DNA fragment
also amplified which was not used for cloning. |
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| RGA amplified from cDNA |
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| Total RNA Isolation |
| |
| Approximately 35 to 40 μg of RNA was estimated from 100 mg
of S.288 leaf tissue. The RNA quality was found good as indicated by
agarose gel separation. mRNA was purified and quality was checked
on agarose gel. |
| |
| RGA amplified from cDNA using non-degenerate primers has
yielded desire fragment of 500 bp. Some unspecific DNA fragment also
amplified which was not used for cloning. |
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| Cloning |
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| Cloning of above amplified RGA fragments were electroeluted from
the agarose gel and quantified small quantity on agarose gel. Cloning
was performed and positive clones were screened first on blue white
selection plates with high transformation efficiency 8 x 107 indicates
the successive ligation reaction, second screening with re-patching
on selection plates, and third screening by isolation of plasmid from
selected clones and amplification with M13 universal primer which
amplify the region of plasmid containing cloned insert. |
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| Sequence analysis |
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| Sequences of these clones were analyzed as mentioned above and
discussed in the next section (Table 3, 4, 5). |
| |
|
Table 3: Details of NCBI BLASTN sequence results of RGAs isolated from genomic DNA using non degenerate primers. |
|
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Table 4: Details of NCBI BLASTN sequence results of RGAs isolated from genomic DNA using degenerate primer. |
|
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Table 5: Details of NCBI BLASTN sequence results of RGAs isolated from cDNA using non-degenerate primer. |
|
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| Comparison of RGAs with known resistance genes |
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| Only those sequences which have respective domains of Ploop
(GVGKTT) and GLPL (GLPLAL) at both sides, compared with
Arabidopsis thaliana disease resistance protein RPP8 (Accession No.
AF089710.1), Capsicum chacoense disease resistance protein BS2
(Accession No. AF202179.1), Lycopersicon esculentum resistance
complex protein I2C-1 (Accession No. AF004878.1), Arabidopsis
thaliana NBS/LRR disease resistance protein RFL1 (Accession No.
AF074916.1), Arabidopsis thaliana Col-0 resistance to Pseudomonas syringae RPS2 (Accession No. U12860.1), Lactuca sativa resistance protein candidate RGC2B (Accession No. AF113948.1), Linum usitatissimum alternatively spliced rust resistance L6 gene (Accession
No. U27081.1), Linum usitatissimum rust resistance protein M gene
(Accession No. U73916.1), Arabidopsis thaliana disease resistance
protein RPP1-WsA gene (Accession No. AF098962.1) and resistance
genes NBS region. Phylogenetic tree was constructed to observe the
distance with existing R genes (Figure 2 ). |
| |
|
Figure 2: Phylogenetic Tree constructed with other known resistance genes
for(A) RGAs isolated using degenerate primers Ploop4 and GLPL4 from
genomic DNA (B) RGAs isolated using non-degenerate primers Ploop-Cof and
GLPL-Cof from cDNA (C) RGAs isolated using non-degenerate primers Ploop-
Cof and GLPL-Cof from genomic DNA. |
|
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| Discussion |
| |
| Sequence of all these RGA clones were analyzed at NCBI database
for vector contamination at (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Edited sequence of clones were analyzed at National Centre for
Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/) and
sol genomic network (SOL) (http://solgenomics.net/) data bases for
sequence homology of the isolated clones using the BLAST (Basic Local
Algorithms Sequence Tools). BLASTN sequence analyzed data was
tabulated, which explains the homology with resistance like proteins.
Sequence analysis was performed using DNA Star software. Duplicate
sequences, which have primers at both sides, were taken as single
sequence for further analysis. Sequences which have primer sequence
deletion for single nucleotide were corrected using DNA Star software.
All sequences were analyzed at SeqMan (DNA Star) to find the contig
and SNP’s present. |
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| RGA isolation from genomic DNA using degenerate primer |
| |
| RGAs were tried to isolate using 16 combination of degenerate
primers used on genomic DNA, only one pair (Ploop4 with GLPL4)
resulted in the amplified product of expected size. Eleven randomly
selected clones were sequenced and analyzed. Out of these 11 clones,
8 showed homology to RGAs. Size of these clones varied from 507 to
531 bp, indicating that they are related to disease resistance genes. The
remaining 3 clones did not have significant homology to any RGA or
any known sequence in both data bases searched. The BLASTN result
details were shown at (Table 4). CBC 185 which is 528 bp, CBC 188
which is 531 bp, CBC 191 which is 530 bp has 99% matches with Coffea
RGA clone accession number EF566686.1. CBC 186 which is 525 bp clone has 96% homology with Coffea arabica partial ORF for disease
resistance like protein accession number AJ298884.1. CBC 189 which
is 530 bp clone has 99% homology with Coffee RGA clone putative for
NBS domain resistance protein gene accession number EF566680.1.
Similarly CBC 193 which is also a 527 bp clone has 98% homology
with Coffea arabica partial ORF for disease resistance like protein
(NBS clone) accession number AJ298884.1. These sequence multiple
alignment comparison suggest different range of RGA class. |
| |
| These clones sequence were analysed for duplication and to search
SNP present in their sequence. CBC 188 and CBC 189 are duplicate
clones having identical sequence data, therefore CBC 188 taken
for the studies. After assembled these sequences in SeqMan DNA
Star six contig found. First contig containing 4 sequences CBC 183,
CBC 185, CBC 191 and CBC 188. CBC 185, CBC 188 and CBC 191
have difference in first and last 25 bp positions. CBC 185 has single
nucleotide polymorphism (SNP) at sequence position 228. Similarly
CBC 188 has SNP at position 249 bp and 462 positions. CBC 191 has SNP present at sequence position 251 bp. Second contig contain two
sequences CBC 186 and CBC 193. The other contigs have one sequence
each, Contig 3 has CBC 187, Contig 4 has CBC 184, Contig 5 has CBC
192 and Contig 6 have CBC 190. |
| |
| The other set of degenerate primer used, failed to give any
amplification with coffee DNA suggests there is difference in the
sequence of primer binding region. This made the primers all 36
combinations un-usable with coffee samples. |
| |
| RGAs isolated from genomic DNA using non-degenerate
primer |
| |
| RGAs isolated from genomic DNA using Ploop-Cof and GLPLCof
primer were sequenced and analyzed. Out of seven clones (CBC
7, CBC8, CBC 138, CBC 139, CBC140, CBC 141, CBC142 and CBC
143) searched; five clones sequence has a match with RGAs. BLASTX
for protein database search indicate all these clones have one class of
RGA. NCBI BLASTN data were tabulated (Table 3), which indicates that most among the five have 98% match with C.arabica/C.canephora disease resistance like protein. Clones CBC7 which is 500bp, CBC
139 which is 486bp , CBC141 which is 523 and CBC 143 which is
486bp has 98% match with the Coffea arabica partial ORF for disease
resistance-like protein, accession number AJ298884.1. These clones
were BLASTX at Gene bank protein database had a match with disease
resistance like protein accession number CAC82597.1. CBC 142 the
only sequence has 98% identity with accession number AY606825.1
at nucleotide database and accession number CAC82609.1 at protein
data base which is disease resistance-like protein identified from Coffea
canephora. Although CBC 8 which is 340bp a short fragment clone has
matched with Coffea arabica microsatellite DNA. Multiple alignment
comparison of these RGA sequences indicates that they share similarity
to class A of disease resistance NBS-LRR genes. CBC 140 clone sequence
which has expected size DNA fragment insert of 528 bp but failed to
match with any known sequence at both database. Only those sequence
has complete sequence were included for further studies. |
| |
| To look for contigs among these sequences were analyze with
SeqMan DNA Star. After removing the duplicate clone sequences,
which have both primers sequences were analyze for contigs. CBC
140 and CBC 142 have identical sequence therefore CBC 140 taken for
study which will represent for CBC 142. Similarly out of CBC 139 and
CBC 143 which are identical sequence, only CBC 139 taken for studies
which will represent for CBC 143. CBC 7 and CBC 141 have difference
at sequence at position 199, and they also have single nucleotide
addition at position 502. Therefore CBC7, CBC 139, CBC 140 and
CBC 141 were assembled and found two contigs. First contig have
three sequences CBC 7, CBC 140 and CBC 141. CBC7 and CBC 141
has almost identical sequence except for SNP present at position 199,
and one nucleotide addition present in CBC 141 which is missing in
CBC 7 at sequence position 506. Second contig has only one sequence
CBC 139. |
| |
| RGAs isolated from cDNA using non-degenerate primer |
| |
| RGAs isolated from cDNA prepared with RNA of Coffea arabica
Cv S.288 to understand the expression profile. The results were
shown with best NCBI/ SGN nucleotide BLAST at (Table 5). RGAs
were isolated using Ploop-Cof and GLPL-Cof primers from cDNA
first strand. Amplified product of desire range were sequenced and
analyzed at NCBI/SGN database. Thirty two randomly picked clones
were sequenced and analyzed. Out of 32 clones, 22 clones showed
homology with RGAs, size of clones varied in the range of 480 bp to
525 bp. Out of these 22 clones, 18 (CBC 9 to 19, CBC 158, 160, 165, 166,
168, 169 and 172) clones sequence have homology with Coffea species
and other 4 (CBC 133, 134, 135, 136) clones sequence have homology
with other plant species RGAs. The remaining clones among them 5
(CBC 163, 164, 167, 170,171) have homology with coffee sequences but
they are not shown any homology with RGAs. Four clones sequence
(CBC 138, 159, 161,162) data is short read therefore not included in
studies. One clone (CBC 137) has homology with other plant species
which is also not included in studies. CBC 9, CBC 10, CBC 160, CBC
165, CBC 166 and CBC 172 have 98% homology with Coffea arabica,
disease resistance like protein accession number AJ 298884.1. CBC
11, CBC 12, CBC 13, CBC 14, CBC 15, CBC 16, CBC 17, CBC 18 and
CBC 19 have 92% homology with Coffea arabica, disease resistance
like protein accession number CAC82597.1. CBC 133, CBC 134 and
CBC 136 which are 330 bp clones have 100% homology with Cucumis
melo mRNA-3 gene for NBS-LRR type resistance protein accession
number AB191232.1. CBC 168 and CBC 169 which are 263 and 425 bp
respectively have 96% homology with Coffea arabica disease resistance like protein accession number AY606825.1. CBC163, CBC 167 and CBC
171 which are clones of 427 bp, 590 bp and 389 bp sizes respectively
have 92% homology with Coffea canephora sequence accession number
SGN-U353589. These clones have primer sequence in the both side
which indicate the full clone sequence but not the expected size. CBC
135 which is 362 bp clone has 100% homology with Musa acuminata NBS-LRR resistance protein-like accession number AY739270.1.
CBC 137 which is 410 bp clone has 78% homology with Myxococcus
xanthus DK 1622, complete genome accession number CP000113.1.
CBC 164 which is 427 bp clone has 98% homology with Coffea arabica
microsatellite accession number AY102435.1. CBC 136 which is 330 bp
clone has 100% homology with Cucumis melo mRNA-3 gene for NBSLRR
type resistance protein accession number AB191232.1. CBC 158
which is 513 bp clone has 93% homology with Coffea canephora disease
resistance like protein accession number AY 606826.1. CBC 167 which
is 590 bp clone has 92% homology with Coffea canephora accession
number SGN-U353389. |
| |
| These clone sequences were analyze at SeqMan DNA Star and
found eight contigs. First contig contains nine sequences CBC 10, CBC
11, CBC 14, CBC 15, CBC 16, CBC 17, CBC 19, CBC 160 and CBC 165.
These sequences have SNP’s at position 66, 91, 123, 139, 144, 200, 236,
246, 277, 285, 350, 351, 368, 375, 376, 468 and 495 sequence positions. |
| |
| Contig 2 has three sequences CBC 134, CBC 133 and CBC 136.
These sequences have 100 % identity with each other, therefore CBC
133 sequence taken for studies. Now contig 2 has only one sequence as
CBC 133. Contig 3 and contig 4 has one sequence each as CBC 158 and
CBC 171 respectively. On the other hand Contig 5 has two sequences
as CBC 163 and CBC 164. Contig 5 sequences have some insertion/
deletion of sequences. Contig 6, 7, 8 have one sequence each as CBC
167, CBC 137 and CBC 135 respectively. |
| |
| Comparison of RGAs with known resistance genes |
| |
| RGAs sequences were compared with existing R genes. RGAs
phylogenetic tree analysis suggest that these RGAs are close to
existing Arabidopsis thaliana disease resistance protein RPP8,
Capsicum chacoense disease resistance protein BS2 and Lycopersicon
esculentum resistance complex protein I2C-1. TIR and non TIR-NBSLRR
sequences are distinguishable by amino acid motifs internal
to their NBS domains. While motifs such as the Ploop, Kin-1a, and
GLPLA signature are present in both classes, motifs RNBS-A-TIR
(LQKKLLSKLL) and RNBS-D-TIR (FLHIACFF) are found exclusively
in TIR class, while RNBS-A-non TIR (FDLxAWVCVSQxF) and RNBSD-
nonTIR (CFLYCALFPED) are found in the non-TIR class (Meyers
et al. 1999). The RGAs sequences obtained in current study were
analyzed for the respective domains for identity of TIR and non-TIR
class. Based on these, it may be concluded that all the RGAs isolated in
this study belongs to nonTIR sub class. Sreenath et al. 2002 and Noir et
al. 2001 also reported in earlier studies for nonTIR RGAs from coffee. |
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| Acknowledgements |
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| The authors are grateful to Dr. Jayarama, Director of Research, Central
Coffee Research Institute, for carrying out this work. Author is grateful to U.G.C.
for providing the fellowship. |
| |
|
| References |
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