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 non-degenerate 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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Claims 11
Claim · model-written summary
Seven RGA were isolated from S.288 genomic DNA using non-degenerate primers.
- Taxon
- Coffea Arabica
Coffea arabicat0 - Material
- S.288e1
- Treatment
- non-degenerate primerse1
- Outcome
- RGAe1
- Value
- Sevene1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e1“Seven RGA isolated from genomic DNA of S.288 using non-degenerate primers”
Claim · model-written summary
Eleven more RGA were isolated from S.288 genomic DNA using degenerate primers.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Material
- S.288e2
- Treatment
- degenerate oligo-nucleotide primerse2
- Outcome
- RGAe2
- Value
- elevene2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e2“eleven more RGA were isolated from S.288 genomic DNA with degenerate oligo-nucleotide primers”
Claim · model-written summary
Thirty-two RGA were isolated from cDNA of S.288.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Material
- cDNA of S.288e3
- Outcome
- RGAe3
- Value
- thirty twoe3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e3“thirty two RGA isolated from cDNA of S.288”
Claim · model-written summary
Fifteen RGA clones were isolated from cDNA of rust race I infected leaves sampled 24 hours after infection.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Organ / stage
- leafe4
- Treatment
- rust race I infected leaf sample for 24 hourse4
- Interactor
- rust race Ie4pathogen
- Outcome
- RGA clonese4
- Value
- Fifteene4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e4“Fifteen RGA clones isolated from cDNA prepared from rust race I infected leaf sample for 24 hours”
Claim · model-written summary
C.arabica RGA showed a high level of similarity with C.canephora RGA by BLASTN.
- Taxon
- C.arabicaCoffea arabicae5
- Outcome
- high level of similaritye5
- Comparator
- C.canephora RGAe5
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e5“BLASTN result showed these C.arabica RGA has a high level of similarity with C.canephora RGA”
Claim · model-written summary
RGA of length below 475 bp or above 530 bp had either no match with any RGA or matched microsatellites.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Outcome
- RGAe6
- Value
- below 475 bp or above 530 bpe6
- Qualifier
- has either no match with any RGA or they match with microsatellitee6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e6“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”
Claim · model-written summary
One independent sequence from genomic DNA gave no BLASTN result.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Outcome
- independent sequencee7
- Value
- onee7
- Qualifier
- which has not given any BLASTN resulte7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e7“There is one independent sequence from genomic DNA and four independent sequences from cDNA which has not given any BLASTN result”
Claim · model-written summary
Four independent sequences from cDNA gave no BLASTN result.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Outcome
- independent sequencese7
- Value
- foure7
- Qualifier
- which has not given any BLASTN resulte7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e7“There is one independent sequence from genomic DNA and four independent sequences from cDNA which has not given any BLASTN result”
Claim · model-written summary
Some of the unmatched sequences may belong to a new type of RGA.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Outcome
- new type of RGAe5
- Qualifier
- primer sequence with them that indicatese5
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e5“these sequences has primer sequence with them that indicates these may be belong to new type of RGA”
Claim · model-written summary
The RGA reported in this study are mainly of the class A type.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Outcome
- RGAe6
- Qualifier
- mainly from the class A typee6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e6“The RGA reported in current study are mainly from the class A type of RGA”
Claim · model-written summary
A difference in expression profile might be due to sequence-level differences between RGA isolated from DNA and cDNA.
- Taxon
- Coffea ArabicaCoffea arabicat0
- Outcome
- difference in expression profilee7
- Qualifier
- difference present at sequence levele7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“(Coffea Arabica L. Cv S.288)”
e7“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”