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CHAPTER 4: RESULTS
4.1. Population genetic structure of turmeric (Curcuma longa) and its response to
Pythium aphanidermatum
4.1.1. Rhizome rot infection analysis
The statistical analysis on disease intensity showing the relative reaction of 62 turmeric
genotypes across 11 populations in response to Pythium aphanidermatum revealed large
and significant variation in the degree of disease incidence in the overall distribution of
the number of plants in all turmeric accessions within the 11 populations. However, no
significant difference was observed between isolates over the disease index scores
(Table 4.1). The two isolates were found equally aggressive or ineffective exhibiting
similar disease index (DI) score for any given isolate-accession combination. The
aggressiveness of each isolate was very much dependent on specific isolate accession
combinations.
Total 4.1: Analysis of variance for the distribution over percentage of infection, testing the
effect of turmeric genotypes across 11 populations and Pythiumapanidermatum isolates
Source df Sum of squares Mean squares F
Between isolates 1 108.98 108.98 0.0812
Between populations 10 52943.86 5294.39 3.947
Isolates x genotypes 102 1156.82 11.34 0.0008
Residual 10 13413.21 1341.32
Total 123 67622.87
The mean DI values ranged from 0.00% to 76.2% among the turmeric accessions. All
the six turmeric accessions from KR population had a disease index ≤ 10% and were
considered resistant. Similarly, six out of seven accessions from MK population and
five accessions from NP population exhibited the lowest disease index values. On the
other hand, turmeric accessions from CTK and CTN population represented the highest
DI values (58.35% to 76.2%). Turmeric accessions from KP, DK, KJ and MB
75
populations also showed >20% DI and categorized as susceptible. Two turmeric lines
from Kandhamal (KM) population (KM2 and KM5) and one genotype from Nayagarh
(NG) population (NG4) revealed ≤ 20% DI although the rest of the turmeric lines were
found highly susceptible. The numbers of accessions from NG and KM populations
were also distributed across the DI classes. Of the 12 accession in the two populations, 1
accession was in first class, 2 accessions were in second class, 2 accessions were in
class III and 3 accessions were in class IV while rest of the four accessions were
grouped within class VI.
4.1.2. Overall genetic diversity in Curcuma longa
We surveyed 62 turmeric plants from 11 populations using a set of 29 molecular
markers. A total of 286 loci were presented from 29 primer (Table 4.2 to 4.5).
Combinations among 62 individuals of the 11 populations. Of these, 186 (65.03%) were
polymorphic The highest percentage of polymorphism was obtained with primer SSR1
(100%) followed by ISSR primer (GA)9T (90.9%). Similarly, RAPD primer OPN6
resulted in only three monomorphic bands (zero polymorphism) followed by OPA20
with only 16.6% polymorphic loci. Among the AFLP markers, the polymorphism
ranged from 52.9% in primer combination M-CAG x E-ACA to 55.5% in primer
combination M-CAT x E-ACA. At the population level, the percentage polymorphic
loci (PPL) ranged from 8.36% in the Dhenkanal (DK) population to 27.87% in
Mayurbhanj (MB) population with an average of 14.47%. All the populations had a PPL
<50%. Assuming Hardy-Weinberg equilibrium, the species wide diversity as measured
by Nei‟s unbiased gene diversity (He) across the total 62 sampled plants was
0.089±0.081. The average species wide observed heterozygosity (Ho) was 0.061±0.042
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Table 4.2: List of primers used for RAPD amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC
value.
Primer Primer sequence % GC content Annealing
Temperature
Total no. of
loci NPL (%) PPL
No. of fragments
amplified Rp PIC
A4 AATCGGGCTG 60 37 9 7 77.7 513 16.52 0.14
A11 CAATCGCCGT 60 37 10 7 70 499 16.08 0.29
A18 AGGTGACCGT 60 37 9 6 66.6 438 14.1 0.32
A3 AGTCAGCCAC 60 37 10 8 80 601 19.4 0.05
A20 GTTGCGATCC 60 37 6 1 16.6 362 11.68 0.05
D18 GAGAGCCAAC 60 37 7 3 42.8 428 13.78 0.04
D20 ACCCGGTCAC 70 37 7 5 71.4 325 10.46 0.37
N6 GAGACGCACA 60 37 3 0 0 186 6 0
N16 AAGCGACCTG 60 37 7 5 71.4 426 13.74 0.04
N4 GACCGACCCA 70 37 11 8 72.7 519 16.72 0.34
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Table 4.3: List of primers used for ISSR amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC
value.
ISSR Primer Primer sequence % GC
content
Annealing
Temperature
Total no.
of loci NPL (%) PPL
No. of fragments
amplified Rp PIC
(AGG)6 AGGAGGAGGAGGAGGAGG 66 55 6 5 83.3 354 11.42 0.09
(GA)9T GAGAGAGAGAGAGAGAGAT 42.8 51 11 10 90.9 686 22.12 0.13
(GAC)5 GACGACGACGACGAC 66.6 45 9 5 55.5 541 17.44 0.06
(GACA)4 GACAGACAGACAGACA 50 43 5 3 60 279 9.02 0.12
(GTG)5 GTGGTGGTGGTGGTG 66.6 45 7 5 71.4 414 13.34 0.09
(GTGC)4 GTGCGTGCGTGCGTGC 75 51 7 4 57.1 417 13.46 0.07
(CAA)5 CAACAACAACAACAA 33.3 35 3 2 66.6 176 5.68 0.1
(GGA)4 GGAGGAGGAGGA 66.6 35 7 4 57.1 413 13.32 0.09
(AG)8T AGAGAGAGAGAGAGAGT 47.1 45 11 7 63.6 621 20.02 0.15
(GT)8A GTGTGTGTGTGTGTGTA 47.1 45 8 5 62.5 463 14.92 0.12
T(GA)9 TGAGAGAGAGAGAGAGAGA 47.4 51 10 4 40 590 19.04 0.09
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Table 4.4: List of primers used for SSR amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC
value.
Primer Primer sequence % GC
content
Annealing
Temperature
Total no. of
loci NPL (%) PPL
No. of fragments
amplified Rp PIC
CBT2 F:TCCTCCCTCCCTTCGCCCACTG
R: GATGTTCGCCATGGCTGCTCC 65.9 55 6 4 66.6 346 11.18 0.13
CBT3 F:ATCAGCAGCCATGGCAGCGACC
R: AGGGGATCATGTGCCGAAGGCC 63.6 55 10 7 70 566 18.22 0.16
ClSSR1 F: ATGAACCCCTTCGGTCACC
R: TGTAACTCCTCTCGCCGTATG 55 60 7 7 100 373 12.04 0.26
ClSSR2 F: CCAAGTGCCCTATCCTAACA
R: CCTGGAAACCTGAATCCATTA 46.3 55 22 15 68.2 1300 41.9 0.05
ClSSR3 F: ACAGCACTCAACAACAGGAGA
R: CCTGGAAACCTGAATCCATTA 45.2 65 23 19 82.6 1349 43.52 0.09
Table 4.5: List of primers used for AFLP amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC
value.
Primer Primer sequence % GC
content
Annealing
Temperature
Total no.
of loci NPL
(%)
PPL
No. of fragments
amplified Rp PIC
MCAG-EACA GATGAGTCCTGAGTAACAG 50 56 17 9 52.9 1006 30.9 0.07
GACTGCGTACCAATTCACA
MCAT-EACA GATGAGTCCTGAGTAACAT 44.7 56 18 10 55.5 1084 34.98 0.06
GACTGCGTACCAATTCACA
MCAT-EAAC GATGAGTCCTGAGTAACAT 44.7 56 20 11 55 1202 38.78 0.06
GACTGCGTACCAATTCAAC
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(Table 4.6) The average population-level expected heterozygosity was 0.051±0.033
ranging from 0.027±0.019 in DK population to 0.114±0.109 in MB population.
Likewise, the population level observed heterozygosity ranged from 0.21±0.008 to
0.103±0.036 with an average of 0.045±0.022. Similar values were calculated for the
Shannon diversity index (I), which varied from 0.042±0.039 in population DK to
0.166±0.072 in population MB. The average Shannon diversity index (I) within
populations and species levels was
Table 4.6: Summary of genetic variation statistics for the combination of markers (RAPD,
ISSR, AFLP and SSR) among the genotypes of Curcuma longa with respect to their
distributions into 11 groups.
Population
ID
Sample
size
Na Ne He I Ho NPL PPL
CTK 5 1.125 (0.332) 1.064
(0.174)
0.042
(0.22)
0.065
(0.012)
0.037
(0.012) 36 12.54
CTN 5 1.087 (0.282) 1.043
(0.141)
0.028
(0.013)
0.044
(0.014)
0.023
(0.009) 25 8.71
KP 5 1.098 (0.297) 1.047
(0.147)
0.032
(0.027)
0.049
(0.051)
0.029
(0.009) 28 9.76
DK 5 1.084 (0.277) 1.039
(0.131)
0.027
(0.019)
0.042
(0.039)
0.021
(0.008) 24 8.36
NG 6 1.115 (0.320) 1.051
(0.150)
0.034
(0.018)
0.055
(0.032)
0.031
(0.010) 33 11.50
KM 6 1.174 (0.380) 1.068
(0.151)
0.049
(0.032)
0.079
(0.043)
0.042
(0.012) 50 17.42
KJ 5 1.157 (0.364) 1.126
(0.303)
0.069
(0.052)
0.098
(0.030)
0.056
(0.026) 45 15.68
MB 5 1.279(0.449) 1.202
(0.347)
0.114
(0.109)
0.166
(0.072)
0.103
(0.036) 80 27.87
KR 6 1.185 (0.389) 1.127
(0.280)
0.074
(0.059)
0.108
(0.063)
0.069
(0.025) 53 18.47
MK 7 1.139 (0.347) 1.066
(0.193)
0.042
(0.012)
0.066
(0.041)
0.034
(0.013) 40 13.94
NP 7 1.160 (0.367) 1.095
(0.233)
0.058
(0.037)
0.087
(0.048)
0.051
(0.019) 46 16.03
Mean 5.63 1.15 (0.363) 1.084
(0.312)
0.051
(0.033)
0.078
(0.043)
0.045
(0.022) 41.81 14.61
Total 62 1.173 (0.301) 1.119
(0.236)
0.089
(0.081)
0.132
(0.107)
0.061
(0.042)
Na= Observed number of alleles; Ne= Effective number of alleles; He= Nei‟s gene diversity; I=
Shannon‟s Information index; Ho= observed heterozygosity; NPL= Number of polymorphic
loci; PPL= Percentage of polymorphic loci
80
0.078±0.043 and 0.132±0.107 respectively. The MB and DK populations yielded the
highest and lowest values respectively for all these gene diversity estimates (Table 4. 6).
The dice similarity coefficient among all analyzed turmeric genotypes from 11
populations varied from as close as 1.0 to as diverse as 0.71. Dendrogram based on
UPGMA analysis classified the 62 genotypes under two major clusters (Figure 4.1).
Cluster-A consisted of 50 accessions while cluster B grouped rest of the 12 turmeric
genotypes. Cluster-A included the genotypes representing the entire sampled plants
from Cuttack-2 (CTN), Dhenkanal (DK), Kandhamal (KM), Nayagarh (NG), Koraput
(KR), Malkangiri (MK) and Nabrangpur (NP) populations and all but one from Cuttack-
1 (CTK) and Kendrapada (KP) populations. On the other hand, Cluster-B included five
samples each from Keonjhar (KJ) and Mayurbhanj (MB) population. Interestingly,
CTK5 from Cuttack population and KP3 from Kendrapada population also grouped
under cluster B along with turmeric genotypes from Keonjhar population. Bootstrap
analysis higher support at lower branch points but represented a value less than 50 at
higher branch points in the dendrogram.
4.1.3. Molecular variance and gene flow
Analysis of Molecular Variance (AMOVA) based on the two gene clusters showed that
majority of genetic variation occurred within populations (60.68%), while the variation
between the clusters was 39.31% (Table 4.7). AMOVA analysis based on individual
marker data also revealed similar results with negligible differences. However, the
genetic differentiation among 11 populations of C. longa was high and significant with
Fst = 0.382, P<0.001 and Gst = 0.486.
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Figure4.1 UPGMA dendrogram of 62 Curcuma longa individuals generated by the NJOIN module of NTSYSpc based on DICE genetic
distance from molecular data. Major clusters are indicated by alphabets. Bootstrap values >50 are given at the branch points.
82
Table 4.7: Summary of analysis of molecular variance (AMOVA) based on (a) RAPD, (b)
ISSR, (c) AFLP and (d) SSR profiling markers among the genotypes of Curcuma longa.
Levels of significance are based on 1000 iteration steps. Source of
variation
Degree of
freedom
Variance
component Percentage of variation P-value
RAPD
Among populations 10 5.932 33.82 < 0.001
Among genotypes 61 9.161 67.22 < 0.001
ISSR
Among populations 10 15.799 35.63 < 0.001
Among genotypes 61 27.361 64.37 < 0.001
SSR
Among populations 10 24.021 39.11 < 0.001
Among genotypes 61 36.083 60.99 < 0.001
AFLP
Among populations 10 18.211 31.46 < 0.001
Among genotypes 61 27.036 68.54 < 0.001
COMBINATION
Among populations 10 6.134 39.32 < 0.001
Among genotypes 61 9.019 60.68 < 0.001
The estimated gene flow, Nm (Nm = 0.5(1-Gst)/Gst) was found to be 0.529. This
suggest that the gene flow among turmeric populations throughout the entire
distribution region is highly restricted and the genetic differentiation is highly
significant.
4.1.4. Population structure in Curcuma longa
A Bayesian model based clustering method was implemented to further test the
population structure with regards to turmeric. Under an admixed model without prior
information, STRUCTURE calculated that the log likelihood of the data (LnP(D)) was
found to be greatest at K=2 (Figure 4.2A). For K>2, LnP(D) meagrely increased but
mostly represented a plateaued plot. ∆K reached its maximum value when K=2 (Figure
4.2 B) suggesting that all populations fell into one of the two clusters. The geographic
83
distribution of turmeric populations corresponded well with the two genetically distinct
clusters. The red cluster included all the genotypes form Kendrapada (KP), Nayagarh
(NG), Cuttack (CTK & CTN), Malkangiri (MK), Nabrangpur (NP), Koraput (KP),
Dhenkanal (DK) and Kandhamal (KM) while the remaining genotypes from Keonjhar
(KJ) and Mayurbhanj (MJ) were grouped in the green cluster (Figure 4.2C).
Furthermore, one individual each from Cuttack (CTK) and Kendrapada (KP) population
grouped within the green cluster. This result shows identical splitting with that of
UPGMA tree. The two dimensional principal coordinate analysis (PCoA) plot showed
that the first principal coordinate accounts for 24.8% of total variation and separates the
turmeric accessions from the Keonjhar (KJ) and Mayurbhanj (MJ) populations from rest
of the other populations analyzed in this study (Figure 4.3). The second principal
coordinate with 16.3% of total variation separated majority of turmeric accessions of
CTK, CTN, DK and KP population from that of KR, MK and NP populations. PCoA
analysis revealed broad overlap amongst the individuals of KM and NG populations.
However, in accordance with STRUCTURE, KJ and MJ populations were separated
from all other populations.
84
Figure4.2: STRUCTURE analysis of Curcuma longa populations based on molecular data. A)
The relationship between the number of cluster (K) and the estimated likelihood of data
(LnP(D)). A model based clustering of 62 genotypes using STRUCTURE without prior
knowledge about the populations and under an admixed model calculated that LnP (D) was
greatest when K = 2. (B) The relationship between K and ΔK., i.e. ΔK reaches its maximum
when K = 2, suggesting that all genotypes fall into one of the 2 clusters. (C) Grouping of
genotypes when K = 2. The genotypes were more likely clustered with respect to one of the 2
clusters. Genotypes from different clusters are represented with different colours: cluster 1(red)
and cluster 2 (blue).
85
Figure4.3: PCA-Principal coordinate analysis of the eleven sampled populations based
on genetic distances amongst all sampled individuals.
86
4.2. Development of sequence tagged diagnostic markers (SCAR/STS) linked to
rhizome rot resistance
4.2.1. Identification of ISSR marker linked with rhizome rot resistance
The rhizome rot resistant and susceptible genotypes of Curcuma longa were screened in
a bulked segregant analysis approach to identify ISSR markers associated with the
resistance. Forty ISSR primers were used to identify polymorphisms between the
susceptible bulks and six susceptible individuals and the resistant bulk and five
resistantindividuals. In total, 38 out of the 40 primers yielded 319 reproducible ISSR
(Table 4.8) bands ranging from 200bp to 3500bp. The number of ISSR bands per primer
varied from 4 to 12 with an average of 8 bands per primer. Majority of the ISSR bands
were identical among the genotypes while 98 bands were found to be polymorphic.
Although each primer generated multiple polymorphic bands, only ISSR2 could amplify
a differential band linked to resistance locus among the turmeric genotypes (Figure 4.4).
ISSR2 with the primer sequence 5‟-GAGAGAGAGAGAGAGAGAT-3‟ [(GA)9T]
resulted in a clear band of ≈1200bp (ClRSM- Curcuma longa resistance specific marker)
found in resistant bulk and all the five resistant individual while absent in the six
susceptible plants of C. longa.
4.2.2. Development of STS marker linked with rhizome rot resistance
The resistant specific fragment corresponding to ISSR marker (GA)9T amplified in the
five resistant individuals were cloned into pTZ57R/T vector and sequenced. Alignment
of the ClRSM sequences from the five resistant individuals of C. longa revealed high
homology. The five fragments have the length of 1203bp and showed variation at only
five nucleotide positions (Figure 4.5). Comparison of deduced ClRSM sequences
against the nucleotide and EST databases at the NCBI homepage showed no significant
87
homology between ClRSM and coding sequences of any structural genes. Further, the
sequences
Table 4.8: Primers used for ISSR analysis in Curcuma longa L.
Primer code Oligonucleotide
name Sequence (5‟-3‟)
Annealing
temp (0C)
No. of
bands
amplified
ISSR1 T(GA)9 TGAGAGAGAGAGAGAGAGA 51 9
ISSR2 (GA)9T GAGAGAGAGAGAGAGAGAT 51 9
ISSR3 (GGA)4 GGAGGAGGAGGA 35 8
ISSR4 (GTG)5 GTGGTGGTGGTGGTG 45 8
ISSR5 (GAC)5 GACGACGACGACGAC 45 7
ISSR6 (CAA)5 CAACAACAACAACAA 35 Smear
ISSR7 (AGG)5 AGGAGGAGGAGGAGG 55 9
ISSR8 (GACA)4 GACAGACAGACAGACA 43 6
ISSR9 (GTGC)4 GTGCGTGCGTGCGTGC 51 12
ISSR10 (AT)8T ATATATATATATATATT 34 4
ISSR11 (AT)8G ATATATATATATATATG 36 7
ISSR12 (AT)8C ATATATATATATATATC 36 8
ISSR13 (TA)8A TATATATATATATATAA 34 Smear
ISSR14 (TA)8C TATATATATATATATAC 36 9
ISSR15 (TA)8G TATATATATATATATAG 36 9
ISSR16 (AG)8T AGAGAGAGAGAGAGAGT 50 4
ISSR17 (GA)8T GAGAGAGAGAGAGAGAT 50 7
ISSR18 (GT)8A GTGTGTGTGTGTGTGTA 50 7
ISSR19 (GT)8T GTGTGTGTGTGTGTGTT 50 8
ISSR20 (AG)8C AGAGAGAGAGAGAGAGC 47 11
ISSR21 (AG)8G AGAGAGAGAGAGAGAGG 47 12
ISSR22 (GA)8C GAGAGAGAGAGAGAGAC 47 9
ISSR23 (GA)8A GAGAGAGAGAGAGAGAA 45 8
ISSR24 (CT)8T CTCTCTCTCTCTCTCTT 45 8
ISSR25 (CT)8A CTCTCTCTCTCTCTCTA 45 4
ISSR26 (CT)8G CTCTCTCTCTCTCTCTG 47 9
ISSR27 (CA)8T CACACACACACACACAT 45 6
ISSR28 (CA)8A CACACACACACACACAA 45 12
ISSR29 (CA)8G CACACACACACACACAG 47 10
ISSR30 (TG)8A TGTGTGTGTGTGTGTGA 45 6
ISSR31 (TG)8C TGTGTGTGTGTGTGTGC 47 4
88
ISSR31 (TG)8G TGTGTGTGTGTGTGTGG 47 9
ISSR33 (AC)8T ACACACACACACACACT 45 9
ISSR34 (GT)8YC GTGTGTGTGTGTGTGTYC 51 13
ISSR35 (GA)8YC GAGAGAGAGAGAGAGAYC 51 10
ISSR36 (GT)8YA GTGTGTGTGTGTGTGTYA 53 6
ISSR37 (GT)8YG GTGTGTGTGTGTGTGTYG 51 12
ISSR38 (GA)8YT GAGAGAGAGAGAGAGAYT 49 10
ISSR39 (AG)8YT AGAGAGAGAGAGAGAGAYT 55 11
ISSR40 (AG)8YC AGAGAGAGAGAGAGAGAYC 53 9
319
Figure 4.4: ISSR amplification patterns of five resistance and six susceptible
germplasm of turmeric obtained with the primer ISSR2 [(GA)9T]. The arrow
represents the putative fragment of 1200bp present in the resistant bulk (RB)
as well as five resistance individuals. M, 100bp ladder plus; RB, resistance
bulk; SB, Susceptible bulk; R1-R5 represents the resistant individuals CBT-
Cl23, CBT-Cl63, CBT-Cl87, CBT-Cl114 and CBT-Cl136 respectively; S1-
S6 represents the susceptible genotypes CBT-Cl09, CBT-Cl26, CBT-Cl35,
CBT-Cl57, CBT-Cl73 and CBT-Cl121 respectively.
89
CBT-Cl136 GAGAGAGAGAGAGAGAGATCCCCCCTTTTTCCGAACACCTCCTTCCTCCCTTTATACATCAAGAGCTTCCTTGAAAGTATGACCAGTTCTGCCAAAACATGGTCTTGAGAAGACAACCAA 120
CBT-Cl114 GAGAGAGAGAGAGAGAGATCCCCCCTTTTTCCGAACACCTCCTTCCTCCCTTTATACATCAAGAGCTTCCTTGAAAGTATGACCAGTTCTGCCAAAACATGGTCTTGAGAAGACAACCAA 120
CBT-Cl87 GAGAGAGAGAGAGAGAGATCCCCCCTTTTTCCGAACACCTCCTTCCTCCCTTTATACATCAAGAGCTTCCTTGAAAGTATGACCAGTTCTGCCAAAACATGGTCTTGAGAAGACAACCAA 120
CBT-Cl23 GAGAGAGAGAGAGAGAGATCCCCCCTTTTTCCGAACACCTCCTTCCTCCCTTTATACATCAAGAGCTTCCTTGAAAGTATGACCAGTTCTGCCAAAACATGGTCTTGAGAAGACAACCAA 120
CBT-Cl63 GAGAGAGAGAGAGAGAGATCCCCCCTTTTTCCGAACACCTCCTTCCTCCCTTTATACATCAAGAGCTTCCTTGAAAGTATGACCAGTTCTGCCAAAACATGGTCTTGAGAAGACAACCAA 120
************************************************************************************************************************
CBT-Cl136 TCACATCCGCATTTAATGCTTGGGTAGGTTATTCATTGGAGAGAGAGAGATCGTCTTGACAAGTGACCTCTCCCATTAATGCTTGACACAGTTGTTTAGTAGAGAGAGAAGATCATTTCT 240
CBT-Cl114 TCACATCCGCATTTAATGCTTGGGTAGGTTATTCATTGGAGAGAGAGAGATCGTCTTGACAAGTGACCTCTCCCATTAATGCTTGACACAGTTGTTTAGTAGAGAGAGAAGATCATTTCT 240
CBT-Cl87 TCACATCCGCATTTAATGCTTGGGTAGGTTATTCATTGGAGAGAGAGAGATCGTCTTGACAAGTGACCTCTCCCATTAATGCTTGACACAGTTGTTTAGTAGAGAGAGAAGATCATTTCT 240
CBT-Cl23 TCACATCCGCATTTAATGCTTGGGTAGGTTATTCATTGGAGAGAGAGAGATCGTCTTGACAAGTGACCTCTCCCATTAATGCTTGACACAGTTGTTTAGTAGAGAGAGAAGATCATTTCT 240
CBT-Cl63 TCACATCCGCATTTAATGCTTGGGTAGGTTATTCATTGGAGAGAGAGAGATCGTCTTGACAAGTGACCTCTCCCATTAATGCTTGACACAGTTGTTTAGTAGAGAGAGAAGATCATTTCT 240
************************************************************************************************************************
CBT-Cl136 GACAAGTCACATCCCAAGTGATGTTTGATTATTAGAGAGAGATCATTTATGATGGGACTGTTGAGCCGAGTGGGCTTGGCAGAATTGAGCTAAGTGTTGAGTGGCCTGGGTGGAGGTGTC 360
CBT-Cl114 GACAAGTCACATCCCAAGTGATGTTTGATTATTAGAGAGAGATCATTTATGATGGGACTGTTGAGCCGAGTGGGCTTGGCAGAATTGAGCTAAGTGTTGAGTGGCCTGGGTGGAGGTGTC 360
CBT-Cl87 GACAAGTCACATCCCAAGTGATGTTTGATTATTAGAGAGAGATCATTTATGATGGGACTGTTGAGCCGAGTGGGCTTGGCAGAATTGAGCTAAGTGTTGAGTGGCCTGGGTGGAGGTGTC 360
CBT-Cl23 GACAAGTCACATCCCAAGTGATGTTTGATTATTAGAGAGAGATCATTTATGATGGGACTGTTGAGCCGAGTGGGCTTGGCAGAATTGAGCTAAGTGTTGAGTGGCCTGGGTGGAGGTGTC 360
CBT-Cl63 GACAAGTCACATCCCAAGTGATGTTTGATTATTAGAGAGAGATCATTTATGATGGGACTGTTGAGCCGAGTGGGCTTGGCAGAATTGAGCTAAGTGTTGAGTGGCCTGGGTGGAGGTGTC 360
************************************************************************************************************************
CBT-Cl136 GGATGGCAGAGCGGAGCTGCCGGATGGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGTCGGATAGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGCCGGATAGCAGTGCTGA 480
CBT-Cl114 GGATGGCAGAGCGGAGCTGCCGGATGGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGTCGGATAGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGCCGGATAGCAGTGCTGA 480
CBT-Cl87 GGATGGCAGAGCGGAGCTGCCGGATGGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGTCGGATAGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGCCGGATAGCAGTGCTGA 480
CBT-Cl23 GGATGGCAGAGCGGAGCTGCCGGATGGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGTCGGATAGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGCCGGATAGCAGTGCTGA 480
CBT-Cl63 GGATGGCAGAGCGGAGCTGCCGGATGGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGTCGGATAGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGCCGGATAGCAGTGCTGA 480
************************************************************************************************************************
CBT-Cl136 GCTGCCGAATAGCAGTGCTGAGCTGCCGGGTCGAATGAGCCAAGTCCTAGAGGGGGGCTATAACACCCATGTGGCCGAATGGGCGATTCGGAGACGCGACCGAAGAGATAACTTGACTTC 600
CBT-Cl114 GCTGCCGAATAGCAGTGCTGAGCTGCCGGGTCGAATGAGCCAAGTCCTAGAGGGGGGCTATAACACCCATGTGGCCGAATGGGCGATTCGGAGACGCGACCGAAGAGATAACTTGACTTC 600
CBT-Cl87 GCTGCCGAATAGCAGTGCTGAGCTGCCGGGTCGAATGAGCCAAGTCCTAGAGGGGGGCTATAACACCCATGTGGCCGAATGGGCGATTCGGAGACGCGACCGAAGAGATAACTTGACTTC 600
CBT-Cl23 GCTGCCGAATAGCAGTGCTGAGCTGCCGGGTCGAATGAGCCAAGTCCTAGAGGGGGGCTATAACACCCATGTGGCCGAATGGGCGATTCGGAGACGCGACCGAAGAGATAACTTGACTTC 600
CBT-Cl63 GCTGCCGAATAGCAGTGCTGAGCTGCCGGGTCGAATGAGCCAAGTCCTAGAGGGGGGCTATAACACCCATGTGGCCGAATGGGCGATTCGGAGACGCGACCGAAGAGATAACTTGACTTC 600
************************************************************************************************************************
CBT-Cl136 TTTAGGCCTTTAGGGATAGACCGTGACGGGAGCCGACCGAACCCATGGATGAGGCTCGAATTAGACTCGAGTCAAGTCTGGGTCCTCCAAAATACTCACCGGAACAGTTATAATTATTTT 720
CBT-Cl114 TTTAGGCCTTTAGGGATAGACCGTGACGGGAGCCGACCGAACCCATGGATGAGGCTCGAATTAGACTCGAGTCAAGTCTGGGTCCTCCAAAATACTCACCGGAACAGTTATAATTATTTT 720
CBT-Cl87 TTTAGGCCTTTAGGGATAGACCGTGACGGGAGCCGACCGAACCCATGGATGAGGCTCGAATTAGACTCGAGTCAAGTCTGGGTCCTCCAAAATACTCACCGGAACAGTTATAATTATTTT 720
CBT-Cl23 TTTAGGCCTTTAGGGATAGACCGTGACGGGAGCCGACCGAACCCATGGATGAGGCTCGAATTAGACTCGAGTCAAGTCTGGGTCCTCCAAAATACTCACCGGAACAGTTATAATTATTTT 720
CBT-Cl63 TTTAGGCCTTTAGGGATAGACCGTGACGGGAGCCGACCGAACCCATGGATGAGGCTCGAATTAGACTCGAGTCAAGTCTGGGTCCTCCAAAATACTCACCGGAACAGTTATAATTATTTT 720
************************************************************************************************************************
(Figure 4.5 )
90
CBT-Cl136 GTATGGTGTGATCTGAGCGAAAGAGCGACTAGCTTTTCGATACGTTGAAGAAAAATGATTTTGGCACCGACAGAAATAAGCTCCAAGAAGAAGAGGAAGGAAATTTATACATGCTGCCCG 840
CBT-Cl114 GTATGGTGTGATCTGAGCGAAAGAGCGACTAGCTTTTCGATACGTTGAAGAAAAATGATTTTGGCACCGACAGAAATAAGCTCCAAGAAGAAGAGGAAGGAAATTTATACATGCTGCCCG 840
CBT-Cl87 GTATGGTGTGATCTGAGCGAAAGAGCGACTAGCTTTTCGATACGTTGAAGAAAAATGATTTTGGCACCGACAGAAATAAGCTCCAAGAAGAAGAGGAAGGAAATTTATACATGCTGCCCG 840
CBT-Cl23 GTATGGTGTGATCTGAGCGAAAGAGCGACTAGCTTTTCGATACGTTGAAGAAAAATGATTTTGGCACCGACAGAAATAAGCTCCAAGAAGAAGAGGAAGGAAATTTATACATGCTGCCCG 840
CBT-Cl63 GTATGGTGTGATCTGAGCGAAAGAGCGACTAGCTTTTCGATACGTTGAAGAAAAATGATTTTGGCACCGACAGAAATAAGCTCCAAGAAGAAGAGGAAGGAAATTTATACATGCTGCCCG 840
************************************************************************************************************************
CBT-Cl136 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATTGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCCAAGTTGACCC 960
CBT-Cl114 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATTGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCAAAGTTGACCC 960
CBT-Cl87 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATTGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCAAAGTTGACCC 960
CBT-Cl23 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATTGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCAAAGTTGACCC 960
CBT-Cl63 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATGGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCAAAGTTGACCC 960
*********************************************************.***************************************************.**********
CBT-Cl136 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAACCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080
CBT-Cl114 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCTAATCTAAACCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080
CBT-Cl87 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAGCCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080
CBT-Cl23 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAACCCTTAGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080
CBT-Cl63 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAACCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080
*************************************************:*******.*****:********************************************************
CBT-Cl136 GTCAATTACTTGGCCTGAATTCCCCGAAAAAAACGCTTTGCCCGGACCCAATTTAAAGCCCCCCCGGGTTAAAAAAAAAGAAAGGGGAAGCGAAGGCTTCCTTTATCTCTCTCTCTCTCTCTC 1203
CBT-Cl114 GTCAATTACTTGGCCTGAATTCCCCGAAAAAAACGCTTTGCCCGGACCCAATTTAAACCCCCCCCGGGTTAAAAAAAAAGAAAGGGGAAGCGAAGGCTTCCTTTATCTCTCTCTCTCTCTCTC 1203
CBT-Cl87 GTCAATTACTTGGCCTGAATTCCCCGAAAAAAACGCTTTGCCCGGACCCAATTTAAACCCCCCCCGGGTTAAAAAAAAAGAAAGGGGAAGCGAAGGCTTCCTTTATCTCTCTCTCTCTCTCTC 1203
CBT-Cl23 GTCAATTACTTGGCCTGAATTCCCCGAAAAAAACGCTTTGCCCGGACCCAATTTAAACCCCCCCCGGGTTAAAAAAAAAGAAAGGGGAAGCGAAGGCTTCCTTTATCTCTCTCTCTCTCTCTC 1203
CBT-Cl63 GTCAATTACTTGGCCTGAATTCCCCGAAAAAAACGCTTTGCCCGGACCCAATTTAAACCCCCCCCGGGTTAAAAAAAAAGAAAGGGGAAGCGAAGGCTTCCTTTATCTCTCTCTCTCTCTCTC 1203
***************************************************************************************************************************
Figure 4.5: Multiple sequence alignment of the ClRSM sequences isolated from five turmeric germplasm resistant to
Pythiumaphanidermatum. The shaded regions indicate the nucleotide base variation among the five sequences.
91
also revealed no open reading frame and multiple stop codons existed in all the three
reading structures.
Based on the complete sequence data of ClRSM, a pair of STS primers (ClSTS-F and
ClSTS-R) was designed (Figure 4.6). The primer pair was used to amplify the five
resistant and four susceptible genotypes of turmeric to confirm their validity. PCR with
ClSTS-F (5‟-CCTTTTTCCGAACACCTCCT-3‟) forward sequence and reverse is
CISTS–R. (5‟CTTTCGCTCAGATCACACCA-3‟) resulted in the amplification of a
720bp fragment (ClSTS) only in the resistant and not in the susceptible individuals of
turmeric (Figure 4.8A). Accurate amplification of a 720bp fragment in the five resistant
genotypes proved the stability of the designed SCAR marker and suggests that ClSTS
amplification during ISSR-PCR is independent to the base changes in the primer
binding sites. Further, 720bp fragment of ClSTS was also obtained with wild Curcuma
zedoaria genotype Cze512-11 resistant to P. aphanidermatum while no amplification
was found in two susceptible wild samples- Curcuma zedoaria Cze102-03 and
Curcuma amada Cam1703 genotypes. This suggests that the specific DNA region of
ClSTS is conserved across wild and cultivated Curcuma species that confers resistant to
P. aphanidermatum. Southern hybridization was performed to determine the nature of
the STS sequence within the turmeric genome. Genomic DNA of the resistant turmeric
line CBT-Cl23 and susceptible line CBT-Cl09 digested with EcoRI, HindIII, XbaI or
SacI was subjected to southern blotting using a digoxigenin labeled ClSTS fragment as
probe. DNA digested with different enzymes revealed single band only with the
resistant genotype CBT-Cl23 in the southern blots (Figure 4.7). This specifies that
ClSTS is represented by a single copy locus in the turmeric genome.
92
Figure 4.6: DNA sequence of the Curcuma longa resistance specific marker ClRSM.
The underlined region represents the annealing position of ISSR2 [(GA)9T]. The arrow
with the shaded region indicates the position of primers ClSTS-F and ClSTS-R for
amplifying the sequenced tagged sites marker ClSTS.
93
Figure 4.7: Southern blot analysis of STS marker ClSTS. Restriction enzymes
used are EcoRI, HinDIII, XbaI and SacI. M, Molecular weight marker; R,
resistant genotype CBT-Cl23; S, susceptible genotype CBT-Cl09.
Hybridization patterns of the genomic DNA digested with all the four
enzymes revealed single band only in the resistant genotype.
94
4.2.3. Application of ClSTS marker to diagnose rhizome rot resistance in turmeric
Fifteen turmeric accessions collected from different agroclimatic zones of Odisha, India
with unknown response to rhizome rot disease were tested for the presence or absence
of the STS marker ClSTS. PCR amplification of the isolated DNA from the 15
individuals with STS primer resulted in single expected DNA band of 720bp in ten
accessions Cl13-07, Cl23-11, Cl27-03, Cl51-06, Cl63-07, Cl96-03, Cl126-17, Cl69-01,
Cl87-19 and Cl29-17. The rest of the five accessions Cl75-15, Cl102-13, Cl42-12,
Cl33-09 and Cl134-05 did not result in the expected PCR product (Fig 4.8 B). Thus, the
amplification pattern of ClSTS suggested that ten out of the 15 unknown turmeric
accessions exhibit resistant response against rhizome rot. An inoculation experiment
was carried out with the fifteen turmeric accessions to determine the associations of
rhizome rot susceptibility with the absence of ClSTS locus. Pseudostems of the fifteen
turmeric accessions were inoculated with fungal zoospore suspension, kept under green
house conditions and observed for disease symptoms (Figure 4.9). Ten turmeric
accessions with positive amplification of ClSTS showed no symptoms of infection,
either foliar or rhizomic. In contrast to them, the rest of the five genotypes without
ClSTS locus showed significant levels of foliar decline and rhizome rot score. The
pseudostems of the five genotypes were completely wilted and their rhizomes were
decayed within four week of inoculation. These results indicate that the five turmeric
accessions are indeed susceptible to P. aphanidermatum infection.
95
Figure 4.8: (A) Analysis of STS marker ClSTS in turmeric genotypes either
resistant or susceptible to rhizome rot. ClSTS amplified a single 720bp band in
five resistant Curcuma longa genotypes CBT-Cl23, CBT-Cl63, CBT-Cl87,
CBT-Cl114, CBT-Cl136 and one resistant Curcuma zedoaria genotype Cze512-
11. No amplification was found with four susceptible C. longa genotypes CBT-
Cl09, CBT-cl26, CBT-cl57, CBT-cl121, one susceptible C zedoaria genotype
Cze102-03 and one susceptible C amada genotype Cam17-03. (B) STS analysis
carried out using ClSTS marker in 15 unknown turmeric accessions listed in
table 1. Sample that amplified a single 720bp band with ClSTS was identified as
resistant while the samples with no amplification were detected as susceptible to
rhizome rot. M, 100bp ladder; R, resistant genotype CBT-Cl23; S, susceptible
genotype CBT-Cl09; 1, Cl13-07;2, Cl23-11; 3, Cl27-03; 4, Cl29-17; 5, Cl33-09;
6, Cl42-12; 7, Cl51-06; 8, Cl63-07; 9, Cl69-01; 10, Cl75-15;11, Cl87-19;12,
Cl96-03; 13, Cl102-13; 14, Cl126-17; 15, Cl134-05.
96
Figure 4.9: Rhizome rot inoculation experiment with resistant genotype CBT-Cl23 and
the identified turmeric accession Cl75-15 (Plant no 10) susceptible to P.
aphanidermatum. A) CBT-Cl23 plant inoculated with P. aphanidermatum 40 days PI.
B) Disease symptoms of Cl75-15 (Plant no 10) inoculated with P. aphanidermatum 40
days PI. C) Rhizome of rot resistant CBT-Cl23 genotype inoculated with P.
aphanidermatum 40 days PI. D) Decayed rhizome of Cl75-15 (Plant no 10) inoculated
with P. aphanidermatum 40 days PI.
97
4.3. Cloning and characterization of resistance related candidate gene sequences
from wild Curcuma spp.
4.3.1. Amplification and cloning of RGCs of the NBS-type from wild Curcuma spp.
Using the three R-gene specific degenerate primers, a ≈530bp fragment was amplified
between the P-loop and GLPLA motifs of the NBS region of the NBS-LRR class of R-
genes from the three wild turmeric genotypes. An additional band of 100/250/300bp
was also observed in a few samples. Different PCR conditions, including the
concentrations of template DNA, Mg2+ and Taq DNA polymerase, were tested to
further confirm the PCR amplification. Although the 530-bp band could be reproduced
under all conditions tested, the DNA bands of other sizes could not. Therefore, the 530-
bp band was cloned and sequenced. Nine amplicons of 530-bp in size (Figure 4.10)
were cloned and 38 clones were isolated that comprised of 10, 17 and 11 clones
respectively from Curcuma aromatica, Curcuma angustifolia and Curcuma zedoaria.
BLASTx search revealed 21 (nine, five and seven from Curcuma aromatica, Curcuma
angustifolia and Curcuma zedoaria respectively) out of 38 clones (Table 4.9). having
high homology to the NBS domain of known R-genes or RGCs from other plant
species. These sequences have been submitted in the Gene Bank database
(http://www.ncbi.nlm.nih.gov) with accession number from JN426969 to JN426989.
The rest of the 17 clones showed homology to putative polyprotein and hypothetical
leucine-rich repeats and were excluded from further analysis.
4.3.2. Sequence analysis of RGCs from wild Curcuma spp.
The 21 nucleotide sequences were translated using ExPaSy translate tool and the
deduced amino acid sequences that showed a high level of sequence similarity with
more than 100 amino acids to the NBS regions of disease resistance R-genes were
published in the GenBank database. The level of identity of these sequences
98
Figure 4.10: Amplification of 530 bp fragment using a degenerate primer of
wild relatives of Curcuma.
99
Table 4.9: Results of the search for similarity between wild turmeric NBS-
sequences and GenBank accessions carried out using the BLASTX algorithm
Curcuma
NBS
clone
GenBank protein
accession from
Zingiberaceae
showing the
highest similarity
Amino
acid
identity
(%)
E
value
Other GenBank
protein accession
showing the
highest similarity
Amin
o acid
identit
y (%)
E
value
Cap11 Kaempferia galanga
NBS-LRR protein
(ADZ73428)
98% 2e-
123
Disease resistance
like protein, Coffea
arabica
(CAC82610)
51% 4e-35
Cap12 Kaempferia galanga
NBS-LRR protein
(ADZ73428)
98% 5e-
122
Disease resistance
like protein, Coffea
arabica
(CAC82610)
53% 3e-36
Cap21 Kaempferia galanga
NBS-LRR protein
(ADZ73428)
99% 2e-
123
Resistance like
protein, Vitis davidii
(ABS50350)
52% 2e-35
Cap22 Kaempferia galanga
NBS-LRR protein
(ADZ73431)
98% 5e-
124
Resistance like
protein, Vitis davidii
(ABS50350)
52% 7e-39
Cap23 Kaempferia galanga
NBS-LRR protein
(ADZ73429)
98% 1e-
126
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
55% 1e-38
Cap24 Kaempferia galanga
NBS-LRR protein
(ADZ73430)
98% 3e-
129
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
60% 3e-37
Cap31 Kaempferia galanga
NBS-LRR protein
(ADZ73428)
96% 3e-
120
NBS-LRR type
resistance protein
[Cynodon x
magennisii]
(ADC54190)
60% 1e-37
Cap32 Kaempferia galanga
NBS-LRR protein
(ADZ73430)
97% 1e-
121
NBS-LRR type
resistance protein
[Cynodon x
magennisii]
(ADC54190)
59% 1e-36
Cap33 Kaempferia galanga
NBS-LRR protein
(ADZ73431)
89% 2e-
126
NBS-LRR type
resistance protein
[Cynodon x
magennisii]
(ADC54190)
56% 2e-36
Can11 Kaempferia galanga
NBS-LRR protein
ADZ73429
89% 2e-
112
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
56% 4e-35
Can12 Kaempferia galanga
NBS-LRR protein
87% 5e-
107
NBS containg R-
gene [Musa
56% 5e-32
100
(ADZ73428) acuminata subsp.
malaccensis]
(ABY75804)
Can21 Kaempferia galanga
NBS-LRR protein
(ADZ73428)
86% 2e-
104
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
55% 8e-34
Can22 Kaempferia galanga
NBS-LRR protein
(ADZ73429)
88% 1e-
108
NBS-LRR type
resistance protein
[Cynodon x
magennisii]
(ADC54190)
56% 2e-32
Can31 Kaempferia galanga
NBS-LRR protein
(ADZ73429)
88% 2e-
107
NBS-LRR type
resistance protein
[Cynodon x
magennisii]
(ADC54190)
54% 3e-30
Czp11 Kaempferia galanga
NBS-LRR protein
(ADZ73429)
87% 4e-
109
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
57% 3e-36
Czp12 Kaempferia galanga
NBS-LRR protein
(ADZ73431)
86% 9e-
106
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
57% 5e-36
Czp21 Kaempferia galanga
NBS-LRR protein
(ADZ73428)
85% 3e-
103
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
63% 1e-35
Czp22 Kaempferia galanga
NBS-LRR protein
(ADZ73429)
93% 2e-
123
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
58% 2e-39
Czp23 Kaempferia galanga
NBS-LRR protein
ADZ73429
89% 2e-
112
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
59% 5e-39
Czp31 Kaempferia galanga
NBS-LRR protein
(ADZ73430)
87% 3e-
109
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
59% 1e-38
Czp32 Kaempferia galanga
NBS-LRR protein
(ADZ73428)
87% 3e-
104
NBS containg R-
gene [Musa
acuminata subsp.
malaccensis]
(ABY75804)
59% 3e-36
101
to RGCs isolated from other plant species ranged from 51% between Cap11 and NBS-
LRR resistance like protein from Coffea arabica (e-value: 4e-35) to 63% between
Czp21 and NBS-containing disease resistance-like protein from Musa acuminata subsp.
malaccensis (e-value: 1e-35). The level of sequence identity of isolated sequences with
RGCs isolated from other zingiberaceous species ranged from 85% between Czp21 and
resistance like protein KRGC1 from Kaempferia galanga (e-value: 3e-103) to 99%
between Cap21 and Kaempferia galanga resistance like protein KRGC4 (e-value: 1e-
04). BLASTP searches of deduced amino acid sequences of the 21 clones revealed the
presence of NB-ARC (nucleotide binding and similarity to Apaf-1, R genes and Ced-4)
domain and significant homology to well characterized RGCs and R-genes from
angiosperms. Analysis of the sequences using the ORF finder at the NCBI server
(http://www.ncbi.nlm.nih.gov/projects/gorf) revealed that all the 21 sequences could be
translated into open reading frame (ORF) with more than 100 amino acids. Southern
blot analysis of selected resistance gene candidates resulted in hybridization pattern
consisting of multiple bands for each of the three RGC tested (Figure 4.11) suggesting
that RGCs occurs as multiple copies in the turmeric genome. The RGCs Cap12, Czp11
and Can12 showed the hybridization pattern representing seven, six and five copies in
the genome of Curcuma aromatica, Curcuma zedoaria and Curcuma angustifolia
respectively. Such types of RGCs attribute to be a member of the multi resistance gene
family.
Analysis of the putative amino acid sequences of the wild Curcuma RGCs using
CLUSTALX program revealed the presence of appropriately located consensus P-
loop/Kinase-1a (GGVGKTT), kinase-2 (LLVFDDVW), RNBS-B (GSRVIVTTRI) and
GLPL motifs characteristic of the NBS domain of R-genes (Figure 4.12). The non-TIR
102
Figure 4.11: Southern hybridization of three selected resistance gene candidates
Cap12, Czp11 and Can12 of Curcuma aromatica, Curcuma angustifolia and
Curcuma zedoaria plants respectively. The restriction enzymes used are (1)
EcoRI and (2) HindIII. Molecular weight marker values are indicated in the left
side of each blot. Hybridization pattern consisting of multiple bands was realized
for each of the three RGC tested with both EcoRI and HindIII suggesting that
the RGCs belongs to a multi-copy gene family in the turmeric genome.
103
Figure 4.12: CLUSTAL_X multiple alignment of deduced amino acid sequences of
the NBS region of wild Curcuma sequences and analogous domains in known R-
genes. RPS2 (A54809) confers resistance to Pseudomonas syringae in Arabidopsis
thaliana, N (U15605) confers resistance to Tobacco mosaic virus in Nicotiana
tabaccum, L6 (U27081) confers resistance to Melampsora lini in Linum usitatissimum
and Pi-ta (AAK00132) confers resistance to Magnaporthe grisea in rice. Conserved
motifs are shaded and indicated above the alignment. The arrows indicate the position
and orientation of degenerate primers.
104
motif (WVxxIRELAYDIEDIVDxY) associated with non-TIR subclass of NBS
sequences was also present in the N-terminal region of all the 21 wild Curcuma
sequences. In addition, the analysis showed a tryptophan (W) residue at the end of
kinase-2 motif, which is also a characteristic feature of non-TIR subclass of NBS-LRR
R-genes. Sequence comparisons of the wild Curcuma RGCs (using the region spanning
the P-loop and GLPLA motifs of the NBS domain) revealed a high amino acid identity
among them. The amino acid identity ranged from 85% (between Czp11 and Cap12,
Cap21) to 99% (between Cap21 and Cap11; between Cap22 and Cap23, Cap24, Cap31
and Cap32; between Can11 and Can12, Can21; between Can22 and Can31; between
Czp11 and Czp12, Czp21). Similarly, the amino acid identity between wild Curcuma
sequences and known R-genes varied from 41% between Can11, Can12, Can21, Can31
and Linium L6 (U27081) to 53% between Cap23 and Arabidopsis RPS2 (A54809).
Likewise, the amino acid identity among R-genes utilized in the study ranged from 42%
(between RPS2, N and L6) to 53% (between L6 and N).
4.3.3. Phylogenetic analysis of wild turmeric RGCs
A phylogenetic tree was constructed using the Neighbor Joining algorithm implemented
in the Molecular Evolutionary Genetics Analysis (MEGA) software package version 2.1
to examine the relationships of wild Curcuma RGCs among themselves and to the R-
genes and RGCs from other plant species. The analysis was performed using only the
NBS domain (P-loop to GLPLA) as it contains numerous conserved motifs that
facilitate proper alignment. The resulting tree consisted of two major branches- TIR and
non-TIR NBS-LRR disease resistance proteins (Figure 4.13). The TIR branch consisted
of seven known TIR-NBS-LRR genes namely L (AAD25976), L6 (U27081), M
(P93244), P2 (AAK28806), N (U15605), RPP4 (NM117790) and RPP1 (NM114316).
105
Figure 4.13: Neighbour joining tree based on amino acid sequence alignment of resistance gene candidates isolated from wild Curcuma
spp., other genotypes of Zingiberaceae and NBS sequences of R-genes from other plant species. Numbers on the branches indicate
the percentage of 1000 bootstrap replications supporting the particular nodes.The bold branches indicate the TIR-NBS cluster.
106
All the wild Curcuma RGCs clustered with the non-TIR subclass of NBS sequences,
which supports our earlier results that the turmeric RGCs lacks a TIR domain at the N-
terminal region. The wild Curcuma RGCs were distributed into four classes. 12 out of
21 RGCs were in class A and 3 RGCs each in class B, C and D. The distribution of wild
Curcuma RGCs in different branches of the non-TIR-NBS cluster reflects a high level
of sequence divergence for these sequences. However, in all the subtrees, the Curcuma
RGCs were clustered with RGCs of known function from other members of the family
Zingiberaceae such as Zingiber officinale and Kaempferia galanga.Thus, the 21
sequences identified from three taxa can be considered as resistance gene candidates
based on their high level of sequence identity to R-genes/RGCs and presence of true
open reading frames with desired conserved motifs.
4.3.4. Transcript expression analysis of the wild turmeric RGCs
Total RNA was isolated from the healthy plants of three wild turmeric genotypes either
resistant or susceptible to Pythium aphanidermatum and subjected to RT-PCR to study
the transcript expression profiles. Cap12 and Can12 resulted in constitutive
amplification of PCR product in both resistant and susceptible accession of Curcuma
aromatica and Curcuma angustifolia. However, in case of Curcuma zedoaria, the
mRNA encoding Czp11 expressed in the plant resistant to Pythium aphanidermatum but
not in plants susceptible to the fungal pathogen (Figure 4.14). Similar results were
alsoobtained with cultivated turmeric (Curcuma longa L.); Czp11 was detected in as
many as seven resistant accessions (CBT no. C106, C109, C110, C122, C123, C134,
C137) but not in susceptible lines (data not shown). This suggests a transcriptional
variance of Czp11 might account for resistance against Pythium aphanidermatum.
Control reactions were set up to ensure that the PCR products were not amplified from
107
Figure 4.14: RT-PCR analysis of three selected resistance gene candidates Cap12,
Czp11 and Can12 in root tissue of Curcuma aromatica, Curcuma zedoaria and
Curcuma angustifolia plants resistant (R1, R2, R3) or susceptible (S1, S2, S3)
respectively to Pythium aphanidermatum. Cap12 and Can12 showed a constitutive
expression in both resistant (R1, R3) and susceptible (S1, S3) plants of Curcuma
aromatica and Curcuma angustifolia while Czp11 expression was realized only in
resistant line of Curcuma zedoaria (R2) but not in susceptible line (S2). C+ denotes a
positive control and show the expected ≈300bp fragment amplified from turmeric actin
1 cDNA.C- denotes the negative control which show no amplification of turmeric actin
1 amplification due to lack of reverse transcription. M denotes the 1kb ladder plus
molecular weight marker.
108
genomic DNA. PCR was done carrying a positive control with actin-specific primers
and negative control with actin primers but with RNA instead of cDNA as the template.
Amplification of the expected cDNA fragment (≈300bp) in the positive control for Actin
1 gene and no amplification in the negative control confirmed that the mRNA samples
were free of genomic DNA contamination.