This study performed a comparative transcriptomic analysis of resistant and susceptible cotton genotypes at early and late stages of infection by the root-knot nematode Meloidogyne incognita. RNA sequencing of infected and uninfected root samples identified differentially expressed genes between resistant and susceptible lines. Two genes involved in defense against the nematode were found to be overexpressed at a resistance quantitative trait locus (qMi-C11), and two plant receptor genes were overexpressed at another locus (qMi-C14). Ongoing work includes time-series analysis of gene expression in resistant, susceptible, and near-isogenic lines to identify genes responsible for resistance mechanisms.

1. Background
▪ Southern root-knot nematode (Meloidogyne
incognita; RKN) is the most important soil-borne
pathogen of Upland cotton (Gossypium hirsutum).
▪ Host resistance is the only feasible measure to
safeguard cotton stands/yields against RKN
infestation.
▪ Two major quantitative trait loci (qMi-C11 and qMi-
C14) in resistant germplasm display different
mechanisms of curtailing nematode pathogenicity.
▪ qMi-C11 interferes with galling while qMi-C14 affects
egg production.
▪ Additive-by-additive epistasis between qMi-C11 and
qMi-C14 results in transgressive resistance in QTL-
stacked lines.
Objective
▪ To perform comparative transcriptomics analysis between
susceptible and resistant genotypes at an early stage (12
days after infection) and a late stage (after 25 days) of RKN
development, which coincide with the establishment of
feeding site (i.e., galling) and egg production, respectively, to
identify differentially expressed genes.
Summary
▪ Sequencing of six cDNA libraries corresponding to RKN-
inoculated and mock-inoculated (untreated) root samples of
susceptible and resistant genotypes produced over 157
million paired-end reads, 76% of which were mapped to the
Gossypium hirsutum genome (Figure 1).
▪ Host responses to nematode infection and development is
remarkably different in resistant and susceptible lines (Figure
2).
▪ Key genes involved host-parasite interactions are enriched in
response to nematode infection (Figure 3).
▪ Based on RNA-seq analysis and qPCR of genes in the QTL
region, two plant defense genes (Gh_A11G2835,
Gh_A11G3090) at qMi-C11 and two plant receptor genes
(Gh_D02G0257, Gh_D02G0259) at qMi-C14 were found to be
overexpressed in response to nematode parasitism (Table 1,
Figure 4).
Ongoing and future works
▪ Time series analysis of the transcriptional responses of
resistant (QTL-stacked), susceptible and near-isogenic lines.
▪ Functional genomics and reverse genetics studies on
candidate genes.
A comparative transcriptomic study of Southern root-knot nematode
resistance in Upland cotton
1Sameer Khanal, 1,2Pawan Kumar, 1,3Mychele Batista da Silva, 1Rippy Singh, 4Robert L. Nichols, 5Richard F Davis, 1Peng Chee
1Cotton Molecular Breeding Laboratory, Department of Crop and Soil Sciences, University of Georgia, Tifton, GA; 2USDA-ARS, Salinas, CA; 3University of California, Davis, CA; 4Cotton Incorporated, Cary,
NC; 5USDA-ARS, Tifton, GA
M-120 RNR Cocker 201
CH11
BC7F2
F1
BC7F1
CH14
X
Cocker 201X
Fig. 5 Marker assisted selection (MAS) for the development
of near isogenic lines (NILs) carrying qMi-CH11 and qMi-
CH14. M-120 RNR is the source of resistant QTLs. Cocker 201
is the susceptible recipient.
Seven backcrosses
X
MAS to develop NILs
C201 chromatin
QTL from M-120
M-120 introgression
Fig. 6 Graphical Genotypes using Variant Call from RNA-seq data. CH11 isoline
mapped 168 variant alleles (16 INDELs and 152 SNPs) in chromosome 11, while
CH14 isoline mapped 128 variant alleles (10 INDELs and 118 SNPs) in chromosome
14.
CH-11 isoline
Chromosome A11
CH-14 isoline
Chromosome D02
* marker distances in MB
0
20
40
60
80
100
120
140
160
180
200
4DAI 8DAI 12DAI 16DAI 20DAI 25DAI
A
B
A
B
A
AB
BC
C
A
AB
B
C
A
A
B
B
Coker 201
CH14
CH11
M-120
Fig. 7 Time-course study of nematode infections in resistant (M-120) and
susceptible (C201) and near-isogenic lines (CH11 and CH14). Letters represent
significance of grouping. Histological work done by Mychele Batista da Silva.
RNA-seq analysis is being done.
Days after infection (DAI)
Numberofnematodes
Ongoing works
Pictures: Damage caused by Southern root-knot nematodes.
Picture credit: Peng Chee
Figure 1. Average number of mapped and
unmapped sequences at early (E), late (L) and
control (C) sampling time point.
Figure 2. Venn diagram illustrating differentially expressed genes in C201 and M120 at
(a) early (b) late stage of RKN infection
Figure 3. GO enrichment terms. (a) biological process, (b) molecular function, (c) Cellular
components
Results
0
0.5
1
1.5
2
2.5
3
Early Late
Gh_D02G0257
M-120 C201
0
0.5
1
1.5
2
2.5
3
Early Late
Gh_D02G0259
M-120 C201
0
0.5
1
1.5
2
2.5
3
3.5
4
Early Late
Gh_A11G3090
M-120 C201
Figure 4. Quantitative real-time PCR (qRT-PCR) of candidate root-knot nematode (RKN) resistance genes at early and
late infection time-points [i.e., 12 days after inoculation (DAI) and after 25 DAI, respectively). Charts show mean fold-
change in expression after RKN infection with error bars representing 95 % confidence interval of the mean. Asterisks
mark significant fold-change in expression in inoculated samples compared to non-inoculated plants and as
determined by t-test of ΔCt values (P ≤ 0.05) using two biological replicates.
* * *
*
* *
*
*
*
a. b. c.
Table 1. Differentially expressed genes (DEGs) in qMi-C11 (Gh_A11G3090 and Gh_A11G2835) and qMi-C14
(Gh_D02G0257 and Gh_D02G0259) QTL regions.
Gene ID Gene Name Gene Description M-120 C201 M-120 C201 Early Late
Gh_D02G0257 RLP12 Receptor-like protein 12 1.36 0.11 1.85 -1.92 1.25 3.77
Gh_D02G0259 RLP12 Receptor-like protein 12 1.42 0.00 0.55 -1.07 1.42 1.62
Gh_A11G3090 PUB21 U-box domain-containing protein 21 1.17 -1.04 0.18 -1.62 2.21 1.8
Gh_A11G2835 RGA3 Putative disease resistance protein RGA3 4.33 -2.26 -0.25 -2.26 6.58 2.01
Log2 fold change compared to
non-inoculated controls
Early Late
Log2 fold change in M-120
compared to C201
Numberofreads
(inmillions)
2019 IPBGG Annual Retreat ● May 13-14, 2019 ● Amicalola Falls State Park & Lodge, Dawsonville, GA.