RNA-seq project: presented at Beltwide Cotton Conferences, 2019

1. 1Sameer Khanal, 1,2Pawan Kumar, 1Larissa Guimaraes, 1,3Mychele Batista da Silva, 1Rippy Singh, 4Robert
L. Nichols, 5Richard F. Davis, 1Peng Chee
Comparative Transcriptome Analysis of Near Isogenic Lines for
Root-Knot Resistance QTLs, qMi-C11 and qMi-C14
1Cotton Molecular Breeding Laboratory, Department of Crop and Soil Sciences, University of Georgia, Tifton, GA • 2USDA-ARS,
Salinas, CA; 3University of California, Davis, CA • 5USDA-ARS, Tifton, GA • 4Cotton Incorporated, Cary, NC
The southern root-knot nematode (Meloidogyne incognita; RKN) is the most destructive parasite of
Upland cotton, particularly in regions with light or sandy soil texture. Near immunity level of
resistance within the cultivated gene pool and molecular tagging of major quantitative trait loci
(QTLs) have enabled breeders to deploy host plant resistance as the most practical way of combating
RKN infestation. Advancement and accessibility of next generation sequencing would help identify
genes and pathways involved in RKN-cotton interactions. In the current study, we aim to build-on
our time-course RNA sequencing experiments in resistant (M-120) and susceptible (Coker 201) lines
and extend that to understand transcriptomic landscapes of host-pathogen interactions in isolines
carrying independent introgressions at major RKN resistance QTLs, qMi-C11 and qMi-C14.
M-line series derived from Auburn 623, a root-knot resistance (RKR) source, show two-gene
resistance model, which were tagged with molecular markers to G. hirsutum chromosomes 11 and
14 (Gutiérrez et al. 2010, He et al. 2014, Shen et al. 2006, Ynturi et al. 2006). The QTLs were further
fine-mapped to genomic regions carrying a number of putative disease resistance genes (Kumar et
al. 2016, Shen et al. 2010).
Histopathological study with near isogenic lines showed different mode of actions during distinct
stages of pathogen development (Da Silva et al. 2018*). qMi-C11 either induced an increase in RKN
emigration (at J2 stage) from cotton roots or nematodes failed to develop beyond SJ2 stage, while
qMi-C14 caused nematodes to cease developing beyond J4 stage.
While additive effects at the two QTLs represent major genetic basis of resistance, additive-by-
additive (epistatic) interaction also represents important genetic component of resistance (He et al.
2014).
Difference in mode of actions and the presence of epistatic interaction between the two QTLs make
it prudent to study transcriptomic diversity in near-isogenic lines. Accordingly, the objectives of the
ongoing study are as follows:
1. Identifying differentially expressed genes between resistant (M-120 and isogenic lines carrying
qMi-C11 or qMi-C14) and susceptible genotypes
2. Building genomic data and calling variants (SNPs and INDELs) between resistant and susceptible
genotypes for marker development
3. Identification of candidate resistance genes at the QTL regions using differential expression,
variant call and functional analysis
RNA-seq libraries:
Transcriptome alignment From a total of 72 libraries, 818,690,965 (~800 million) paired-end reads
were generated, of which 675,571,945 (~675 million; ~83%) were concordantly mapped (using
HISAT2) to the G. hirsutum reference (Fig. 3). Based on overall alignment (concordant, discordant
or one of a mate-pair match), approx. 88% of sequences aligned to the reference genome.
Nematode RNA were also recovered from the treated libraries. C-201, the susceptible line, showed
highest proportion of nematode RNA (Fig. 4).
Variant Call analysis:
Fig. 5 shows genome constitution at chromosome 11 and 14 for CH-11 and CH-14 isolines,
respectively.
Abstract
Materials and Methods
Background
Results
References
Da Silva et al. (2018) Resistance QTLs qMi-C11 and qMi-C14 in Cotton have Different Effects on the Development of Meloidogyne incognita, the
Southern Root-Knot Nematode. Plant Disease. https://doi.org/10.1094/PDIS-06-18-1050-RE. *draft version accessible online
Gutiérrez et al. (2010) SSR markers closely associated with genes for resistance to root-knot nematode on chromosomes 11 and 14 of Upland
cotton. Theor Appl Genet 121:1323–1337
He et al. (2014) Re-evaluation of the inheritance for root-knot nematode resistance in the Upland cotton germplasm line M-120 RNR revealed
two epistatic QTLs conferring resistance. Theor Appl Genet 127(6):1343–1351
Kumar et al. (2016) Fine mapping and identification of candidate genes for a QTL affecting Meloidogyne incognita reproduction in Upland
cotton. BMC Genomics 17:567
Development of near isogenic lines Fig. 1 illustrates the development of near isogenic lines differing
for the resistance QTLs qMi-CH11 and qMi-CH14
Time-course experiment Fig. 2 illustrates the time-course experiment with nematode count data.
RNA-seq analysis We are following the ‘new Tuxedo’ protocol reported by Pertea et al. (2016), with
our project specific modifications.
Reference genome and annotation JGI’s reference genome assembly Gossypium hirsutum v1.1
M-120 RNR Cocker 201
qMi-CH11
BC7F2
F1
BC7F1
qMi-CH14
X
Cocker 201X
Fig. 1 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
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ROOT-KNOTTOTALNUMBERS
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Coker 201
CH14
CH11
M-120
Fig. 2 Time-course study of nematode infections in resistant and
susceptible cotton genotypes. Current RNA-seq experiment sampled
nematode treated vs control individuals at these six time points.
Letters represent significance of grouping. Histological work done by
Mychele Batista da Silva.
Days after infection (DAI)
0.00
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NematodeRNA(%of
total)
Days after infection (DAI)
M-120 C-201 CH-11 CH-14
Fig. 4 Southern root-knot RNA detected in RNA-seq libraries
from three resistant (M-120, CH-11 and CH-14) and one
susceptible (C-201) genotypes infected with the nematode. Error
bars represent standard errors from two replications.
C-201 chromatin
QTL from M-120
M-120 introgression
Fig. 5 Graphical Genotypes using Variant Call from RNA-seq data. CH-11 isoline
mapped 168 variant alleles (16 INDELs and 152 SNPs) in chromosome 11, while CH-
14 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
Pertea et al. (2016) Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Proto 11:1650-1667
Shen et al. (2006) QTL mapping for resistance to root-knot nematodes in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source. Theor Appl Genet 113(8):1539–1549
Shen et al. (2010) Fine mapping QMi-C11 a major QTL controlling root-knot nematodes resistance in Upland cotton. Theor Appl Genet 121(8):1623–1631
Ynturi et al. (2006) Association of Root-Knot Nematode Resistance Genes with Simple Sequence Repeat Markers on Two Chromosomes in Cotton. Crop Sci 46:2670–2674
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Paired-endsequences
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aligned concordantly 0 times aligned concordantly exactly 1 time aligned concordantly >1 times
C-201 libraries CH-11 libraries CH-14 libraries M-120 libraries
Fig. 3 Number of mapped and unmapped paired-end reads from quality controlled RNA-seq libraries from nematode resistant and susceptible genotypes.
Sequences were mapped against JGI’s reference genome assembly Gossypium hirsutum v1.1.