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Front. Genet. | doi: 10.3389/fgene.2017.00190

RNA–Seq of plant–parasitic RNA-seq of plant-parasitic nematode Meloidogyne incognita at various stages of its development

Inchan Choi1,  Parthiban Subramanian1, Donghwan Shim2, Byung-Ju Oh1 and  Bum-Soo Hahn1*
  • 1Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, Rural Development Administration, South Korea
  • 2Department of Forest Genetic Resources, National Institute of Forest Science, South Korea

INTRODUCTION
Meloidogyne incognita is a plant–parasitic plant-parasitic nematode which attacks a diverse variety of plants (Mitkowski and Abawi, 2003). The life cycle of this nematode is comprised of five stages namely eggs, J2, J3 and J4 juveniles and adults. Female adults are important in the life cycle as they bear produce eggs which hatch into infective J2 that travels and attaches to plant roots to for continue continuation of its life infective cycle. Among the stages of its life cycle, the J3, J4 and female are sedentary and occur after the motile J2 infects a plant root. It is an obligate plant–parasite plant-parasite and possesses a wide host range of over 100 plants including cotton, tobacco, legumes, vegetable crops, spices and coffee. It also is one of the most widely spread plant pest and has been reported from Asia, Africa, North, Central and South America, the Caribbean, Europe and Oceania (CABI Data Report, 2017).
Hitherto employed methods have made use of nematicides that can lead to environmental pollution and their use has been banned in developed countries (Collange et al., 2011). Molecular studies on the nematode can help us understand the development and physiology of nematode which can be used to develop control strategies in the future. There have been earlier reports on the transcriptome from egg, J2 juveniles of M. incognita and from infected plant tissues (Dubreuil et al., 2007; McCarter et al., 2003; Neveu et al., 2003; Schaff et al., 2007). The present report was aimed at expanding the currently available information on transcriptome of M. incognita. We aimed to study RNA–SeqRNASeq data independently for the endophytic J3, J4 and female stages, as well as exophytic egg and J2 stages of the nematode.
VALUE OF THE DATA
• M. incognita is a notorious plant parasite causing huge economic loss in agricultural crops.
• Five morphologically distinct developmental stages of the nematode were selected to collect samples and respective transcriptomes were sequenced.
• The present data can be used to compare and confirm previously reported partial transcriptome of M. incognita as well be a useful resource to study the stage–wise stage-wise changes occurring in the nematode.
DATA
We used RNA–Seq RNA-seq to compare the transcriptomes of five stages namely eggs, J2, J3, J4 and female of the root-knot nematode. Samples were collected, RNA isolated using LiCl2 method and RNA sequencing was carried out using HiSeq2500 platform from Illumina Inc. RNA integrity number (RIN) for the samples were >7 (8.5, 9, 9.4, 9.4, 7.8 for egg, J2, J3, J4 and female stages respectively). Egg stage consisted of the highest number of clean reads at 56,902,902 and J2, J3, J4 and female stages consisted of 50,762,456; 40,968,532; 47,309,223 and 51,730,234 clean reads, respectively (Table 1). Three runs were performed for each stage and raw sequences were submitted to Sequence Read Archive (SRA). Contigs were obtained by assembly using TRINITY software and data deposited to the NABIC sever (http://nabic.rda.go.kr/) maintained by Rural Development Administration, Korea (Seol et al., 2016). We were able to identify 48,024 contigs in egg stage followed by 61,315 in J2 stage, 60,697 in J3; 48,398 contigs in J4 and 41,489 contigs in female stage. The accession numbers and other related statistics are given in Table 1. The raw data was submitted to NCBI SRA portal and the accession number for egg stage are SRR5684407, SRR5684403 and SRR5684417; J2 stage – SRR5684416, SRR5684412 and SRR5684414; J3 stage – SRR5684413, SRR5684415 and SRR5684404; J4 stage – SRR5684408, SRR5684406 and SRR5684405; female stage – SRR5684410, SRR5684409 and SRR5684411.

Table 1. Summary of sequencing, process, assembly and submission of M. incognita transcriptome at its developmental stages
Attributes Egg J2 J3 J4 Female Combined
Pre-assembly
Total rRaw reads 70,310,943 61,150,827 51,342,127 58,016,948 64,618,511
Total high quality sequences (readsClean reads 56,902,902 50,762,456 40,968,532 47,309,223 51,730,234
NCBI bioproject ID PRJNA390559 PRJNA390559 PRJNA390559 PRJNA390559 PRJNA390559
NCBI biosample IDs SAMN07174878
SAMN07174879
SAMN07174880 SAMN07174881
SAMN07174882
SAMN07174883 SAMN07174884
SAMN07174885
SAMN07174886 SAMN07174965
SAMN07174966
SAMN07174967 SAMN07174968
SAMN07174969
SAMN07174970
NCBI SRA IDs SRR5684407
SRR5684403
SRR5684417 SRR5684416
SRR5684412
SRR5684414 SRR5684413
SRR5684415
SRR5684404 SRR5684408
SRR5684406
SRR5684405 SRR5684410
SRR5684409
SRR5684411
Platform Illumina HiSeq 2500 Illumina HiSeq 2500 Illumina HiSeq 2500 Illumina HiSeq 2500 llumina HiSeq 2500
Number of cleaned reads mapped to genome 27,285,236 25,208,092 19,962,672 24,502,219 26,329,321
Number of tTotal bases of cleaned reads mapped to genome 5,510,011,716 5,090,859,842 4,031,018,384 4,276,893,153 5,317,234,914
Post-assembly
NABIC accession number NN-3882-000001 NN-3881-000001 NN-3883-000001 NN-3884-000001 NN-3885-000001
Number of contigs 48,024 61,315 60,697 48,398 41,489 113,421
N50 (bp) 900 973 780 1,142 1,023 1,338
Number of contigs mapped to genome 37,330 45,981 41,515 40,190 32,531 197,514


MATERIALS AND METHODS
Nematode propagation and collection
Root-knot nematode Meloidogyne incognita was maintained in tomato plants (Solanum lycopersicum var. Rutgers) under greenhouse conditions (25°C, 16/8h day/night period). For collection of the nematode at its developmental stages, a new set batch of tomato plants were infected. Eggs used for infection were collected from infected tomato plants by harvesting and treatment of the infected roots with 10% NaClO for 5 mins followed by continuous washing over a series of filters 100 µM, 25 µM mesh filter to trap the eggs. Around 1,000 eggs were used to infect each plant and the newly infected plants were continuously monitored to confirm the stage of nematodes in the roots.
Sample collection was carried out in the following order. Around two and six weeks after infection presence of J3, J4 and female stage nematodes was identified by manual inspection of root-knots under a stereo microscope (Discovery v12, Zeiss Germany). Eggs samples were collected from egg masses and purified by sucrose gradient centrifugation (35% sucrose, 1,500 rpm and room temperature). J2 samples were obtained by hatching a portion of the collected eggs. Eggs of the nematodes were suspended in sterile double distilled water at 25°C for five days and hatched J2 were filtered using 5–7 KimWipes® onto clean Petri dish placed on a lab table. Nematodes at J3, J4 and female were collected from infected roots around two and six weeks after infection using the following protocol. The harvested roots containing root-knots were washed, chopped and treated with 7.7% cellulase and 15.4% pectinase followed by rinsing in running water and filtering through a 75 µM mesh. For every five roots, 15 mL of cellulase and 30 mL of pectinase was used during enzyme treatment. Rinsing of the roots following enzyme treatment was carried out over a filter (75 µM mesh) and nematodes retained on the surface of the filter were suspended in distilled water and handpicked using a pipette under a stereo microscope. Two hundred milligrams (200 mg) of samples for each stage at egg, J2, J3, J4, and female were collected from the roots of tomato plants and were snap frozen using liquid nitrogen followed by storage at -80°C until further processing.
Sample collection was performed carried out in the following order. Briefly, the collection of nematodes started with J3 nematodes which were collected first followed by J4, and females. Egg masses were harvested at the end of which one part was used to collect the eggs and another part of the eggs were hatched to collect J2 juveniles. To explain indetail, Aat the end of six weeks after infection presence of J3 stage nematodes was identified by manual inspection of washed and crushed root-knots under a stereo microscope (Discovery v12, Zeiss Germany). Nematodes at J3, J4 and female were collected from infected roots using the following protocol. The harvested roots containing root-knots were washed, chopped and treated with 7.7% cellulase and 15.4% pectinase followed by rinsing in running water and filtering through a 75 µM mesh. For enzyme treatment, the mixture consisted ofFor every five roots, 15 mL of cellulase and 30 mL of pectinase at a ratio of 15 mL and 30 mL respectively for five rootswas used during enzyme treatment. Rinsing of the roots following enzyme treatment was carried out over a filter (75 µM mesh) and nematodes retained on the surface of the filter were suspended in distilled water and handpicked using a pipette under a stereo microscope. Eggs samples were collected as mentioned above and purified by sucrose gradient centrifugation (35% sucrose, 1,500 rpm, room temperature). J2 samples were obtained by hatching a portion of the collected eggs. Eggs of the nematodes were suspended in sterile double distilled water at 25°C for five days and hatched J2 were filtered using 5–7 KimWipes® onto clean Petri dish placed on a lab table. Two hundred milligrams (200 mg) of samples for each stage at egg, J2, J3, J4, and female were collected from the roots of tomato plants and were snap frozen using liquid nitrogen followed by storage at -80°C until further processing.
Total RNA extraction, library preparation and RNA-SeqRNA-seq
To obtain high–throughput transcriptome data of M. incognita, we used Illumina–based NGS sequencing. RNA was extracted from nematodes collected at five developmental stages, i.e. egg, J2, J3, J4 and Fe (female) with three technicalindependent replicates at each stage. Total RNA from the samples was quantified using Nanodrop spectrophotometer (ThermoFisherThermo Scientific), quality-assessed by RNA 6000 Nano assay kit (Agilent) and Bioanalyser2100 (Agilent). NGS sequencing libraries were generated from one microgram of total RNA using TruSeqTruseq RNA Sample Prep Kit (Illumina) according to the manufacturer's protocol. In brief, the poly–A containing RNA molecules were purified using poly–T oligo attached magnetic beads. After purification, the total poly (A)+ RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved mRNA fragments were reverse transcribed into first strand cDNA using random primers. Short fragments were purified with a QiaQuick PCR extraction kit and resolved with elution buffer for end reparation and addition of poly (A). Following this, the short fragments were connected with sequencing adapters. Each library was separated by adjoining distinct multiplex identifier (MID) tag. The resulting cDNA libraries were then paired-end sequenced (2x101 bp) with Illumina HiSeq™ 2500 system.
Preprocess, de novo assembly and mapping
Complete paired–end paired end sequences were obtained as individual fastq files (forward and reverse) from the images by CASAVA v.1.8.2 base calling software with ASCII Q-score (offset 33). Adaptor sequences, minimum length (90 bp), bad fraction (>10%) and low quality bases with PHERED scores (Q) ≤ 20, were removed using CLC genome cell (v.4.0).
We cleaned paired–end reads using prinseq–vlite v0.19.3 with parameters (-min_len 50 -min_qual_score 10 -min_qual_mean 20 -derep 14 -trim_qual_left 20 -trim_qual_right 20) (Schmieder and Edwards, 2011). De novo transcriptome assembly was carried out using the clean paired–end paired-end reads with the Trinity assembly pipeline (Trinity Release v2.0.2) using default parameters (Grabherr et al., 2011). The final outputs from TRINITY assembly were clustered by cd-hit-est as a set of FASTA sequences (contigs) for each stage of the nematode (Fu et al., 2012). The contigs were mapped against the reference of M. incognita genome (ASM18041v1) using Burrows–Wheeler Aligner (BWA) (Li and Durbin, 2009). And then,This was followed by data was summarizedsummarization with bamtools stats option and python script. The summary of the transcriptomes of various stages of M. incognita are listed in Table 1. The assembled transcriptomes can be used for applications such as gene discovery or comparison of M. incognita transcriptome with other plant-parasitic nematodes to understand the molecular origin of their parasitism.

Keywords: Plant parasite, Root-knot nematode, Developmental stages, RNA-Seq, Transcriptome

Received: 26 Jun 2017; Accepted: 14 Nov 2017.

Edited by:

Graziano Pesole, Università degli studi di Bari Aldo Moro, Italy

Reviewed by:

Tirza T. Doniger, Bar-Ilan University, Israel
Claudia Ghigna, Institute of Molecular Genetics National Research Council (IGM-CNR), Italy
Francesca De Luca, Istituto per la Protezione Sostenibile delle Piante (CNR), Italy  

Copyright: © 2017 Choi, Subramanian, Shim, Oh and Hahn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Bum-Soo Hahn, Rural Development Administration, Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, 370, Nongsaemyeong-Ro, Wansan-Gu, Jeonju, 54874, South Korea, bshahn@korea.kr