Selection and validation of suitable reference genes for quantitative real time PCR analysis of gene expression studies in patchouli under Meloidogyne incognita attack and PGPR treatment

Journal Pre-proof Selection and validation of suitable reference genes for quantitative real time PCR analysis of gene expression studies in patchouli under Meloidogyne incognita attack and PGPR treatment

Bitupon Borah, Marine Hussain, Sawlang Borsingh Wann, Brijmohan Singh Bhau PII:

S2452-0144(20)30039-X

DOI:

https://doi.org/10.1016/j.genrep.2020.100625

Reference:

GENREP 100625

To appear in:

Gene Reports

Received date:

8 September 2019

Revised date:

21 January 2020

Accepted date:

6 February 2020

Please cite this article as: B. Borah, M. Hussain, S.B. Wann, et al., Selection and validation of suitable reference genes for quantitative real time PCR analysis of gene expression studies in patchouli under Meloidogyne incognita attack and PGPR treatment, Gene Reports (2018), https://doi.org/10.1016/j.genrep.2020.100625

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© 2018 Published by Elsevier.

Journal Pre-proof Selection and validation of suitable reference genes for quantitative real time PCR analysis of gene expression studies in patchouli under Meloidogyne incognita attack and PGPR treatment Bitupon Borah1,2,*, Marine Hussain1,2, Sawlang Borsingh Wann1, and Brijmohan Singh Bhau# 1

Biological Sciences & Technology Division, CSIR-North East Institute of Science & Technology, Jorhat 785006, Assam, India 2

Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India.

#

Department of Botany, Central University of Jammu, Rahya-Suchani (Bagla), Samba-181 143, Jammu, Jammu & Kashmir, India

Bitupon Borah - [email protected] Marine Hussain - [email protected]

Brijmohan Singh Bhau – [email protected]

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Sawlang Borsingh Wann - [email protected]

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E-Mail ID of authors:

Corresponding author

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E-mail address: [email protected] (Bitupon Borah)

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Journal Pre-proof ABSTRACT Patchouli (Pogostemon cablin) is an economically important aromatic plant and is often vulnerable to nematode attack (Meloidogyne incognita). The application of PGPR has tremendously changing disease management approach in plants and a systematic understanding the molecular mechanism behind it is needed to control over the disease more precisely. Since, qRT-PCR is the most frequently used molecular tools in studying molecular mechanism but is greatly dependent upon suitable reference or housekeeping genes. This study is aim to select suitable reference genes for qRT-PCR experiments in patchouli under the condition of nematode attack and PGPR treatments. Expression pattern of six reference genes (Actin, EF1, ACP, COX3, GAPDH and Pat18S) were statistically analyzed and validated using different algorithms viz., ΔCt, BestKeeper, GeNorm, NormFinder

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and RefFinder. The result showed that the Actin and ACP are the most suitable reference genes and the combination of ACP & GAPDH gene pairs can also be used whenever the experiment needed.

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Keywords: Pogostemon cablin; Meloidogyne incognita; PGPR; qRT-PCR; Reference gene.

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Journal Pre-proof 1. Introduction Patchouli (Pogostemon cablin Benth) belonging to Lamiaceae family is an economically important aromatic and medicinal plant with enormous industrial potentials. The plant favors the hot tropical and subtropical climatic conditions and has been extensively cultivated in countries like Indonesia, Malaysia, India and China (Mahanta et al., 2007). The essential oils from patchouli contains patchouli alcohol which has great commercial applications including fixative in perfumery industries, cosmetics, incense stick production and food flavoring industries with some added therapeutic properties like anti-depressant, anti-inflammatory, antiseptic, aphrodisiac, insecticides etc. (Bunrathep et al., 2006). However, despite the great economic value and extensive

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cultivation nematode disease caused by root-knot nematode (Meloidogyne incognita) has reduced the crop productivity to a great extent resulting in a considerable loss in oil yield (Bhau et al., 2016). Many strategies have been employed for the management of M. incognita infection such as use of chemical nematicides,

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resistance plant selection and application of Plant Growth Promoting Rhizobacteria (PGPR) (Barker 1998; Mahajan, 1978; Noweer and Susan, 2005; Molinari, 2011). PGPR provides defense to the host plant against

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plant pathogens through the induction of systemic resistance. Systemic resistance is generally categorized into

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induced systemic resistance (ISR), which is triggered by rhizobacteria expressing JA/ET-responsive genes and on the other hand systemic acquired resistance (SAR) by other agencies where mostly SA responsive genes were

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involved (Pieterse et al., 2014). A greater understanding of the SA, JA and ET signaling pathways and how they modulate each other will provide insight into the mechanisms underlying the activation and regulation of

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defense responses and made it easy to deploy new strategies to produce plants with more resistance.

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Gene expression study is generally performed to understand the function of genes involved in SA, JA and ET pathways. Quantitative real-time PCR (qRT-PCR) is currently the most accurate technique for gene expression analysis by quantifying the amount of messenger RNA (mRNA). It has several advantages over northern blotting and in situ hybridization like high sensitivity, wide dynamic range, reproducibility and less labor intensive since no post-PCR processing is required (Nolan et al., 2006; Penna et al., 2011). However, the qRT-PCR reliability is influenced by various factors, such as amount of sample, integrity of the RNA, reverse transcription efficiency, efficiency of PCR amplification, cDNA quality and all these factors are greatly dependent upon normalization (Derveaux et al., 2010). To reduce the potential bias of these factors, normalization of internal reference genes are used to obtain accurate biologically meaningful expression values (Thellin et al., 1999). The reference genes are generally expressed in uniform levels inside the cells under (Hu et al., 2009) however, depending upon the different experimental conditions, tissues types and life cycle an organism the levels of the expression of these genes may vary greatly (Gutierrez et al., 2008). Therefore, it is

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Journal Pre-proof necessary to verify the stability of the reference genes which can be used under specific experimental conditions of interest (Gueanin et al., 2009). However, to the best of our knowledge, no detailed analysis of qRT-PCR reference genes has been carried out in P. cablin plants subjected to nematode infections and PGPR application. In this study, six housekeeping genes viz., Actin (ACT), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acyl carrier protein (ACP), Patchouli 18S ribosomal RNA (Pat18S rRNA) and cytochrome c oxidase (COX) were analyzed for their compatibility to use as a reference gene in root knot nematode infected and PGPR treated patchouli plants across a time course upto 30 day after inoculation (DAI). The stability and ranking of the candidate reference genes were evaluated with the four different algorithms viz., the comparative

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ΔCt method, geNorm, NormFinder and Bestkeeper. Finally, RefFinder was used to give a comprehensive final

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rank for each of the candidate gene based on the result generated from the four algorithms.

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Journal Pre-proof 2. Material and methods 2.1. Plant material and growing conditions Three months old patchouli plantlets were planted in earthen pot containing loamy soil under a greenhouse where temperature and relative humidity were maintained at 28 °C / 18 °C ± 2 °C and 60-70% respectively. 2.2. Preparation of bacterial inoculums We selected two rhizobacterial strains isolated from patchouli rhizosphere after initial screening. The

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selected bacterial strains were BG2 (Pseudomonas putida; Genbank acc no. KU312064.1) and BC1 (Bacillus cereus; Genbank acc no. KX762284). The bacterial strains were grown in LB (Luria-Bertani) broth and incubated at 30 ± 2 °C for 36 hours. The fully grown bacterial cultures were diluted to a final concentration of

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107CFU ml-1 using 1% sugar solution additionally 1% carboxymethyl cellulose was added for attachment.

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2.3. Preparation of nematode inoculums

Meloidogyne incognita infected patchouli plants were collected from the CSIR-NEIST experimental

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field. Galled roots were collected from the infected patchouli plants. Collection of nematode eggs from the galled roots was carried out according to the method described by Atamian et al. (2012). Infectious J2 stage

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larvae of M. incognita were obtained by incubating the nematode eggs for 2 -3 days at 28 °C. Numbers of nematodes were estimated under a light compound microscope in the suspension and patchouli plantlets of three

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2.4. Experimental design

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months old were inoculated with 1500 J2 stage M. incognita larvae per plant by drenching with water.

Complete randomized design was opted for the experiment taking 10 replicates for each treatment. The treatment includes: (a) Control plant (no nematode) (b) nematode inoculated plant (c) Nematode inoculated + BG2 bacterized plant (d) Nematode inoculated + BC1 bacterized plant (e) Nematode inoculated + 0.3% carbofuran and (f) Nematode inoculated + BG2 + BC1 bacterized plant. Matured young leaves tissues were collected at the time intervals of 1, 7, 14, 21 and 30 days after inoculation (DAI). 2.5. Total RNA extraction and cDNA synthesis Total RNA was extracted from the leave samples using the TRIzol® Reagent (Invitrogen TM, USA) according to the manufacturing protocol. Possible DNA contamination was removed from the extracted RNA samples by treating sample with DNase I (RNase-free) (InvitrogenTM, USA) enzyme. 10 μg of RNA was mixed with 1 μl (2U) of DNase and incubated at 37 °C for 30 minutes.

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Journal Pre-proof High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems™) was used to synthesize the first-strand cDNA. Breifly, 1µg of RNA template was mixed with 10 μl of 2X RT buffer and 1 μl of 20X enzyme mix with a final volume make up to 20 μl using nuclease free water. 2.6. Selection of candidate reference genes and primer design The most common reference genes in other plants were: Actin, EF1, ACP, COX3, GAPDH and Pat18S (Table 1). In present study, primers for Actin, Pat18S and GAPDH were manually designed using the sequence available in the NCBI database. For EF1, COX3 and ACP where nucleotide sequences are not available; primers were designed by multiple sequence alignment for the conserved region of closely related species and subjected

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to PCR amplification, since most of the reference genes were conserved housekeeping genes. The amplified product was then sequenced, checked with primer BLAST and final primer set was designed. The primers were designed according to the following conditions: Tm (melting temperature) between 50 and 65°C (optimum Tm

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of 55°C); primer length between18-22 base pairs (bp); GC content of 40–60% and the size of the PCR product was chosen between 100 and 500 bp. Further the primers were checked in oligo-calculator for GC content or

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2.7. qRT- PCR analysis

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presence of any palindromic sequence.

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qRT-PCR for the candidate reference genes was performed in StepOnePlus™ Real time PCR System (Applied Biosystems™, USA) taking SYBR Green (Thermo Fisher Scientific, USA) as reporter dye. The 20 µl

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reaction mixture contained 2 μL cDNA (5 times diluted), forward and reverse primer (10 pmol each) and nuclease free water was added to make up the volume. Negative controls were prepared accordingly for the

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primer pair of each gene by replacing the template with water. PCR conditions were set with an initial denaturation period of 10 minutes at 95 °C followed by 40 cycles of 95 °C for 15sec, 50 °C for 15 sec. Melt curve was analyzed at the end with and temperature increase of +0.5 °C beginning from 50 °C and ending at 95 °C. A standard curve was prepared using a 10-fold dilution series upto 5 times ([1/1], [1/10], [1/10 2], [1/103] and [1/104]) of the cDNA from all the tested samples as a template to calculate the amplification efficiencies (E) of the primers (Radonic et al., 2014). 2.8. Analysis of gene expression stability The expression stability of the six candidate reference genes were analyzed under different experimental setups using the four common statistical software algorithms viz. comparative ΔCt, (Silver et al., 2006) geNorm (Vandesompele et al., 2002), BestKeeper (Pfaffl et al., 2004) and NormFinder (Andersen et al.,

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comprehensive

ranking

of

the

six

reference

genes

was

obtained

by

RefFinder

(http://150.216.56.64/referencegene.php?type=reference) using raw Ct values. Comparative ΔCt method determines the stability of the candidate reference genes by calculating the change in the average standard deviation of Ct values for a particular gene when compared to all the other genes being assessed. The lowest standard deviation is considered to the highest stability. Analysis of expression stability by the geNorm and Normfinder requires relative quantitative values rather than Ct values. The Ct values were first transformed to relative quantities by using the formula 2 ΔCt, where, ΔCt = the minimum Ct value – Ct value of samples. The geNorm employs geometric mean to determine

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the best reference genes by calculating expression stability value (M) for each gene. Lower the M value implies higher stable expression. geNorm calculates the optimal number of reference genes by pairwise variation (V) with a threshold of 0.15. NormFinder uses ANOVA to calculate the expression stability value (SV) of the genes

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with the lowest stability value (SV) has the most stable expression (Andersen et al., 2004).

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In contrast to geNorm and Normfinder; BestKeeper uses pairwise correlations and only requires the raw Ct values for calculating and identifying the stability of candidate reference genes. Here the raw Ct values

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along with the PCR efficiency data were loaded and based on the calculation of standard deviation (SD), the percentage of covariance (CV), and the correlation coefficient (r). The results of stable of reference genes were

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generated. The higher the SD and CV values correspond to the least stable reference genes (Pfaffl et al., 2004). Reference genes with lower (<1) SD values were considered as stable and suitable internal reference for qRT-

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PCR (Xiao et al., 2005). The final comprehensive ranking of the reference genes was generated by RefFinder a

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web-based comprehensive tool using the raw Ct values.

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Journal Pre-proof 3. Results 3.1. Confirmation of primer specificity and efficiency From the agarose gel electrophoresis it was confirmed the single amplicon of the PCR product with the

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expected size ranged from 179 to 273 bp (Fig. 1).

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Fig. 1. Agarose gel electrophoresis (2.0%) indicates amplification of a single PCR product of the

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expected size for ACTIN, EF, SLEEPER, GAPDH1, GAPDH2, TUB, UBI, DSK2A, CYC, and PLA). Melt curve analysis indicated one single peak that corresponds to a specific melting temperature for all

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the 6-candidate genes (Fig. 2).

Fig. 2. Melting curves of the genes show single peaks.

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Journal Pre-proof The amplification efficiencies (E) and the linear correlation coefficients (R 2) of these reference genes were found between 94.54% to 107.97 and 0.990 to 0.997 respectively (Table 1).

Gene name Accession Primer sequence No

GU385981 GCTGCTGCAACAAGATGGACGC (F) HQ694771 CCTTGTACCAGTCGAGGTTGGT (R)

Beta Actin KP676600

ACP

COX3

GAPDH

base

length

pairs

(bp)

22

21

CGAGAGCAATGTAGGCTAGC (R)

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KF887919 GGCGAGAAACGCACTGCTGAAGTA(F) 24 CCTTGGAAGGATCAACTTTCTCG (R)

23

KT179401 CCACGTTGGAAGGACATCATACC(F) KT179395 CCTTCCCCGCGAGTATAGCAT (R) KT179394 KT179393

23

KP676601

GTGCACTCCATCACCGCTACT (F)

(%)

189

2.08

107.97

0.990

206

2.03

102.77

0.996

209

2.04

104.17

0.996

273

1.95

94.54

0.997

21

179

1.96

95.88

0.996

219

2.01

100.91

0.992

21 20 21

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CGATGGTTCACGGGATTCTGC (R)

n factor

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TCGCCGTTCGGACCAAATAA (F)

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FJ980282

R2

22

CGACTCTGGTGATGGTGTCAG(F)

GTGGGTACACGGAATGCCATA(R)

Pat18S

Product Amplificatio Efficiency

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EF1

No of

Table 1 Primer sequence of the candidate reference gene and their characteristics.

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3.2. Expression profiles of the selected reference genes

The overall differences in the expression level of all the 6 candidate reference genes were compared by

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plotting the medium Ct values in the box-plot chart (Fig. 3). The mean Ct values of the candidate reference

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genes at all the time intervals revealed that a minimum of 27.98±1.78 (ACP) (mean ± SD) and a maximum of 35.67±0.49 (GAPDH) for highest and lowest expression levels. The remaining genes showed moderate expression levels with Ct ranging between 30.44±2.39 to 32.23±3.07. The coefficients of variation (CV) of the six reference genes were found to be 9.48% (EF1), 7.87% (Actin), 6.38% (ACP), 10.94% (COX3), 12.09% (Pat18S) and 1.37% (GAPDH).

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Fig. 3. Expression levels (Ct values) for the six candidate reference genes. (Lines across the Box-plot

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graph of Ct values represent the median values. Lower and upper boxes show the 25th percentile to the 75th

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percentile. The whisker caps represent the maximum and minimum values).

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3.3. GeNorm Analysis

The output of GeNorm is a variable result across all the time periods under study. For day 1 and day 21,

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ACP/GAPDH gene pair showed most stable combination of reference gene with M value of 0.2353 and 0.1781 respectively. EF1 and COX3 were found to be the most unstable reference gene from day1 to day 21 with M

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value ranging from 0.4411 to 1.4063. In contrast to this EF1/ACP gene pair showed highest stability with a M value of 0.2027 and when considered across all the time intervals the result showed a considerably high M value

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(M >> 1.5) for all the candidate reference genes with the highest M value found for ACP with 3.0559 and lowest for the COX3/Pat18S gene pair with the M value of 1.6093 which is greater than the threshold limit of 1.5 and hence none of the all 6 candidate reference genes were considered for stable expression according to GeNorm analysis (Fig. 4).

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Fig. 4. Gene expression stability values (M) and ranking of 6 reference genes as assayed by GeNorm

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The V2/3 value of the reference genes were found below 0.15 in 1 DAI, 7 DAI and 21 DAI with 0.077,

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0.087 and 0.131 respectively) suggested that two reference genes can be used for normalization if necessary (Fig. 5). In DAI 21 and DAI 30, V3/4 value was found to be lower than 0.15 suggesting that three reference

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genes were sufficient for accurate normalization. When all the time periods were considered, no pair of genes

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were found to have pairwise variation value greater than 0.15 is not recommended.

Fig. 5. Pairwise variation (V) of the candidate reference genes calculated by geNorm. Vn/Vn+1 value were used for decision of the optimal number of reference genes.

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Journal Pre-proof 3.4. NormFinder analysis The stability values obtained from NormFinder software for all the 6 candidate reference genes were plotted in the graph (Fig. 6). In 1 DAI GAPDH was found to be the most stable gene for both GeNorm and NormFinder. COX3 and EF1 were found to be the most unstable candidate reference gene for all the time periods. ACP was found to be the most stable gene at DAI 14 to DAI 30. But in contrast to this ACP was found to be the most unstable and Actin as the most stable candidate reference gene when all the time periods were

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considered all together.

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3.5. BestKeeper analysis

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Fig. 6. Expression stability analysis of reference genes assayed by NormFinder software.

The stability values of the six reference genes as calculated by BestKeeper were presented in Table 2. The BestKeeper ranked GAPDH and ACP at the top of the list as most stably expressed genes among the six candidate reference genes. GAPDH showed highest stability in 1DAI, 7DAI and when all time periods were considered together with stability values of 0.134, 0.044 and 0.371 respectively. On the other hand ACP showed highest stable expression in 14DAI, 21DAI and 30DAI with stability value of 0.184, 0.071 and 0.201 respectively. According to BestKeeper result COX3 and EF1 were found to be the most unstable genes from 1DAI to 21DAI with stability value ranging from 0.372 to 0.781 for COX3 and 0.379 to 2.6 for EF1. When all the time periods were considered together Pat18S was found to be the most unstable candidate reference gene with stability value of 3.367. 3.6. ΔCt method

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Journal Pre-proof Comparative ΔCt analysis evaluates the variability in the candidate housekeeping gene expression

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based on the standard deviation and their comparisons were shown in box plot graph (Fig. 7).

Fig. 7. Comparative ΔCt approach to housekeeping gene selection. ΔCt variability in candidate

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housekeeping gene comparisons are shown as medians (lines). Lower and upper boxes show the 25th percentile

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to the 75th percentile. The whisker caps represent the maximum and minimum values. Based on ΔCt analysis, GAPDH and ACP were identified as the most stably expressed reference genes

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from 1DAI to 30DAI. GAPDH showed higher stability in 1DAI with 0.46 and ACP was found to be the most stable candidate reference gene from 7 DAI to 30DAI with stability value ranging from 0.73 to 1.02. COX3 and

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EF1 were found to be the most unstable genes from 1DAI to 30DAI and ACP was found to be the most unstable gene when all the time periods were considered together with stability value of 3.59 whereas Actin stands out as best candidate reference gene with a stability value 2.60 (Table 2).

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1 DAI

Delta Ct 14 DAI

7DAI

1 2 3 4 5 6

Genes Name GAPDH ACP ACTIN Pat18S EF COX

Stability Value 0.46 0.47 0.53 0.54 0.69 0.92

Genes Name ACP Pat18S GAPDH ACTIN EF COX

Stability Value 1.02 1.04 1.04 1.06 1.29 2.99

1 2 3 4 5

GAPDH ACP ACTIN Pat18S EF

0.134 0.157 0.195 0.438 0.443

GAPDH ACP ACTIN Pat18S COX

0.044 0.107 0.203 0.383 0.781

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COX

0.71

EF

2.6

21 DAI

Genes Name ACP ACTIN Pat18S GAPDH EF COX

Stability Genes Value Name 0.55 ACP 0.56 GAPDH 0.68 ACTIN 0.69 Pat18S 0.72 COX 0.72 EF BESTKEEPER ACP 0.184 ACP ACTIN 0.252 GAPDH Pat18S 0.336 ACTIN GAPDH 0.339 Pat18S COX 0.372 COX EF

0.379

EF

30 DAI

ALL

Stability Value 0.64 0.72 0.74 0.75 0.94 1.39

Genes Name ACP EF GAPDH ACTIN Pat18S COX

Stability Value 0.73 0.81 0.84 0.85 0.90 1.18

Genes Name ACTIN GAPDH COX Pat18S EF ACP

Stability Value 2.60 2.87 2.90 3.11 3.28 3.59

0.071 0.236 0.259 0.325 0.55

ACP ACTIN GAPDH EF Pat18S

0.201 0.241 0.43 0.458 0.559

GAPDH ACP ACTIN EF COX

0.371 1.527 2.049 2.374 3.055

0.943

COX

0.945

Pat18S

3.367

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Ranking Order

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Table 2 Stabilities of candidate reference genes ranked by Delta Ct and BestKeeper.

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3.7. RefFinder

According to the analysis from RefFinder (Table 3), the most stable genes were GAPDH (1DAI), ACP

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(7DAI to 30DAI) and Actin (All), whereas COX3 and EF1 were found to be the least stable from 1DAI to

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30DAI. ACP was found the least stable candidate reference gene when all the time periods were taken together.

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Delta CT BestKeeper Normfinder Genorm Recommended comprehensive ranking

ACP GAPDH Pat18S ACP | GAPDH ACP

Pat18S ACP EF

Delta CT BestKeeper Normfinder Genorm Recommended comprehensive ranking

ACP ACP ACP ACTIN | ACP ACP

ACTIN ACTIN ACTIN

Delta CT BestKeeper Normfinder Genorm Recommended comprehensive ranking

ACP ACP ACP Pat18S | GAPDH ACP

Delta CT BestKeeper Normfinder Genorm Recommended comprehensive ranking

ACP ACP ACP EF | ACP ACP

EF ACTIN EF

ACTIN GAPDH ACTIN COX | Pat18S ACTIN

GAPDH ACP GAPDH

Delta CT BestKeeper Normfinder Genorm Recommended comprehensive ranking

COX COX COX COX COX

DAI 14 Pat18S Pat18S Pat18S GAPDH Pat18S

GAPDH GAPDH GAPDH Pat18S GAPDH

EF COX COX EF COX

COX EF EF COX EF

DAI 21 ACTIN ACTIN GAPDH ACP Pat18S

Pat18S Pat18S Pat18S ACTIN ACTIN

COX COX COX COX COX

EF EF EF EF EF

DAI 30 GAPDH GAPDH GAPDH GAPDH GAPDH

ACTIN EF ACTIN ACTIN ACTIN

Pat18S Pat18S Pat18S Pat18S Pat18S

COX COX COX COX COX

ALL COX ACTIN COX ACTIN COX

Pat18S EF Pat18S EF Pat18S

EF COX EF GAPDH EF

ACP Pat18S ACP ACP ACP

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GAPDH GAPDH ACTIN

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EF EF GAPDH EF EF

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ACTIN

GAPDH

EF

GAPDH

6 COX COX COX COX COX

ACTIN Pat18S ACTIN Pat18S ACTIN

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GAPDH

5 EF Pat18S EF EF EF

DAI 7 GAPDH ACTIN ACP ACTIN Pat18S

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Method Delta CT BestKeeper Normfinder Genorm Recommended comprehensive ranking

Ranking Order (Better--Good--Average) DAI 1 1 2 3 4 GAPDH ACP ACTIN Pat18S GAPDH ACP ACTIN EF GAPDH ACP Pat18S ACTIN ACP | GAPDH ACTIN Pat18S GAPDH ACP ACTIN Pat18S

Table 3 Comprehensive ranking of candidate reference genes analyzed by RefFinder.

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Journal Pre-proof 4. Discussion Plant response to disease resistance is a complex cascade of many interlinking cellular regulatory mechanism and it is very difficult to understand primarily because of the complexity and incomplete knowledge of the molecular pathways. Patchouli being a commercially important plant has been greatly threatened by the attack of M. incognita and is a major bottleneck for its cultivations (Bhau et al., 2016). An accurate gene expression analysis of the defence related genes can help us to better understanding of the molecular mechanism underlying in host defense against M. incognita. Many of the previous study were carried out to control the nematode disease in patchouli as well as understanding the molecular pathways linking to the production of its essential oil (Borah et al., 2018; Deguerry et al., 2006; Yu et al., 2014). In recent years importance has been

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given to PGPR for sustainable agricultural method of disease control and enhancement of yield. PGPRs are providing benefits to the host plants by modulating gene expression in the host cells by producing some active

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metabolites. So, in order to understand these expressional profiles qRT-PCR is generally used which is the most powerful and commonly used molecular biology tool now a day. However, qRT-PCR reliability is greatly

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dependent upon internal reference gene selection as inappropriate use of reference genes could create deviation

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in results and hinders the characterization of the expression profile of the genes (Thellin et al., 1999). Therefore, it is essential to accurate selection of suitable reference gene for transcriptional, which is expressed stably in

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different experimental conditions. In recent times many studies have been performed in identifying suitable reference genes as internal control (Wang et al., 2017). However, no study has been conducted to select

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appropriate reference genes in M. incognita infected and PGPR treated P. cablin. In this study, we aimed to find out the suitable reference genes for the normalization of relative gene expression data in P. cablin under M.

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incognita attack and PGPR treatments for different time periods of post inoculation. In the process of evaluating stable reference gene it has been recommended that at least two statistical algorithms should be used (Xiao et al., 2014). This bias can be minimized by analyzing the data by using the four commonly used complementary statistical strategies viz., geNorm, NormFinder, BestKeeper, and comparative ΔCt to select the best reference gene and finally RefFinder for a comprehensive ranking. Previously Actin and Pat18S gene were used as internal controls in patchouli (Yu et al., 2014) but it may not be suitable in other experimental properties since no single housekeeping gene is universal (Artico et al., 2010). In this study, six housekeeping genes viz., Actin, GAPDH, EF1, ACP, COX3 and Pat18S were selected and their expression were examined across five different time periods. The result showed discrepancies in the selection of reference genes among all the four algorithms and this is possible because each algorithm follow different calculation (Zhang et al., 2016). In this study the ACP, GAPDH and Actin gene were found to be the most stable gene and

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Journal Pre-proof COX3 and EF1 were found continuously least stable across all the time periods. The Actin gene was often used as a reference gene in several studies (Chandna et al., 2012) and here also Actin showed most stable when all the time periods were considered together as suggested by RefFinder. The 18S gene is a small subunit of eukaryotic ribosomes (40S) has been commonly used as a reference gene for RT-qPCR normalization (Kozera and Marcin, 2013; Wilson and Doudna, 2012) but in some other study 18S gene showed least stable and inappropriate for gene expression analyses (Die et al., 2010; Fan et al., 2013). This study also suggests us that the Pat18S gene is not suitable as reference gene and showed moderate stability across all the experimental conditions. The GAPDH gene encodes the key enzyme involved in the glycolysis, gluconeogenesis and cell prolifereation and commonly used as a reference gene in many studies (McNulty and Toscano, 1995; Plaxton, 1996). GAPDH

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gene was found to be an unpredictable reference gene in many cases where it showed as the most stable reference gene and in some other cases as least stable (Bustin, 2000; Huis et al., 2010; Tong et al., 2009). Similarly, in the present study GAPDH showed less variation expression level and holds first rank in 1DAI

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across all the algorithms used. ACP (Acyl Carrier Protein) is a co-factor for various metabolic pathways

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synthesizing fatty acids, phospholipids and other signaling molecules (Byers and Gong, 2007; Crosby and Crump, 2012). Earlier study showed ACP as an average reference gene with lower stability value (Chandna et

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al., 2012; Zhang et al., 2015) however in this study ACP was found the most stable gene in all the selected algorithms as well as all the experimental time periods except when it was evaluated for all the time periods

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together where it was ranked the last. On the other hand we found that EF1 as unsuitable reference gene as it

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ranks continuously lower along with COX3 as compared to other genes.

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5. Conclusion

In this study the six commonly used reference genes were selected and subjected for the expression analysis using qRT PCR and then analyzed using five different algorithms viz., ΔCt, BestKeeper, GeNorm, NormFinder and RefFinder. After analyzing and considering the entire parameters of experimental properties Actin and ACP genes are found as the most stable reference genes that can be used for the further gene expression experiments using qRT-PCR in patchouli under the condition of nematode attack and PGPR treatments.

Acknowledgements The authors are thankful to CSIR, Govt. of India, New Delhi for financing the network project (BSC0117). BSB & SB are thankful to Dr. Rakesh Pandey, Microbial Technology & Nematology Division of CSIRCIMAP, Lucknow for imparting training on nematology.

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Journal Pre-proof Conflict of interest The authors have no conflicts of interest to declare.

References Andersen, C.L., Jensen, J.L., Orntoft, T.F., 2004. Normalization of real-time quantitative reverse transcriptionPCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245-5250. Artico, S., Nardeli, S.M., Brilhante, O., Grossi-de-Sa, M.F., Alves-Ferreira, M., 2010. Identification and

quantitative RT-PCR data. BMC Plant Biol. 10, 49-61.

ro of

evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time

Atamian, H.S., Roberts, P.A., Kaloshian, I., 2012. High and Low Throughput Screens with Root-knot

-p

Nematodes Meloidogyne spp. JoVE. (61), e3629, doi:10.3791/3629.

Barker, K.R., Koenning, S.R., 1998. Developing sustainable systems for nematode management. Ann. Rev.

re

Phytopathol. 36, 165-205.

lP

Bhau, B.S., Borah, B., Ahmed, R., Phukon, P., Gogoi, B., Sarmah, D.K., Wann, S.B., 2016. Influence of rootknot nematode infestation on antioxidant enzymes, chlorophyll content and growth in Pogostemon cablin

na

(Blanco) Benth. IJEB. 54, 254-261.

Borah, B., Ahmed, R., Hussain, M., Phukon, P., Wann, S.B., Sarmah, D.K., Bhau, B.S., 2018. Suppression of

ur

root-knot disease in Pogostemon cablin caused by Meloidogyne incognita in a rhizobacteria mediated

Jo

activation of phenylpropanoid pathway. Bio. Control. 119, 43-50. Bunrathep, S., Lockwood, G.B., Songsak, T., Ruangrungsi, N., 2006. Chemical constituents from leaves and cell cultures of Pogostemon cablin and use of precursor feeding to improve patchouli alcohol level. Sci. Asia. 32, 293-296. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169-193. Byers, D.M., Gong, H., 2007. Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649-662. Chandna, R., Augustine, R., Bisht, N.C., 2012. Evaluation of Candidate Reference Genes for Gene Expression Normalization

in Brassica

juncea using

Real

Time

Quantitative

RT-PCR.

PLoS

ONE.

7(5),

e36918. https://doi.org/10.1371/journal.pone.0036918

18

Journal Pre-proof Crosby, J., Crump, M.P., 2012. The structural role of the carrier protein active controller or passive carrier. Nat. Pro. Rep. 29, 1111-1137. Deguerry, F., Pastore, L., Wu, S., Clark, A., Chappell, J., Schalk, M., 2006. The diverse sesquiterpene profile of patchouli, Pogostemon cablin, is correlated with a limited number of sesquiterpene synthases. Arch. Biochem. Biophys. 454, 123-136. Derveaux, S., Vandesompele, J., Hellemans, J., 2010. How to do successful gene expression analysis using realtime PCR. Method. 50, 227-230. Die, J.V., Roman, B., Nadal, S., Gonzalez-Verdejo, C.I., 2010. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta. 232, 145-153.

ro of

Fan, C., Ma, J., Guo, Q., Li, X., Wang, H., Lu, M., 2013. Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis). PLoS ONE. 8(2), e56573. doi: 10.1371/journal.pone.0056573. Gueanin, S., Mauriat, M., Pelloux, J., Van, W.O., Bellini, C., Gutierrez, L., 2009. Normalization of qRT-PCR

-p

data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J.

re

Exp. Bot. 60, 487- 493.

Gutierrez, L., Mauriat, M., Guenin, S., Pelloux, J., Lefebvre, J.F., Louvet, R., Rusterucci, C., Moritz,

lP

T., Guerineau, F., Bellini, C., Van Wuytswinkel, O., 2008. The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in

na

plants. Plant Biotechnol. J. 6, 609-618.

Hu, R., Fan, C., Li, H., Zhang, Q., Fu, Y.F., 2009. Evaluation of putative reference genes for gene expression

Jo

1186/1471-2199-10-93.

ur

normalization in soybean by quantitative real-time RT-PCR. BMC Mol. Biol. 10 (93), https://doi.org/10

Huis, R., Hawkins, S., Neutelings, G., 2010. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol. 10, 71-85. Kozera, B., Marcin, R., 2013. Reference Genes in Real-Time PCR. J. Appl. Genetics. 54, 391-406. Mahajan, R., 1978. Efficacy of carbofuran for control of M. incognita on okra. Indian J. Nematol. 8, 91-92. Mahanta, J.J., Chutia, M., Sarma, T.C., 2007. Study on weed flora and their influence on patchouli (Pogostemon cablin Benth.) oil and patchoulol. J. Plant Sci. 2, 96-101. McNulty, S., Toscano, W., 1995. Transcriptional regulation of glyceraldehyde-3- phosphate dehydrogenase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. Biophys. Res. Comm. 212, 165-171. Molinari, S., 2011. Natural genetic and induced plant resistance as a control strategy to plant-parasitic nematodes alternative to pesticides. Plant Cell Reports. 30, 311-323.

19

Journal Pre-proof Nolan, T., Hands, R.E., Bustin, S.A., 2006. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559-1582. Noweer, E.M.A., Susan, A.A.H., 2005. Effect of Different Management Practices for Controlling Root-Knot Nematode Meloidogyne incognita on Squash. Egyp. J. Phytopathol. 33, 73-81. Penna, I., Vella, S., Gigoni, A., Russo, C., Cancedda, R., Pagano, A., 2011. Selection of Candidate Housekeeping Genes for Normalization in Human Postmortem Brain Samples. Inter. J. Mol. Sci. 12, 54615470. Pfaffl, M.W., Trichopad, A., Prgomet, C., Neuvians, T.P., 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: Best Keeper-Excel-based tool using pair-wise

ro of

correlations. Biotechnol. Lett. 26, 509-515. Pieterse, C.M.J., Zamioudis, C., Berendsen, R.L., Weller, D.M., Van, W.S.C.M., Bakker, P.A.H.M., 2014. Induced Systemic Resistance by Beneficial Microbes. Ann. Rev. Phytopathol. 52, 347-375.

-p

Plaxton, W.C., 1996. The organization and regulation of plant glycolysis. Ann. Rev. Plant Physiol. Plant Mol.

re

Biol. 47, 185-214.

Radonic ,A., Thulke, S., Mackay, I.M., Landt, O., Siegert, W., Nitsche, A., 2014. Guideline to reference gene

lP

selection for quantitative real-time PCR. Biochem. Biophys. Res. Comm. 313, 856-862. Silver, N., Best, S., Jiang, J., Thein, S. L., 2006. Selection of housekeeping genes for gene expression studies in

na

human reticulocytes using real-time PCR. BMC Mol. Biol. 7, 33. doi:10.1186/1471-2199-7-33. Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T., Igout, A., Heinen, E.,

ur

1999. Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75, 291-295.

Jo

Tong, Z., Gao, Z., Wang, F., Zhou, J., Zhang, Z., 2009. Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol. Biol. 10, 71 doi:10.1186/1471-2199-10-71 Vandesompele, J., Preter, K.D., Pattyn, F., Poppe, B., Roy, N.V., Paepe, A.D., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, 1-11. Wang, K., Niu, Y., Wang, Q., Liu, H., Jin, Y., Zhang, S., 2017. Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus. Peer J. 26, 5:e3260. doi: 10.7717/peerj.3260. Wilson, D.N., Doudna, C.J.H., 2012. The Structure and Function of the Eukaryotic Ribosome. Cold Spring Harb. Perspect. Biol. 4(5), a011536 doi: 10.1101/cshperspect.a011536 Xiao, X., Ma, J., Wang, J., Wu, X., Li, P., & Yao, Y., 2015. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR. Front. Plant Sci. 5, 788. doi:10.3389/fpls.2014.00788

20

Journal Pre-proof Xiao, X.L., Wu, X.M., Ma, J.B., Li, P.B., Li, T.T., Yao, Y.N., 2015. Systematic assessment of reference genes for RTqPCR across plant species under salt stress and drought stress. Acta Physiol. Plant. 37, 186. https://doi.org/10.1007/s11738-015-1922-8 Yu, Z.X., Wang, L.J., Zhao, B., Shan, C.M., Zhang, Y.H., Chen, D.F., Chen, X.Y., 2014. Progressive Regulation of Sesquiterpene Biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-Targeted SPL Transcription Factors. Mol. Plant. 8, 98-110. Zhang, Q.L., Zhu, Q.H., Liao, X., Wang, X.Q., Chen, T., Xu, H.T., Wang, J., Yuan, M.L., Chen, J.Y., 2016. Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus. Sci. Rep. 6, 37549. doi:10.1038/srep37549

ro of

Zhang, Y., Maximova, S.N., Guiltinan, M.J., 2015. Characterization of a stearoyl-acyl carrier protein desaturase

Jo

ur

na

lP

re

-p

gene family from chocolate tree, Theobroma cacao L. Front. Plant Sci. 6, 239-261.

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Journal Pre-proof Figure legends Fig. 1 Agarose gel electrophoresis (2.0%) indicates amplification of a single PCR product of the expected size for ACTIN, EF, SLEEPER, GAPDH1, GAPDH2, TUB, UBI, DSK2A, CYC, and PLA). Fig. 2 Melting curves of the genes show single peaks. Fig. 3 Expression levels (Ct values) for the six candidate reference genes. (Lines across the Box-plot graph of Ct values represent the median values. Lower and upper boxes show the 25th percentile to the 75th percentile. The whisker caps represent the maximum and minimum values). Fig. 4 Gene expression stability values (M) and ranking of 6 reference genes as assayed by GeNorm Fig. 5 Pairwise variation (V) of the candidate reference genes calculated by geNorm. Vn/Vn+1 value were used

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for decision of the optimal number of reference genes. Fig. 6 Expression stability analysis of reference genes assayed by NormFinder software. Fig. 7 Comparative ΔCt approach to housekeeping gene selection. ΔCt variability in candidate housekeeping

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gene comparisons are shown as medians (lines). Lower and upper boxes show the 25th percentile to the 75th

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percentile. The whisker caps represent the maximum and minimum values.

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Journal Pre-proof Abbreviations PGPR, Plant Growth Promoting Rhizobacteria; ISR, induced systemic resistance; SAR, systemic acquired resistance; SA, Salicylic acid; JA, jasmonic acid; ET; ethylene; qRT-PCR, quantitative realtime

Polymerase

chain

reaction;

mRNA,

messenger

ribonucleic

acid;

cDNA,

complementary deoxyribonucleic acid; DAI, day after inoculation; ΔCt, delta cycle threshold; BLAST,

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Basic Local Alignment Search Tool; ANOVA, analysis of variance.

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Journal Pre-proof Author statement

Bitupon Borah: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft

Marine Hussain: Writing - Review & Editing, Software, Investigation, Resources.

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Sawlang Borsingh Wann: Visualization, Writing - Review & Editing.

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Brijmohan Singh Bhau: Supervision, Project administration, Funding acquisition

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Journal Pre-proof Highlights 

Six candidate reference genes (Actin, EF1, ACP, COX3, GAPDH and Pat18S) were statistically validated by five different algorithms viz., ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder.



ACP and ACTIN were found as suitable reference genes for qTR-PCR gene expression experiment in patchouli under M. incognita attack and PGPR treatments, whereas COX3 and EF1 were found unsuitable.

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ACP/GAPDH was showed the best combination of reference gene pairs.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7