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Philippine Journal of Science148 (S1): 183-191, Special Issue on GenomicsISSN 0031 - 7683Date Received: 19 Mar 2019

Genome-guided Molecular Characterization of Oil Genes in Coconut (Cocos nucifera L.)

Anand Noel C. Manohar1,3*, Darlon V. Lantican1,3, Melvin P. Dancel1,3, Don Emanuel M. Cardona1,3, Alissa Carol M. Ibarra1, Cynthia R. Gulay1,3,

Alma O. Canama1, Roanne R. Gardoce1, and Hayde F. Galvez1,2

1Genetics Laboratory, Institute of Plant Breeding, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna 4031 Philippines

2Institute of Crop Science, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna 4031 Philippines

3Philippine Genome Center – Program for Agriculture, Livestock,Fisheries and Forestry (PGC-Agriculture), University of the Philippines Los Baños,

College, Laguna 4031 Philippines

Coconut oil is a major source of medium chain fatty acids (MCFAs), which are health-promoting plant compounds. The MCFAs of coconut oil have been reported to exhibit various health properties such as antioxidant, antibacterial, antiviral, and cardiovascular benefits brought about by the multi-functionality of these complex MCFAs. Six (6) candidate genes involved in oil and MCFA synthesis were identified in the general seed oil biosynthetic pathway. The candidate gene sequences were mined using local BLAST in the coconut genome assembly constructed based on 15× PacBio® and 50× Illumina® MiSeq sequence reads of CATD coconut variety. Scaffolds harboring the candidate genes were mapped based on sequence homology alignment. Gene structures of all genes were elucidated using evidence-based and ab initio prediction algorithms. The coding DNA sequences of KasII and KasIII in coconut were characterized. These MCFA genes have not been characterized nor reported in coconut. Gene-specific PCR primers were designed targeting the coding regions of each gene. PCR conditions were optimized to mine natural allele variants across 48 established coconut varieties in the Philippines through EcoTILLING (Ecotype Targeting Induced Local Lesions IN Genomes).Asingle nucleotide polymorphism (SNP) on the lysophosphatidic acid acyltransferase genes (LPAAT) was detected in the ‘West African Tall’(WAT) and ‘Aguinaldo Tall’ (AGDT) varieties. The partial LPAAT gene sequences of WAT and AGDT were cloned and sequenced in order to characterize the SNP. Based on the identified SNPs, robust DNA markers may be developed for high-throughput screening and selection of favorable alleles in genomics-assisted coconut breeding for outstanding high-quality oil producing varieties.

Keywords: coconut, EcoTILLING, genomics-assisted breeding, in silico gene prediction, medium- chain fatty acid, SNP markers

*Corresponding Author: [email protected]

INTRODUCTION

The coconut (Cocos nucifera L.), dubbed as the “Tree of Life,” is the most important palm in the humid tropics, with approximately 12 million hectares planted with the

crop in around 86 countries. In the Philippines, coconut is a major agricultural crop grown in around 3.6 million hectares of farmland or 26% of the country’s agricultural lands. The country produces an average of 13.8 million tonnes of harvest per year derived from 340 million bearing trees. As the second largest producer and exporter

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of coconut in the world, the industry provides employment to approximately 1.4 million Filipino coconut farmers who comprise 21.7% of the total number of farmers directly engaged in agriculture (FAO 2018). Every part of the coconut palm – from the leaves, all the way down to the roots – have various economic uses. Copra oil (or coconut oil), the coconut’s most economically important product, used to be the most-traded vegetable oil in the world (Purseglove 1985). However, there is a remarkable global decline in the importance of coconut oil when used for food based on consolidated values/reports from 1961 and 2011. One of the major reasons for this is the stigma associated with the presence of long-chain fatty acids (Nayar 2017).

MCFAs and long-chain fatty acids comprise around 83.92% of the total composition of copra oil, of which lauric acid (C12:0) is the most predominant (Padolina et al. 1987). The high proportion of saturated fatty acids in coconut oil has been previously reported to increase cholesterol levels in the body, thus increasing the risk of cardiovascular diseases (Mensink et al. 2003). However, this has been scientifically discredited by numerous researches demonstrating several health benefits from consuming high amounts of MCFAs in coconut oil, particularly lauric acid extracted from the fruit endosperm (Blackburn et al. 1989a, b; Kintanar and Castro 1988; Müller et al. 2003; Dayrit et al. 2008; Orsasova et al. 2015). MCFAs in coconut oil exhibit synergistic cardioprotective, anti-thrombotic, anti-atherosclerotic, anti-bacterial, anti-dermatophytic, and anti-viral effects (Kaunitz et al. 1958, Bach and Babayan 1982, Scalfi et al. 1991, Fushiki et al. 1995). One of the primary objectives in coconut breeding is to develop outstanding coconut varieties not only based on yield/productivity but also with improved copra oil quality by increasing MCFAs and short-chain fatty acid content. To develop varieties with improved fatty acid profile, the coconut oil biosynthesis pathway must be elucidated. The general oil biosynthetic pathway in seeds involves various organelles and extensive lipid trafficking – requiring several enzymes. It starts with the synthesis of fatty acids in the plastids, while the triacylglycerides (TAGs) synthesized by LPAAT are assembled outside the plastid – particularly in both smooth and rough endoplasmic reticulum (Chapman and Ohlrogge 2012). Assembly of fatty acids is initiated by the acyl carrier protein (ACP) via a cycle of reactions that elongate the acyl chain at a rate of two carbon atoms per cycle. The processed fatty acid can either be hydrolyzed by FatB or further elongated by KasII (Li-Beisson et al. 2010). However, genes involved in the synthesis of MCFAs in coconut oil are not yet well-characterized at the gene structure level.

With the advent of next-generation sequencing technologies and bioinformatics, a coconut genome assembly has been constructed from the 15X PacBio® and 50X Illumina® MiSeq sequence data of ‘Catigan Green Dwarf’ (CATD) coconut (Lantican et al. 2019). Using the appropriate bioinformatics tools, these genes can be characterized at the genome level with the aid of the genome assembly. Allele variations of these genes across various ecotypes can be detected by EcoTILLING. EcoTILLING is a modification of the Targeting Induced Local Lesions IN Genomes (TILLING) protocol used for the discovery of SNPs in natural populations instead of chemically-induced mutant populations. It was first used to identify and map SNPs detected in various Arabidopsis ecotypes (Comai et al. 2004). A similar approach was performed in the assessment of fatty acid elongase 1 (FAE1) gene among Brassica species for the development of DNA markers associated with seed erucic acid (Wang et al. 2010). Identified polymorphisms using this technique will be screened further for potential use in marker-assisted breeding of coconut to improve copra oil quality by manipulating any or a combination of these coconut oil genes.

This paper reports coconut genes involved in coconut endosperm oil biosynthesis that were characterized in the CATD genome through in silico gene prediction. The validation of the genes by EcoTILLING is also presented and discussed, including its potential application in the development of gene-markers in coconut marker-assisted breeding procedures.

MATERIALS AND METHODS

Gene Mining Candidate genes for variant detection were selected based on their roles in the general endosperm oil biosynthesis pathway of plant seeds (Bates et al. 2013). These genes are acyl-ACP thioesterase A and B (FatA and FatB); β-ketoacyl-ACP synthases I, II, and III (KasI, KasII, and KasIII); and LPAAT genes. Equivalents of these genes were mined in the Elaeis guineensis genome assembly available in the NCBI database (https://www.ncbi.nlm. nih.gov/). These are deposited E. guineensis genes with NCBI accession nos. XM_010929397.2, AF147879.2, XM_010917679.2, XM_010923620.2, XM_010907910.2, and XM_010910594.1. The nucleotide sequences of the genes were downloaded and, through local BLASTn (e-value = 1e–150) analysis, the gene homologs on the CATD coconut genome assembly (Lantican et al. 2019) were mined using the E. guineensis sequences as a query. Assembly scaffolds harboring the genes were identified for subsequent analysis.

Manohar et al.: Genome-guided Molecular Characterization of Oil Genes in Coconut

Special Issue on Genomics

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Gene AnnotationThe sequence scaffolds harboring the coconut candidate oil genes were subsequently extracted from the CATD genome assembly using a local Perl script (github.com/aubombarely/GenoToolBox/blob/master/SeqTools/FastaExtract). Annotation was performed via ab initio, cDNA/EST, and homology-based approaches – and integrated by the MAKER annotation pipeline (Cantarel et al. 2008). The gene structures were predicted on the scaffold by ab initio gene prediction using AUGUSTUS and SNAP programs (Korf 2004) – revealing exon-intron junctions, ORFs, and UTRs. These sets of predictions were merged with BLASTx, BLASTn, and exonerate alignments of oil biosynthesis gene homologs deposited at the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank/).

Plant MaterialsForty-eight (48) accessions of coconuts established at the Institute of Plant Breeding, University of the Philippines Los Baños (IPB-UPLB) were used in this study. The accessions included 24 Dwarf and 24 Tall coconut varieties from both local and foreign sources, which were obtained from the germplasm collection of the Philippine Coconut Authority - Zamboanga Research Center (PCA-ZRC) through the Coconut Genomics Project 8 of IPB-UPLB. The list of the varieties used is presented in Table 1.

Genomic DNA ExtractionLeaf samples from the 48 Philippine coconut varieties were harvested from the mini-germplasm collection of the Genetics Laboratory in IPB-UPLB. These are among the selected accessions from the PCA-ZRC coconut genebank being established at IPB-UPLB for the coconut genomics project 8 (Galvez 2016). Genomic DNA from each variety was extracted using the modified Doyle and Doyle (1990) protocol described by Cardona et al. (2015). Each accession consists of 4–10 individual genomic DNA samples, which were pooled and normalized to 10 ng/μL prior to polymerase chain reaction (PCR) analysis.

PCRThe optimized amplification reaction mixture consisted of 30 ng of DNA template; 1X PCR buffer (10 mM Tris-Cl pH 8.3, 50 mM KCl); 2.0mM MgCl

2 (Vivantis

Technologies); 2.5 mM dNTPs (Promega Aust Ltd); 0.2 μM of forward and reverse primers; and 0.4 U of Taq polymerase (Vivantis Technologies). The samples were amplified through PCR using different temperature profiles based on corresponding optimum temperature in T100™ Thermal Cycler (Bio-rad Laboratories Inc., Hercules, CA, USA). The temperature profiles used in amplifying the samples were similar except for annealing temperatures as presented in Table 2. The amplified PCR

Table 1. Coconut varieties obtained from PCA-ZRC and used to screen for SNPs on identified coconut candidate oil genes.

Code Variety Code Variety

PILD Pilipog Dwarf MVT Markham Talla

CRD Cameron Red Dwarfa KKT Karkar Tall

TACD Tacunan Dwarf AGDT Aguinaldo Tall

MYD Malayan Yellow Dwarfa WAT West African Talla

CATD Catigan Green Dwarf GATT Gatusan Tall

MRD Malayan Red Dwarfa BAYT Baybay Tall

KIAD Kiamba Dwarf SNRT San Ramon Tall

SGD Sri Lanka Green Dwarfa TPKT Tampakan Tall

EGD Equatorial Green Dwarf SCHT Sanchez Mira Tall

BAGD Baguer Dwarf VTT Vanuatu Talla

AROD Aromatic Green Dwarf TAGT Tagnanan Tall

KIND Kinabalan Dwarf BAOT Bago Oshiro Tall

BAND Banga Dwarf GPT Gazelle Tall

MAKD Makilala Dwarf PYT Tahiti Talla

GALD Galas Dwarf VENT Venus Tall

VICD La Victoria Green Dwarf RIT Rennel Island Talla

VIBD La Victoria Brown Dwarf AGAT Agta Tall

TUPD Tupi Dwarf LONT Loong Tall

MAGD Magtuod Dwarf CULT Culaman Tall

TALD Talisay Dwarf KGT Katangawan Tall

SNOD Sto. Niño Dwarf BNGT Banigan Tall

BUSD New Buswang Dwarf SALT Salambuyan Tall

KAPD Kapatagan Dwarf PAGT Panda Tall

SNID San Isidro Dwarf LAGT Laguna TallaForeign accession

Table 2. PCR temperature profile used in amplifying the candidate oil genes in 48 coconut varieties

Step Temperature (°C) Time

Initial denaturation 95 3 min

Denaturation 95 1 min

Annealing 50–60 1 min

Extension 72 2 mins

Final Extension 72 5 mins

Hold 12 ∞

products were electrophoresed in a 1% agarose gel in 1X TBE buffer (90 mM Tris-borate, 2 nM EDTA) at 100 V for 40 min. After electrophoresis, the gel was stained with ethidium bromide for 20–30 min, after which was viewed under ultraviolet (UV) light using the Enduro Gel Documentation System (Labnet International, Inc.).



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EcoTILLING involving natural population was performed upon optimization of PCR amplification conditions. Amplicons of the 48 coconut accessions were mixed in equal parts of the amplicons of the reference genome CATD. Running the admixture in a series of annealing and denaturation causes the formation of heteroduplexes. These were then subjected to a nuclease assay using Cel1, which is composed of 194.4 μL of T-Digest buffer and 1.6 μL of dsDNA cleavage enzyme (Advanced Analytical) for every 96 samples analyzed. Cel1 nuclease specifically cleaves at regions where a mismatch bubble is formed, thus revealing the presence and relative location of an SNP (Barkley and Wang 2008). Resulting fragments were resolved in Fragment AnalyzerTM (Advanced Analytical Technologies, Inc., San Diego, CA, USA). Lanes that showed cleaved fragments were considered for further characterization as SNPs were detected in these regions.

Cloning and SequencingLigation and transformation. Identified genes with variants were cloned using the pGEM-T Easy Vector System (Promega Aust Ltd), following the manufacturer’s instruction. The transformed cells were incubated at 37 °C with shaking for 90 min. A total of 100 and 150 μL of each transformation culture was plated onto prewarmed Luria-Bertani (LB) agar plates with ampicillin (100 μg/mL), overlaid with 50 μL of 100 mM IPTG and 20 μL of 80 mg/mL X-gal. The plates were incubated overnight (16–24 h) at 37 °C.

Colony PCR and subculture. White colonies corresponding to putative transformants were selected. Using a sterile pipette tip, selected colony were pricked and mixed with prepared PCR mixtures containing gene-specific primers. Recommended PCR conditions were used in the PCR analysis. In colonies that exhibited the expected amplicon, a sterile toothpick was used to prick the corresponding bacterial colony and placed in a culture tube containing LB broth with ampicillin (100 μg/mL). The tubes were incubated overnight at 37 °C in a shaking incubator.

Bacterial plasmid isolation. Purelink Plasmid Miniprep Kit (Invitrogen) was used to isolate the plasmids from bacterial cultures following the manufacturer’s instructions. Resulting purified plasmids were sent for double pass sequencing using T7 and SP6 primers.

Sequence Analysis and SNP Marker DevelopmentSequenced clones positive for putative SNPs were analyzed using appropriate bioinformatics tools. Chromas (Technelysium Pty. Ltd.) was used to determine the quality of sequencing by checking the sequencing chromatogram provided. Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/) was used for all pairwise and multiple sequence alignments. Putative SNPs that are located in

intronic regions were characterized by determining any gene regulatory elements present in the sequences. The miRBase (Kozomara and Griffiths-Jones 2014) was used to check for any homologies with microRNAs deposited in the database. Furthermore, PlantTFcat (Dai et al. 2013) was used to categorize transcription factors and/or transcriptional regulators.

RESULTS AND DISCUSSIONAll six (6) candidate MCFA genes were identified in the CATD genome assembly by running a local BLASTn search using E. guineensis cDNA sequences as a query at e-value = 1e–150 (Table 3). High homology with these genes indicates their presence within the CATD assembly. Multiple contig hits for the case of AF147879.2 (EgFatB) and XM_010917679.2 (EgKasI) is indicative of the presence of gene homologs within the genome. This is further supported by the high percent identity among the contigs identified. Corresponding CATD contigs harboring the genes were identified for annotation. The gene structure of each sequence was determined by evidence-based and ab initio gene prediction using MAKER. MAKER is a genome annotation pipeline that makes use of homology-based approaches incorporating BLAST, AUGUSTUS, and SNAP within its pipeline (Cantarel et al. 2008). Using the predicted gene sequence, alignment with the contig revealed exon and introns. It has been observed that most genes (KasII, KasIII, and LPAAT) exhibit large gene lengths and abundant introns. This would explain why very limited information are available for these genes as there is a difficulty in characterizing large genes in the genome level in the conventional approach. A summary of the data gathered is presented in Table 4. The relative positions of exons and introns along the gene are also shown in Figure 1.

Gene-specific primer design is a crucial step in performing any TILLING procedure. Making use of the genome assembly sequences as the basis for primer design ensures the specificity of the primers developed for each gene. For predicted full-length genes greater than 2000 bp, primers with amplicons not exceeding 1500 bp were designed at specific regions of the gene. As for small genes, such as the FatA, primers capturing the whole length of the gene were generated. All primers designed in each gene successfully amplified the target gene region based on estimated size. The optimized annealing temperature for each primer pair ranges from 50 to 60 ºC via gradient PCR using approximately 30 ng/μL of coconut CATD genomic DNA as a template. PCR products were electrophoresed in 1% agarose gels and then stained in ethidium bromide. An electrophoretogram of the amplicons is shown in Figure 2.

EcoTILLING results revealed no significant SNP cleavage



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Table 3. Local BLASTn results of Elaeis guineensis sequences against the CATD reference genome.

Query Scaffold ID % Identity Length Query start

Query end

Subject start

Subject end e-value Bit

score

XM_010929397.2:PREDICTED: Elaeis guineensis oleoyl-acyl carrier protein thioesterase 1, chloroplastic (LOC105049664), mRNA

CONTIG1616_PILON 91.17 419 1 416 711695 711280 4e–158 564

AF147879.2:Elaeis guineensis var. tenera palmitoyl-ac-yl carrier protein thioesterase (PTE) mRNA, complete cds

CONTIG201_PILON

93.32 584 187 770 1158940 1158366 0.0 854

92.57 565 1254 1811 1154251 1153690 0.0 802

CONTIG117_PILON 86.17 564 211 770 1567971 1567414 4e–169 601

XM_010917679.2:PREDICTED: Elaeis guineensis 3-oxoacyl-[acyl-carrier-protein] synthase I, chloroplas-tic-like (LOC105040922), mRNA

CONTIG160_PILON

96.99 432 707 1138 2310007 2309576 0.0 726

97.21 359 348 706 2310894 2310536 2e–171 608

XM_010923620.2:PREDICTED: Elaeis guineensis acyl car-r ie r p ro te in 2 , mi tochondr ia l - l ike (LOC105045364), mRNA

CONTIG4419_PILON 93.10 377 384 759 90586 90960 7e–154 549

X M _ 0 1 0 9 0 7 9 1 0 . 2 : P R E D I C T-ED: Elaeis guineensis acyl carrier protein 3, mitochondrial (LOC105033210), transcript variant X1, mRNA

CONTIG5102_PILON 92.71 645 308 949 49028 48385 0.0 928

XM_010910594.1:PREDICTED: Elaeis guineensis 1-acyl-sn-glycerol-3-phosphate acyltransferase (LOC105035151), transcript variant X2, mRNA

CONTIG19_PILON 91.89 407 1005 1408 318719 318313 1e–158 566

Figure 2. Electrophoretogram of the six coconut candidate oil genes: Lanes 1 – 1 kb Plus DNA ladder (Invitrogen), 2 – FatA, 3 – FatB, 4 – KasI, 5 – KasII, 6 – KasIII, and 7 – LPAAT

Figure 1. A graphical representation of the approximate locations of the exons and introns within each coconut candidate oil gene.

Table 4. Gene structures of the selected coconut candidate oil genes.

Gene code Protein product Size (bp) Predicted transcript length (bp) No. of exons No. of introns

FatA Acyl-ACP thioesterase A 681 416 4 3

FatB Acyl-ACP thioesterase B 5016 1684 6 5

KasI Ketoacyl-ACP synthase I 4443 1808 7 6

KasII Ketoacyl-ACP synthase II 24342 1829 5 4

KasIII Ketoacyl-ACP synthase III 9686 949 7 6

LPAAT Lysophosphatidic acid acyltransferase 28262 1325 11 10



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Figure 3. Chromatogram and capillary gel electrophoretogram of the EcoTILLING results of the LPAAT gene in WAT coconut depicting the cleaved heteroduplex of the PCR amplicon.

Figure 4. A graphical representation of the SNPs mined within the LPAAT amplicon (1400 bp) of WAT and AGDT coconut.

for FatA, FatB, KasI, KasII, and KasIII. However, an SNP was detected in the LPAAT gene of WAT and AGDT coconut varieties at approximately 500–600 bp position of the LPAAT amplicon. Figure 3 shows the chromatogram and gel representative of the EcoTILLING results of WAT. The red peak on the chromatogram corresponds to the homoduplexes, while the orange peaks represent the heteroduplexes formed during the endonuclease digestion process. LPAAT is a major gene responsible for the high lauric acid accumulation in coconut, as previously demonstrated in the genetic transformation experiments of this gene to Brassica rapus (Knutzon et al. 1999). Moreover, recent transcriptome analysis of developing coconut endosperm carried out by Reynolds et al. (2019) confirmed the important role of LPAAT in oil biosynthesis, particularly in the synthesis of TAGs. The predicted sequence length of coconut LPAAT gene is 28,344 bp in reference to the CATD genome assembly. This is shorter than the reported gene sequence of African oil palm by 10,487 bp (Singh et al. 2013). Analysis of its gene structure in coconut should provide an effective reference to targeted gene editing and variation screening

towards improved copra oil quality (Lantican et al. 2019). Liang et al. (2014) has also reported the roles of LPAAT in the fatty acid biosynthesis in coconut using the Kyoto Encyclopedia of Genes and Genomes analysis from suppression subtractive hybridization experiment. However, none has been published regarding the degree of variation of this gene in a natural coconut population.

The SNP was verified through cloning and sequencing of the amplicon of LPAAT from WAT and AGDT coconut DNA samples. Sequence data revealed that a pair of SNPs (T/C and C/A, respectively) – 6 bp apart – is inherent along the approximate Cel1 cleavage site in the partial LPAAT sequence of WAT. Multiple sequence alignment of the gene region is presented in Figure I. The relative position of the SNPs along the gene is shown in Figure 4. Coincidentally, WAT is a popular foreign coconut cultivar known for its high oil yield. The pair of SNPs is located in an intronic segment of the gene and may harbor functional genetic elements that may influence the expression of LPAAT and/or associated genes in the metabolic network (Cooper 2010).



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Moreover, comparative gene analysis was carried out between the CATD and ‘Hainan Tall’ (HAT) LPAAT gene. Multiple sequence alignment was used to check for similarities against the HAT genome assembly by Xiao et al. (2017). The high similarity between the CATD LPAAT (Scaffold ID: CONTIG19_PILON) and HAT LPAAT (Scaffold ID: scaffold1063) was observed at 99.7% homology. Few single base insertions were identified to be present along both genes commonly found in nucleotide base runs (i.e., AAAAA, TTTTT,GTGTGTGT). Interestingly, comparative analysis reveals a sequence discrepancy approximately 9286–10510 bp on the HAT sequence assembly within the predicted first exon region of LPAAT. Upon further characterization on the discrepant locus, it was found out that it is comprised of ambiguous base calls (i.e., NNNN) and runs of adenine bases, which may be attributed to sequencing and/or scaffolding errors. Nonetheless, further validation through wet lab experiments is needed to confirm the polymorphisms detected.

To check for possible affected regulatory elements within the SNP/polymorphic region of coconut LPAAT, its sequence homology was tested against miRbase (www.mirbase.org) to determine any microRNA sequences in the gene segment. Significant homology with miR172 was detected in the presence of the two SNPs. The miR172 microRNA is responsible to modulate the expression of mRNAs coding for APETALA2-like transcription factors in Arabidopsis. APETALA2 is a member of a family of transcription factors AP2/EREBP, which plays a major role in plant hormone regulation, particularly those involved in seed and flower development (Ogawa et al. 2007). Reverse genetics studies conducted by Ohto et al. (2005) also showed that APETALA2 is directly involved in the control of seed mass by changing the ratio of hexose to sucrose during seed development. Using PlantTFcat (plantgrn.noble.org/PlantTFcat/), 154 APETALA2 transcription factor family genes were detected in the annotated CATD coconut genome assembly (Lantican et al. 2019). The results of the transcription factor analysis of the reference CATD coconut genome are shown in Table I.

This study is the first report of natural variation in LPAAT in coconut. The identified SNPs within the LPAAT gene region can be used as a basis in designing a DNA marker potentially associated with improved copra oil quality and/or yield. However, further EcoTILLING using larger population samples i.e., more coconut varieties and individual palms, is needed to validate the genetics of these coconut LPAAT SNPs. There is also a need to validate the correlation of the SNP to copra oil phenotype data in a defined bi/multi-parent genetic mapping population.

CONCLUSIONThe genome sequences and predictive structure of MCFA synthesis-related genes in coconut were characterized using appropriate bioinformatics analysis and in reference to the CATD genome assembly. Gene-specific markers were developed and used to analyze the genetic diversity of at least 48 coconut accessions, Tall and, Dwarf varieties through EcoTILLING and using a high-throughput fragment analyzer platform. All the gene markers positively amplified by PCR the expected fragment sizes of the MCFA-related genes in all the coconut DNA samples. This validates the good quality of the assembly of coconut CATD as the reference genome in this study. EcoTILLING analysis likewise revealed sequence conservation of almost all the MCFA-related genes in the 48 coconut varieties based on the genomic regions tagged by the markers. Except for LPAAT, no potential SNP was detected in the other coconut genes mined in this study. Among the coconut varieties screened, one SNP was detected in the partial sequence (amplicons) of AGDT and WAT coconut varieties. WAT is a foreign coconut accession conserved in the Philippine Coconut Authority (PCA) germplasm, while AGDT is a local variety collection. Although no data is available for AGDT, WAT has been a favorite genetic resource of the PCA breeders for coconut oil yield. The potential association of this sequence variant of LPAAT, specifically in AGDT and/or WAT to improve the economic value of coconut oil, is yet to be validated. In bay thioesterase-transformed rapeseed, further introduction of coconut LPAAT resulted in increased total lauric acid levels (Knutzon et al. 1999).

The LPAAT amplicons in selected palms/individuals of coconut AGDT and WAT were cloned and sequenced, which indeed all returned high homology to the reference CATD sequence except for the characterized SNP. This study has revealed a potential SNP marker that can be used to determine coconut palms with outstanding oil quality. However, phenotypic validation is still needed prior to marker association. Moreover, other exons and/or introns not characterized in this study, as well as promoter regions, of the mined candidate MCFA-related genes may also be screened for potential DNA sequence variation i.e., SNPs and functional association to coconut oil quality.

ACKNOWLEDGMENTSThis study would not be possible without the generous funding from the Department of Science and Technology – Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development through PGC-Agriculture. The authors extend their sincere gratitude to Dr. Antonio C. Laurena, Dr. rer. nat. Lourdes B. Cardenas,



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and Dr. Consorcia E. Reaño for their guidance during the conduct of the study. Also, to the authors’ colleagues at PCA-ZRC – Mr. Ramon L. Rivera, Dr. Susan M. Rivera, and Mr. Ernesto E. Emmanuel – for the source of the plant materials used in this study. Equally, to the co-authors and collaborators in the assembly of the reference genome, the CATD coconut genome, especially Dr. Suzy Strickler and Dr. Lukas A. Mueller of the Boyce Thompson Institute for Plant Research, Ithaca, NY, USA. The high-quality assembly of the reference genome was vital to the success of this study.

STATEMENT ON CONFLICT OF INTERESTThe authors declare no conflict of interest.

NOTES ON APPENDICESThe complete appendices section of the study is accessible at http://philjournsci.dost.gov.ph

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191

Figure I. Clustal Omega alignment.



192

Table I. PlantTFCat results.

Gene models Functional annotation

Cnu_07676-RA protein Name:"Similar to AIL5 AP2-like ethylene-responsive transcription factor AIL5 (Arabidopsis thaliana)" AED:0.17 eAED:0.17 QI:0|0.71|0.5|1|0.85|0.75|8|0|443

Cnu_06751-RA protein Name:"Similar to DREB2C Dehydration-responsive element-binding protein 2C (Oryza sativa subsp. japonica)" AED:0.02 eAED:0.01 QI:0|-1|0|1|-1|1|1|0|195

Cnu_07685-RA protein Name:"Similar to ERF003 Ethylene-responsive transcription factor ERF003 (Arabidopsis thaliana)" AED:0.09 eAED:0.09 QI:73|1|0.5|1|1|1|2|0|151

Cnu_28685-RA protein Name:"Similar to ERF3 Ethylene-responsive transcription factor 3 (Arabidopsis thaliana)" AED:0.21 eAED:0.23 QI:0|-1|0|1|-1|1|1|0|220

Cnu_13245-RA protein Name:"Similar to RAP2-11 Ethylene-responsive transcription factor RAP2-11 (Arabidopsis thaliana)" AED:0.01 eAED:0.01 QI:0|-1|0|1|-1|1|1|0|271

Cnu_06457-RA protein Name:"Similar to AP2 Floral homeotic protein APETALA 2 (Arabidopsis thaliana)" AED:0.12 eAED:0.12 QI:0|0.71|0.75|1|1|1|8|823|510

Cnu_06459-RA protein Name:"Similar to ERF010 Ethylene-responsive transcription factor ERF010 (Arabidopsis thaliana)" AED:0.33 eAED:0.33 QI:0|0|0|0.5|1|1|2|0|137

Cnu_09318-RA protein Name:"Similar to RAP2-13 Ethylene-responsive transcription factor RAP2-13 (Arabidopsis thaliana)" AED:0.07 eAED:0.07 QI:0|0|0|1|0|0.5|2|0|282

Cnu_16629-RA protein Name:"Similar to ERF110 Ethylene-responsive transcription factor ERF110 (Arabidopsis thaliana)" AED:0.01 eAED:0.01 QI:380|1|1|1|1|1|2|0|313

Cnu_07492-RA protein Name:"Similar to ERF091 Ethylene-responsive transcription factor ERF091 (Arabidopsis thaliana)" AED:0.03 eAED:0.02 QI:0|-1|0|1|-1|1|1|0|267

Cnu_07493-RA protein Name:"Similar to PTI5 Pathogenesis-related genes transcriptional activator PTI5 (Solanum lycopersicum)" AED:0.13 eAED:0.13 QI:0|0|0|0.5|1|1|2|0|115

Cnu_08747-RA protein Name:"Similar to AIL1 AP2-like ethylene-responsive transcription factor AIL1 (Arabidopsis thaliana)" AED:0.18 eAED:0.18 QI:194|0.83|1|1|0.83|0.71|7|221|364

Cnu_08831-RA protein Name:"Similar to EREBP1 Ethylene-responsive transcription factor 1 (Oryza sativa subsp. japonica)" AED:0.10 eAED:0.10 QI:0|0.75|0.6|0.8|0.25|0.2|5|1025|338



Cnu_17567-RA protein Name:"Similar to ERF3 Ethylene-responsive transcription factor 3 (Nicotiana tabacum)" AED:0.26 eAED:0.59 QI:0|0|0|1|1|1|2|0|165

Cnu_05776-RA protein Name:"Similar to WRI1 Ethylene-responsive transcription factor WRI1 (Arabidopsis thaliana)" AED:0.12 eAED:0.12 QI:0|0|0|1|1|1|7|0|403

Cnu_08064-RA protein Name:"Similar to CRF2 Ethylene-responsive transcription factor CRF2 (Arabidopsis thaliana)" AED:0.11 eAED:0.11 QI:0|0|0|1|1|1|2|0|238

Cnu_32115-RA protein Name:"Similar to RAP2-11 Ethylene-responsive transcription factor RAP2-11 (Arabidopsis thaliana)" AED:0.26 eAED:-0.21 QI:0|-1|0|1|-1|1|1|0|346

Cnu_17079-RA protein Name:"Similar to ERF019 Ethylene-responsive transcription factor ERF019 (Arabidopsis thaliana)" AED:0.11 eAED:-0.13 QI:0|-1|0|1|-1|1|1|0|206

Cnu_03074-RA protein Name:"Similar to ANT AP2-like ethylene-responsive transcription factor ANT (Arabidopsis thaliana)" AED:0.12 eAED:0.12 QI:0|0.85|0.62|1|0.71|0.62|8|277|674


Cnu_24426-RA protein Name:"Similar to WIN1 Ethylene-responsive transcription factor WIN1 (Arabidopsis thaliana)" AED:0.04 eAED:0.03 QI:0|0|0|1|0|0.5|2|0|203


Cnu_27083-RA protein Name:"Similar to DREB2F Dehydration-responsive element-binding protein 2F (Arabidopsis thaliana)" AED:0.06 eAED:-0.06 QI:0|-1|0|1|-1|1|1|0|352



193

Cnu_00637-RA protein Name:"Similar to DREB2A Dehydration-responsive element-binding protein 2A (Oryza sativa subsp. indica)" AED:0.06 eAED:0.06 QI:258|1|1|1|1|1|2|489|319

Cnu_00788-RA protein Name:"Similar to AP2 Floral homeotic protein APETALA 2 (Arabidopsis thaliana)" AED:0.20 eAED:0.20 QI:0|0.8|0.72|1|0.8|0.90|11|464|474


Cnu_17504-RA protein Name:"Similar to RAP2-7 Ethylene-responsive transcription factor RAP2-7 (Arabidopsis thaliana)" AED:0.30 eAED:0.30 QI:0|0.81|0.75|1|0.72|0.75|12|370|456

Cnu_10507-RA protein Name:"Similar to DREB1A Dehydration-responsive element-binding protein 1A (Arabidopsis thaliana)" AED:0.01 eAED:0.01 QI:0|-1|0|1|-1|1|1|0|237


Cnu_02062-RA protein Name:"Similar to CRF2 Ethylene-responsive transcription factor CRF2 (Arabidopsis thaliana)" AED:0.03 eAED:0.03 QI:0|0.5|0.33|1|0.5|0.66|3|481|466

Cnu_34292-RA protein Name:"Similar to DREB1F Dehydration-responsive element-binding protein 1F (Oryza sativa subsp. japonica)" AED:0.04 eAED:0.03 QI:0|-1|0|1|-1|1|1|0|208



Cnu_11116-RA protein Name:"Similar to ERF118 Ethylene-responsive transcription factor ERF118 (Arabidopsis thaliana)" AED:0.13 eAED:0.13 QI:266|0.66|0.5|1|0|0|4|0|292

Cnu_26511-RA protein Name:"Similar to CRF4 Ethylene-responsive transcription factor CRF4 (Arabidopsis thaliana)" AED:0.09 eAED:0.09 QI:0|-1|0|1|-1|1|1|0|344


Cnu_09067-RA protein Name:"Similar to DREB3 Dehydration-responsive element-binding protein 3 (Arabidopsis thaliana)" AED:0.03 eAED:0.02 QI:0|-1|0|1|-1|1|1|0|227

Cnu_19675-RA protein Name:"Protein of unknown function" AED:0.17 eAED:0.17 QI:0|0|0|0.5|1|1|2|0|243


Cnu_17652-RA protein Name:"Similar to RAP2-3 Ethylene-responsive transcription factor RAP2-3 (Arabidopsis thaliana)" AED:0.13 eAED:0.12 QI:0|0.5|0|0.66|0.5|0.33|3|0|338

Cnu_13415-RA protein Name:"Similar to EREBP1 Ethylene-responsive transcription factor 1 (Oryza sativa subsp. japonica)" AED:0.29 eAED:0.35 QI:0|0.2|0|0.83|0.2|0.5|6|0|677

Cnu_13436-RA protein Name:"Protein of unknown function" AED:0.35 eAED:0.35 QI:0|-1|0|1|-1|1|1|0|177


Cnu_13439-RA protein Name:"Similar to ERF017 Ethylene-responsive transcription factor ERF017 (Arabidopsis thaliana)" AED:0.11 eAED:-0.09 QI:0|0|0|0.5|1|1|2|0|176




Cnu_30526-RA protein Name:"Protein of unknown function" AED:0.21 eAED:0.21 QI:477|1|1|1|0|0.5|2|285|184

Cnu_30527-RA protein Name:"Similar to At1g16060 AP2-like ethylene-responsive transcription factor At1g16060 (Arabidopsis thaliana)" AED:0.44 eAED:0.41 QI:10|0.6|0.66|0.83|1|1|6|0|129

Cnu_32725-RA protein Name:"Similar to ERF4 Ethylene-responsive transcription factor 4 (Nicotiana sylvestris)" AED:0.09 eAED:-0.02 QI:0|-1|0|1|-1|1|1|0|232

Cnu_08572-RA protein Name:"Similar to AP2 Floral homeotic protein APETALA 2 (Arabidopsis thaliana)" AED:0.13 eAED:0.13 QI:408|1|1|1|1|1|10|856|479

Table I continued . . . .



194


Cnu_22517-RA protein Name:"Similar to AP2 Floral homeotic protein APETALA 2 (Arabidopsis thaliana)" AED:0.16 eAED:0.16 QI:554|1|1|1|0.66|0.8|10|73|527


Cnu_06398-RA protein Name:"Similar to BBM2 AP2-like ethylene-responsive transcription factor BBM2 (Brassica napus)" AED:0.09 eAED:0.09 QI:0|0|0|1|0.71|0.87|8|0|710


Cnu_12746-RA protein Name:"Similar to DREB1F Dehydration-responsive element-binding protein 1F (Oryza sativa subsp. japonica)" AED:0.13 eAED:-0.03 QI:0|0|0|0.33|1|1|3|0|291

Cnu_24731-RA protein Name:"Similar to At4g13040 Ethylene-responsive transcription factor-like protein At4g13040 (Arabidopsis thaliana)" AED:0.21 eAED:0.21 QI:0|0|0|0.6|1|1|5|0|190

Cnu_02813-RA protein Name:"Similar to AIL5 AP2-like ethylene-responsive transcription factor AIL5 (Arabidopsis thaliana)" AED:0.14 eAED:0.14 QI:51|1|0.87|1|1|1|8|189|471

Cnu_02837-RA protein Name:"Similar to ERF003 Ethylene-responsive transcription factor ERF003 (Arabidopsis thaliana)" AED:0.14 eAED:0.14 QI:0|1|0|1|1|0.5|2|0|174



Cnu_12538-RA protein Name:"Similar to DREB1E Dehydration-responsive element-binding protein 1E (Oryza sativa subsp. japonica)" AED:0.02 eAED:0.02 QI:0|-1|0|1|-1|1|1|0|212

Cnu_04667-RA protein Name:"Similar to AIL1 AP2-like ethylene-responsive transcription factor AIL1 (Arabidopsis thaliana)" AED:0.15 eAED:0.07 QI:0|0|0|0.85|0.16|0.28|7|0|648

Cnu_18444-RA protein Name:"Similar to At4g13040 Ethylene-responsive transcription factor-like protein At4g13040 (Arabidopsis thaliana)" AED:0.17 eAED:0.17 QI:865|1|0.85|1|0.5|0.42|7|383|121

Cnu_30019-RA protein Name:"Similar to ERF9 Ethylene-responsive transcription factor 9 (Arabidopsis thaliana)" AED:0.10 eAED:-0.04 QI:0|0|0|1|1|1|2|0|236


Cnu_34164-RA protein Name:"Similar to ERF12 Ethylene-responsive transcription factor 12 (Arabidopsis thaliana)" AED:0.25 eAED:-0.15 QI:0|-1|0|1|-1|1|1|0|166


Cnu_09596-RA protein Name:"Protein of unknown function" AED:0.12 eAED:0.12 QI:0|0.5|0|1|1|1|3|0|231

Cnu_09597-RA protein Name:"Similar to RAP2-3 Ethylene-responsive transcription factor RAP2-3 (Arabidopsis thaliana)" AED:0.02 eAED:0.01 QI:0|1|0.5|1|1|0.5|2|407|249

Cnu_15237-RA protein Name:"Similar to PLT2 AP2-like ethylene-responsive transcription factor PLT2 (Arabidopsis thaliana)" AED:0.13 eAED:0.13 QI:0|0|0|1|0.85|0.75|8|0|405

Cnu_23095-RA protein Name:"Similar to CRF4 Ethylene-responsive transcription factor CRF4 (Arabidopsis thaliana)" AED:0.14 eAED:0.30 QI:7|0.5|0.33|1|0.5|0.33|3|0|354




Cnu_25805-RA protein Name:"Similar to ERF1B Ethylene-responsive transcription factor 1B (Arabidopsis thaliana)" AED:0.04 eAED:0.03 QI:0|-1|0|1|-1|1|1|0|190

Cnu_14580-RA protein Name:"Similar to ABI4 Ethylene-responsive transcription factor ABI4 (Arabidopsis thaliana)" AED:0.23 eAED:0.23 QI:0|0|0|0.5|1|1|2|0|216




195

Cnu_22340-RA protein Name:"Similar to ERF019 Ethylene-responsive transcription factor ERF019 (Arabidopsis thaliana)" AED:0.11 eAED:-0.05 QI:0|-1|0|1|-1|1|1|0|158\



Cnu_03309-RA protein Name:"Similar to WRI1 Ethylene-responsive transcription factor WRI1 (Arabidopsis thaliana)" AED:0.12 eAED:0.17 QI:0|0|0|1|0.33|0.71|7|0|404


Cnu_12485-RA protein Name:"Similar to AP2 Floral homeotic protein APETALA 2 (Arabidopsis thaliana)" AED:0.22 eAED:0.15 QI:0|0.83|0.69|0.92|0.75|0.61|13|55|485



Cnu_24714-RA protein Name:"Similar to PTI5 Pathogenesis-related genes transcriptional activator PTI5 (Solanum lycopersicum)" AED:0.05 eAED:0.05 QI:0|-1|0|1|-1|1|1|0|180


Cnu_26361-RA protein Name:"Similar to ANT AP2-like ethylene-responsive transcription factor ANT (Arabidopsis thaliana)" AED:0.09 eAED:0.09 QI:112|0.85|0.75|1|1|0.87|8|319|659

Cnu_06945-RA protein Name:"Similar to DREB1E Dehydration-responsive element-binding protein 1E (Oryza sativa subsp. japonica)" AED:0.11 eAED:-0.14 QI:0|-1|0|1|-1|1|1|0|298







Cnu_08246-RA protein Name:"Similar to ERF5 Ethylene-responsive transcription factor 5 (Nicotiana sylvestris)" AED:0.13 eAED:0.13 QI:0|-1|0|1|-1|1|1|0|213

Cnu_08247-RA protein Name:"Similar to ERF1 Ethylene-responsive transcription factor 1 (Solanum lycopersicum)" AED:0.08 eAED:0.04 QI:0|0|0|1|1|1|2|0|282

Cnu_17812-RA protein Name:"Similar to ERF1B Ethylene-responsive transcription factor 1B (Arabidopsis thaliana)" AED:0.00 eAED:0.00 QI:0|-1|0|1|-1|1|1|0|227

Cnu_12326-RA protein Name:"Similar to AIL1 AP2-like ethylene-responsive transcription factor AIL1 (Arabidopsis thaliana)" AED:0.08 eAED:0.08 QI:0|0|0|0.77|0.5|0.66|9|0|623


Cnu_04041-RA protein Name:"Similar to ERF9 Ethylene-responsive transcription factor 9 (Arabidopsis thaliana)" AED:0.50 eAED:-0.04 QI:0|0|0|0.5|1|1|2|0|196






196

Cnu_18957-RA protein Name:"Similar to RAP2-11 Ethylene-responsive transcription factor RAP2-11 (Arabidopsis thaliana)" AED:0.03 eAED:-0.00 QI:0|-1|0|1|-1|1|1|0|266

Cnu_26752-RA protein Name:"Similar to RAP2-11 Ethylene-responsive transcription factor RAP2-11 (Arabidopsis thaliana)" AED:0.35 eAED:0.22 QI:0|0|0|0.5|1|1|2|0|355

Cnu_06218-RA protein Name:"Similar to ERF105 Ethylene-responsive transcription factor ERF105 (Arabidopsis thaliana)" AED:0.13 eAED:-0.04 QI:0|0|0|1|1|1|2|0|234


Cnu_20463-RA protein Name:"Similar to PTI6 Pathogenesis-related genes transcriptional activator PTI6 (Solanum lycopersicum)" AED:0.11 eAED:0.07 QI:0|-1|0|1|-1|1|1|0|251



Cnu_28657-RA protein Name:"Similar to WRI1 Ethylene-responsive transcription factor WRI1 (Arabidopsis thaliana)" AED:0.13 eAED:0.32 QI:0|0|0|0.71|0.16|0|7|0|325



Cnu_23164-RA protein Name:"Similar to PLT2 AP2-like ethylene-responsive transcription factor PLT2 (Arabidopsis thaliana)" AED:0.30 eAED:0.35 QI:0|0.5|0.28|1|1|1|7|0|414





Cnu_05246-RA protein Name:"Similar to AIL5 AP2-like ethylene-responsive transcription factor AIL5 (Arabidopsis thaliana)" AED:0.16 eAED:0.15 QI:0|0.5|0.22|0.88|0.75|0.66|9|0|468

Cnu_33633-RA protein Name:"Similar to ERF110 Ethylene-responsive transcription factor ERF110 (Arabidopsis thaliana)" AED:0.24 eAED:0.28 QI:0|0.33|0|0.75|1|0.75|4|0|267

Cnu_16571-RA protein Name:"Similar to LEP Ethylene-responsive transcription factor LEP (Arabidopsis thaliana)" AED:0.06 eAED:0.02 QI:0|-1|0|1|-1|1|1|0|281


Cnu_16598-RA protein Name:"Similar to ERF113 Ethylene-responsive transcription factor ERF113 (Arabidopsis thaliana)" AED:0.30 eAED:0.30 QI:41|0|0.33|0.66|0.5|0.66|3|0|182

Cnu_01626-RA protein Name:"Similar to DREB2C Dehydration-responsive element-binding protein 2C (Arabidopsis thaliana)" AED:0.16 eAED:0.16 QI:215|1|1|1|1|1|2|333|321



Cnu_30010-RA protein Name:"Similar to CRF4 Ethylene-responsive transcription factor CRF4 (Arabidopsis thaliana)" AED:0.09 eAED:0.09 QI:0|-1|0|1|-1|1|1|0|326

Cnu_20112-RA protein Name:"Similar to BBM2 AP2-like ethylene-responsive transcription factor BBM2 (Brassica napus)" AED:0.13 eAED:0.11 QI:0|0|0|1|1|1|7|0|719





197

Cnu_01859-RA protein Name:"Similar to EREBP1 Ethylene-responsive transcription factor 1 (Oryza sativa subsp. japonica)" AED:0.02 eAED:0.02 QI:191|1|1|1|0.5|0.33|3|1010|385




Cnu_14805-RA protein Name:"Similar to CRF4 Ethylene-responsive transcription factor CRF4 (Arabidopsis thaliana)" AED:0.06 eAED:0.06 QI:0|0|0|1|1|1|2|0|251

Cnu_23607-RA protein Name:"Similar to WRI1 Ethylene-responsive transcription factor WRI1 (Arabidopsis thaliana)" AED:0.09 eAED:0.09 QI:127|0.8|1|1|0.8|0.83|6|205|339

Cnu_23610-RA protein Name:"Protein of unknown function" AED:0.25 eAED:0.21 QI:0|0.46|0.37|0.81|1|1|16|0|953

Cnu_23915-RA protein Name:"Similar to RAP2-3 Ethylene-responsive transcription factor RAP2-3 (Arabidopsis thaliana)" AED:0.02 eAED:0.02 QI:61|1|0.5|1|1|1|2|0|206

Cnu_24775-RA protein Name:"Similar to WIN1 Ethylene-responsive transcription factor WIN1 (Arabidopsis thaliana)" AED:0.00 eAED:0.00 QI:103|1|1|1|1|1|2|312|205

Cnu_16178-RA protein Name:"Similar to PLT2 AP2-like ethylene-responsive transcription factor PLT2 (Arabidopsis thaliana)" AED:0.16 eAED:0.16 QI:0|0.5|0.28|1|0.83|0.71|7|0|388


Cnu_01025-RA protein Name:"Similar to WRI1 Ethylene-responsive transcription factor WRI1 (Arabidopsis thaliana)" AED:0.16 eAED:0.23 QI:0|0|0|1|0.6|0.66|6|0|298

Cnu_01179-RA protein Name:"Similar to DREB2C Dehydration-responsive element-binding protein 2C (Arabidopsis thaliana)" AED:0.16 eAED:-0.06 QI:0|-1|0|1|-1|1|1|0|259

Cnu_01240-RA protein Name:"Similar to AP2 Floral homeotic protein APETALA 2 (Arabidopsis thaliana)" AED:0.09 eAED:0.09 QI:208|0.71|0.75|1|0.71|0.87|8|349|483

Table I continued…



Cnu_03725-RA protein Name:"Similar to ERF1A Ethylene-responsive transcription factor 1A (Arabidopsis thaliana)" AED:0.41 eAED:0.49 QI:0|0|0|0.5|1|1|6|0|383

Cnu_03727-RA protein Name:"Similar to ERF5 Ethylene-responsive transcription factor 5 (Nicotiana sylvestris)" AED:0.32 eAED:0.27 QI:0|0|0|1|0|0.5|2|0|331

Cnu_03728-RA protein Name:"Similar to RAP2-2 Ethylene-responsive transcription factor RAP2-2 (Arabidopsis thaliana)" AED:0.41 eAED:0.43 QI:0|0|0|0.5|1|1|4|0|332







198

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