Tree Genetics & Genomes (2013) 9:813–827 DOI 10.1007/s11295-013-0600-5
ORIGINAL PAPER
Isolation and characterization of an AINTEGUMENTA-like gene in different coconut (Cocos nucifera L.) varieties from Sri Lanka H. D. Dharshani Bandupriya & J. George Gibbings & Jim M. Dunwell
Received: 20 May 2012 / Revised: 19 December 2012 / Accepted: 7 January 2013 / Published online: 24 February 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Development of an efficient tissue culture protocol in coconut is hampered by numerous technical constraints. Thus a greater understanding of the fundamental aspects of embryogenesis is essential. The role of AINTEGUMENTA-like genes in embryogenesis has been elucidated not only in model plants but also in economically important crops. A coconut gene, CnANT, that encodes two APETALA2 (AP2) domains and a conserved linker region similar to those of the BABY BOOM transcription factor was cloned, characterized, and its tissue specific expression was examined. The full-length cDNA of 1,780 bp contains a 1,425-bp open reading frame that encodes a putative peptide of 474 amino acids. The genomic DNA sequence includes 2,317 bp and consists of nine exons interrupted by eight introns. The exon/intron organization of CnANT is similar to that of homologous genes in other plant species. Analysis of differential tissue expression by real-time polymerase chain reaction indicated that CnANT is expressed more highly in in vitro grown tissues than in other vegetative tissues. Sequence comparison of the genomic sequence of CnANT in different coconut varieties revealed one single nucleotide polymorphism and one indel in the first exon and first intron, respectively, which differentiate the Tall group of Communicated by W.-W. Guo H. D. D. Bandupriya : J. G. Gibbings : J. M. Dunwell (*) School of Biological Sciences, University of Reading, Reading RG6 6AS, UK e-mail:
[email protected] H. D. D. Bandupriya Tissue Culture Division, Coconut Research Institute, Lunuwila, Sri Lanka
trees from Dwarfs. The indel sequence, which can be considered a simple sequence repeats marker, was successfully used to distinguish the Tall and Dwarf groups as well as to develop a marker system, which may be of value in the identification of parental varieties that are used in coconut breeding programs in Sri Lanka. Keywords CnANT gene . Coconut varieties . Indel . Single nucleotide polymorphism
Introduction The coconut palm (Cocos nucifera L.) is an important cash crop grown in more than 90 countries in the tropics. Being a cross-pollinated plant that is propagated only by seed, it exhibits a high level of genetic variation among seedlings. Although plant breeding research has improved the performance of the crop, problems encountered with conventional breeding such as its long life span and high heterozygosity make plant breeding a long, difficult, and expensive process. Vegetative propagation of coconut via tissue culture is a promising way of increasing yield, specifically by propagating elite material. Despite research conducted over several decades, in vitro culture of coconut has not developed sufficiently to allow an efficient commercial clonal propagation method. In recent years, embryogenesis-related research have been focused on the use of molecular methods to better understand the process (Casson et al. 2005; Hecht et al. 2001). Therefore, to remove the obstacles associated with coconut somatic embryogenesis and speed up the process, it is of great importance to be able to use molecular
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markers to identify cultivars, explants, and cultures which show a higher response for somatic embryogenesis at an early stage. During the process of embryogenesis, several genes related to cell differentiation, morphogenesis, desiccation tolerance, and signal transduction are expressed (reviewed by Dunwell 2007; Yang and Zhang 2010). Many studies on embryo specific genes have been carried out using Arabidopsis thaliana, which serves as a model plant (Ishikawa et al. 2003; Long et al. 1996; Xu et al. 2005). A minimum set of ~750 A. thaliana genes are thought to be required in this process from which a subset of 250 genes are required for normal embryo development (McElver et al. 2001; Tzafrir et al. 2004). Extensive research has also identified several transcription factors that are specifically activated during embryogenesis. Among these transcription factor genes involved in embryogenesis, LEAFY COTYLEDON genes [LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2) and FUSCA3 (FUS3)] play an important role in both early and late phases. LEC1 encodes a protein similar to a HAP3 subunit while LEC2 and FUS3 encode proteins similar to the B3 domain (Lotan et al. 1998; Luerssen et al. 1998; Stone et al. 2001). Mutants of LEC genes (LEC1, LEC2, and FUS3) developed somatic embryos at a very low frequency in Arabidopsis, and a total loss of somatic embryo competence was reported with double mutants. This provides evidence that LEC genes are particularly important in the induction of somatic embryogenesis (Gaj et al. 2005). In addition, a variety of experimental evidence indicates that the group of WUSCHEL (WUS) homeodomain protein transcription factors mainly specify the stem cell fate (Mayer et al. 1998) and play a role during embryogenesis. When ectopically expressed, these genes promote somatic embryo development (Gallois et al. 2004; Zuo et al. 2002) by the maintenance of an undifferentiated cell state that responds to different stimuli. Specifically, the MADS-domain transcription factor gene AGAMOUS-LIKE 15 (AGL15) was identified as an embryo expressed gene (Heck et al. 1995; Rounsley et al. 1995). The soybean AGL15 homologue, GmAGL15, was detected in young developing embryos and in somatic embryos. When ectopically overexpressed, GmAGL15 was able to enhance somatic embryo development in soybean (Thakare et al. 2008). In another study, AGL15 was shown to promote somatic embryo development from cultured zygotic embryos (Harding et al. 2003). Related studies have revealed that members of the transcription family APETALA2/Ethylene-responsive element binding protein (AP2/EREBP) have roles in plant development (Elliott et al. 1996; Klucher 1996; Nole-Wilson et al. 2005) especially during embryo formation. To date, members such as APETALA2 (AP2), AINTEGUMENTA (ANT), AINTEGUMENTA-like (AIL) BABY BOOM (BBM),
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PLETHORA1 (PLT1), and PLETHORA2 (PLT2) have shown effects during the reproductive phase of plant development. The Arabidopsis APETALA2 gene regulates embryo size by negatively controlling cell proliferation during seed development (Ohto et al. 2005). Also, it maintains the stem cell niche of the shoot meristem (Schulze et al. 2010). It has been revealed that ANT is required for ovule development and floral organ growth (Elliott et al. 1996; Klucher 1996). Several AIL genes have been shown to be involved in the differentiation of embryogenic stem cells from somatic cells (Morcillo et al. 2007; Tsuwamoto et al. 2010). Likewise, overexpression of the BBM gene induced spontaneous somatic embryos from leaf and cotyledon margins (Boutilier et al. 2002). PLETHORA genes (PLT1 and PLT2) which are closely related to AIL genes (Nole-Wilson et al. 2005; Tsuwamoto et al. 2010) are thought to be regulators of root development and embryo differentiation (Galinha et al. 2007; Aida et al. 2004). Moreover, there is evidence to show redundancy in gene function. For example, It has been shown that AtBBM and PLT2 function redundantly when the mutant phenotypes of bbm and plt2 was studied in early Arabidopsis embryogenesis (Galinha et al. 2007). Furthermore, there are reports on the improvement of the response of plants to embryogenesis in vitro by modifying gene expression via the introduction of AP2/ANT genes (Deng et al. 2009). Although the majority of detailed functional information on the EP2 genes has been obtained from the dicot model Arabidopsis, there is an increasing amount of information now being obtained from monocotyledonous species, particularly from cereals. Examples of this type include phylogenetic studies on maize (Zhuang et al. 2010) and wheat (Zhuang et al. 2011), expression studies in rice (Sharoni et al. 2011, 2012; Qi et al. 2011; Lee and An 2012), and links to domestication traits in wheat (Zhang et al. 2011). The only information from the important group of perennial monocots, the palms, is the identification of an AINTEGUMENTA-like transcription factor gene (EgAP2-1) in oil palm (Elaeis guineensis Jacq.) and study of its expression in meristematic tissues of both zygotic and somatic embryos (Morcillo et al. 2007). In this study, we isolated the full-length cDNA sequence of an ANT-like gene homologue in coconut (C. nucifera L.). The structure of this gene and the encoded protein sequence was compared with previously reported genes of the same family. In addition, the gene sequence was isolated from a range of different coconut varieties from Sri Lanka. CnANT was identified as the coconut ANT homologue and has the same genomic structure as AtBBM, BnBBM1, AtAIL, and Os04g0653600 genes from the same gene subgroup. Sequence comparison of genomic sequence of CnANT in different coconut varieties revealed one SNP and one indel in the first exon and first intron, respectively. These differences allowed the design of
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a molecular marker that discriminated a group of Tall trees from a group of Dwarfs.
Materials and methods Plant material A mature coconut embryo (approximately 100 mg; 14 months after pollination) was isolated from the Sri Lanka “Tall” variety and used for RNA extraction and cDNA synthesis with the aim of isolating the ANT homologue gene. Plant material for DNA comparison in different varieties was obtained from the ex situ coconut gene bank of Coconut Research Institute of Sri Lanka. Seven varieties namely “Typica Tall,” “Nawasi,” “Gon Thembili,” “Pora Pol,” “Bodiri,” and “Kamandala” from the major Typica group (Tall), four varieties namely “Dwarf Green,” “Dwarf Yellow,” “Dwarf Red,” and “Dwarf Brown” from the major Nana (Dwarf) group, and two varieties, “King Coconut” and “Rathran Thembili,” which represented the Aurantiaca group (Liyanage 1958) were used for the analysis. In addition, “San Ramon” (Fernando 1987), an introduced coconut variety from the Philippines, which is used extensively for conventional breeding programs, was also analyzed. For the qRT-PCR analysis, shoot apex, roots, and young leaves were harvested from in vitro grown plantlets supplied by the Tissue Culture Division, Coconut Research Institute, Sri Lanka. An immature inflorescence was collected from −7 maturity stage (considering the youngest open inflorescence as 0; −1 is the inflorescence that opens next). Embryogenic callus used for expression analysis were obtained either from microspore explants or plumule explants. Microspore derived callus and somatic embryo induction using anther explants was carried out as described by Perera et al. (2008). The culture conditions for plumules were according to a method developed at Coconut Research Institute, Sri Lanka by incorporating glutamine in the medium (Vidhanaarachchi et al. 2007). RNA isolation and RT-PCR Total RNA was extracted from each tissue sample using the RNeasy® Plant Kit (Qiagen) according to the manufacturer’s instructions. All RNA samples were treated with DNase I (Qiagen, UK) according to the manufacturer’s instructions to remove DNA contamination. Total RNA was quantified using a NanoDrop® ND-1000 Spectrophotometer, and the quality of the extracted RNA was assessed by 1 % (w/v) agarose gel electrophoresis. Approximately 500 ng of total RNA was used to synthesize first strand cDNA using SensiMix 2 step kit (Quantace, UK) according to the manufacturer’s instructions.
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Cloning of partial sequence of coconut ANT-like cDNA Two primer pairs 2 F (5′-TCT ATC TAC CGC GGC GTC3′) and 4R (5′-ACA AAC TCC TGT CGT GTC A-3′), 4 F (5′-TGA CAC GAC AGG AGT TTG T-3′) and 5R2 (5′-ATT CCA TTC CAA AGA TGG G-3′) were designed based on the most conserved sequences of rice (Accession NM 001060643) and oil palm (Accession AY691196) AINTEGUMENTA-like genes. PCR was performed using 1 μL of cDNA in the presence of primers at 0.3 μM concentration in a 25-μL reaction volume containing 12.5 μL Biomix (Bioline, UK) with the following conditions: initial 2 min denaturation step at 94 °C, 30 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s, followed by 7 min at 72 °C for a final extension. DNA sequencing was carried out with purified PCR products after they were subjected to BigDye terminator cycle sequencing reaction. Rapid amplification of cDNA ends After an internal fragment (1,031 bp) of the coconut ANTlike gene was obtained, sequence determination of the 5′ and 3′ ends was carried out by the Rapid amplification of cDNA ends (RACE) method using the GeneRacer kit (Invitrogen, UK). Approximately 2.2 μg of total RNA from mature zygotic embryo was treated with calf intestinal phosphatase to remove 5′ terminal phosphate of nonfull-length RNA, and then treated with tobacco acid pyrophosphatase to remove the 5′-cap structure of the full-length mRNA which leaves a 5′ phosphate for the ligation to the GeneRacer RNA Oligo (5′- CGA CUG GAG CAC GAG GAC ACU GAC AUG GAC UGA AGG AGU AGA AA-3′) by action of T4 ligase. The ligated mRNA was then reverse transcribed using the GeneRacer Oligo dT primer (5′-GCT GTC AAC GAT ACG CTA CGT AAC GGC ATG ACA GTG(T) 24 -3′) and SuperScriptTM reverse transcriptase to synthesize the complete cDNA carrying the GeneRacer kit specific oligonucleotide sequences at the 5′ and 3′ ends. A touchdown PCR was carried out using a 4R gene specific reverse primer with GeneRacer 5′ primer (5′-CGA CTG GAG CAC GAG GAC ACT GA-3′) to amplify the 5′ end. The amplification parameters were 5 cycles of 94 °C for 30 s and 70 °C for 1 min, 5 cycles of 94 °C for 30 s and 68 °C for 1 min, 25 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. To amplify the 3′ end, a gene specific forward primer 4 F was combined with a GeneRacer 3′ primer (5′-GCT GTC AAC GAT ACG CTA CGT AAC G-3′), and the same PCR conditions were used. Then nested PCR reactions using the GeneRacer 5′ nested primer (5′ GGA CAC TGA CAT GGA CTG AAG TAG AAA-3′) and 3R for the 5′ end, GeneRacer 3′ nested primer (5′-CGC TAC GTA ACG GCA TGA CAG TG-3′) and ANTF (5′-AAC TGG ATT
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ATG CAT GAT GA-3′) primer for the 3′ end were performed. Thirty cycles were run each with 30 s denaturation at 94 °C, followed by 30 s of annealing at 59 °C and 1 min extension at 72 °C. The final extension was at 72 °C for 10 min. Purified 5′ and 3′ RACE products were cloned in the TOPO vector (Invitrogen, UK) and sequenced. Cloning of putative promoter region Genome walking using the Universal GenomeWalker kit (Clontech, UK) was performed to obtain the sequence of the promoter region. The total genomic DNA was extracted from young coconut leaves using a DNeasy Plant Kit (Qiagen, UK) according to the manufacturer’s instructions. Genomic DNA was digested with restriction endonucleases DraI, EcoRV, PvuII, and StuI separately and purified by the phenol/chloroform method. The GenomeWalker adaptor was ligated to the digests, and these were then used to amplify the promoter of the CnANT gene. Two PCR reactions were performed using gene specific primers designed based on the CnANT cDNA sequence. The adaptor primer AP1 (5′-GTA ATA CGA CTC ACT ATA GGG C-3′) and gene specific primer GW1 (5′-GGC GAT GTG CTT CAG TTC AGA GTC GT-3′) were used in the primary PCR. The amplification parameters were 6 cycles of 94 °C for 25 s and 72 °C for 3 min, 32 cycles of 94 °C for 25 s, and 67 °C for 3 min followed by a final extension step at 67 °C for 7 min. A diluted primary PCR product was used as a template for the nested PCR with the adaptor primer AP2 (5′-ACT ATA GGG CAC GCG TGG T-3′) and gene specific GWR2 primer (5′-GAA GGT AGG GCT GGT GGT TGG ATA GC-3′). The following regimes were used for the PCR amplification: six cycles of 94 °C for 25 s and 72 °C for 3 min, 20 cycles of 94 °C for 25 s, and 67 °C for 3 min followed by a final extension step of 67 °C for 7 min. Amplified products were sequenced from both directions on an ABI310 DNA sequencer (Macrogen, Korea). The promoter sequence was then analyzed with the Plant cisacting regulatory DNA elements database [PLACE- (http:// www.dna.affrc.go.jp)] at the Advanced Biosciences Computing Center (Higo et al. 1999). Expression analysis by real-time RT-PCR Two gene-specific primers were designed based on the CnANT gene to amplify a 180 bp coding region. The primer sequences were ANTF1: (5′-CGG TCT CTT CTC CTC TGG TG-3′) and ANTR: (5′-TCG TAA TTC CCT CCA AAT GC-3′). The coconut elongation factor gene was used as the internal control gene (Menssen and Hermeking 2002; Morcillo et al. 2007; Olsvik et al. 2005). cDNA from each tissue sample was used for real-time RT-PCR analysis using SYBR premix Ex taqTM (Takara). Experiments were
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conducted with two biological samples, and the real-time qPCT reactions were performed in triplicate using the CAS-1200 liquid handling system, version 4.7.979 (Corbett Robotics). The real-time RT-PCR was performed on a Rotor-Gene 6000 real-time cycler (software 1.7, Corbett Research). The amplification parameters were one cycle at 95 °C for 1 min, 39 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 8 s. The relative expression level in different tissues was calculated by the standard curve method. Amplification and direct sequence analysis of the full-length CnANT gene and its comparison in different C. nucifera L. varieties from Sri Lanka DNA from the spear leaf of the Tall variety was used to amplify the genomic sequence of CnANT. Primer pairs ANTpro1 (5′-TCC TCA AAG CGT TCA AAT CA-3′) and 3R (5′-CGC CTT CTC CTC CTT ATC-3′), 2 F and 4R, 7 F (5′-GAA CGA CGA GTT AAA TTT GG-3′) and 8R (5′TTG AGC CCC CGA AAC TTA AT-3′), ANT6F (5′-GGA GGC CTA TGA CAT TGC AG-3′), and CnANTR1 (5′-TTC CCA CCC ATC CAT ATC AT-3′) were used to amplify four overlapping regions of CnANT gene. Three independent reactions were conducted for each primer pair. PCR products were checked on agarose gels, purified and sequenced from both directions. Finally, all partial sequences were assembled to obtain the full-length genomic sequence. The exon/intron boundaries were determined by comparing the gene sequence with cDNA sequence. The successful procedure was applied for the other 13 coconut varieties from Sri Lanka. Distinguishing Tall and Dwarf varieties using the simple sequence repeat in the CnANT gene The indel region in the first intron was used to develop a marker system in order to uniquely identify coconut varieties that are used as parents in coconut breeding program in Sri Lanka and their hybrids. Primer3 software was used to design a primer pair (ANTdelF 5′-TAC CTT CTC GAG GCT CTG T-3′ and ANTdelR 5′-TCT TAG AAG CCA TAT GAG AGG-3′) to amplify a sequence which contains the indel region. The actual length of the amplified product for Tall varieties was 104 bp and for Dwarfs it was 92 bp. gDNA isolated from different coconut varieties was used for PCR. The amplification profile was 35 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 45 s and a final extension step at 72 °C for 7 min. PCR products were separated on 3 % agarose (Bioline, UK) in 1 X TAE buffer at 95 v for 3 h. A 10bp ladder (Invitrogen, UK) was used as the molecular weight marker.
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Results Isolation and characterization of CnANT cDNA Two overlapping amplifications of primer pairs 2 F; 4R and 4 F; 5R2 were assembled to obtain a 1,029-bp consensus sequence. Since this is a truncated sequence, 5′ and 3′ RACE were used to clone the full-length cDNA. Amplification of a 446 bp fragment from 5′ RACE and 317 bp fragments from 3′RACE then resulted in a 1,792bp full-length cDNA and named as C. nucifera AINTEGUMENTA-like (CnANT) gene. The predicted amino acid sequence contained 474 amino acids and shared a high degree of sequence similarity with the oil palm ANT-like gene EgAP2-1. CnANT showed a higher sequence similarity within the region of the two AP2 domains, including the sequence linking them, to the members in the ANT group within the AP2 subfamily, which includes AINTEGUMENTA (ANT; (Elliott et al. 1996) BABY BOOM (BBM; (Boutilier et al. 2002), and AIL5 (Tsuwamoto et al. 2010). To identify the presence of previously reported conserved motifs (Kim et al. 2006) in CnANT, sequence similarities were checked after a BLASTP search. The conserved motif sequences of representative ANT-group genes from other species are listed in Table 1 alongside CnANT. All four motifs were identified in the protein encoded by CnANT, providing evidence for CnANT being grouped as an ANT subgroup protein. The 10 amino acid insertion in the AP2 repeat 1 formed the first motif
(EuANT1) as described by Kim et al. (2006). Three other motifs EuANT 2, EuANT 3, and EuANT 4 were identified in the pre-domain region; among them, the EuANT 4 motif appeared just before the first AP2 domain (Kim et al. 2006). Although the CnANT protein sequence, other than the AP2 domain region, did not show large stretches of sequence similarity with other genes in different families, it did show 93 % sequence identity and 94 % sequence similarity with EgAP2-1 which has been isolated from oil palm. The EgAP2-1 protein consists of 478 amino acid residues while the CnANT protein consists of 474 amino acid residues. The major difference between two species was identified as an indel of four amino acids. Interestingly, all four amino acids are glutamines and the indel occurred in a high glutamine repeat area. Whereas oil palm EgAP2-1 contains 10 glutamines starting from amino acid residue 365, coconut CnANT contains only six of them starting from the same position. Apart from this indel, the rest of the sequences were very similar with 448 amino acids from a total of 478 being identical. The ANT-like Os04g0653600_Os (Oryza sativa), another monocot protein, has 57 % identity with the CnANT protein (Fig. 1). The CnANT protein contains Ser-rich (residues 314–325) and Gln-rich (residues 365–370 and 413–417) regions common to other genes such as the Ser-rich region in AIL5 and Gln rich region in PLT1, AIL6, and AtBBM (Nole-Wilson et al. 2005; Seipel et al. 1992) in the same group and are considered as transcription activation sites (Jin and Liu 2008).
Table 1 The conserved motif sequences (as described by Kim et al. 2006) of representative AINTEGUMENTA-like proteins in different species Gene name
Species
euANT1
euANT2
euANT3
euANT4
CnANT_Cn EgAP2-1_Eg Os04g0653600_Os AIL5_At Os06g0657500_Os PLT1_At PLT2_At
Cocos nucifera Elaeis guineensis Oryza sativa Arabidopsis thaliana Oryza sativa Arabidopsis thaliana Arabidopsis thaliana
NSCRREGQSR NSCRREGQSR NSCRREGQSR NSCRREGQSR NSCRREGQSR NSCRREGQSR NSCRREGQSR
WLAFSLS WLAFSLS WLNFSLA WLSFSLS WLSFSLS WLGFPLS WLAFPLS
PKLEDFLG PKLEDFLG PKLEDFLG PKLENFLG PKMEDFLG PKVADFLG PKVADFLG
TFGQR TFGQR SFGQR SFGQR TFGQR TFGQR TFGQR
AIL7_At 06g031120_Sb BBM_At ANT_Rc 04g025960_Sb BBM1_Zm PLT2_Gm BBM1_Bn BBM2_Bn BBM_Mt
Arabidopsis thaliana Sorghum bicolor Arabidopsis thaliana Ricinus communis Sorghum bicolor Zea mays Glycine max Brassica napus Brassica napus Medicago truncatula
NSCRREGQAR NSCRREGQSR NSCKREGQTR NSCRREGQTR NSCRREGQSR NSCRREGQSR NSCRREGQSR NSCKREGQTR NSCKREGQTR NSCRREGQTR
CLTFSLS WLNFSLA WLGFSLS WLGFSLS WLGFSLS WLGFSLS WLSFPLS WLGFSLS WLGFSLS LLGFSLS
PKLEDFLG PKLEDFLG PKLENFLG PKLENFLG PKLEDFLG PKLEDFLG PKVADFLG PKLENFLG PKLENFLG PKLENFLG
TLGQR SFGQR SFGQR TFGQR TFGQR TFGQR TFGQR SFGQR SFGQR TFGQR
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Fig. 1 Alignment of the deduced amino acid sequence of CnANT with two other monocot sequences EgAP2-1 (oil palm) and Os04g06536 (rice) from the same ANT subgroup. Amino acids in dark boxes are identical for all three species whereas light gray boxes represent identical amino acids between the two palm species, coconut and oil palm
Cloning the putative promoter region A genomic DNA fragment of 828 nucleotides upstream of the CnANT gene was identified from genome walking. The transcription start site was determined by 5′ RACE and was located 63 nucleotides upstream of the ATG translation initiation codon of CnANT cDNA. Thus, the first transcribed nucleotide of CnANT was an adenine. A BLASTN search of the putative promoter sequence did not reveal any significant homology in the GenBank database indicating that it is a novel genomic sequence. The structure of the CnANT promoter is shown in Fig. 2. The most proximal TATA box was located at −154 from the putative transcription initiation site. Putative CAAT box sequences were found at −330 and −710. The cisacting element analysis revealed several putative plant cis-acting DNA elements. Among them, an ABAresponsive element (ABRE) (CACGTGGC), a major
cis-acting element involved in ABA-mediated gene expression, was located at position −495. Interestingly, consensus sequences for some other ABA and dehydration responsive elements such as MYC (5′-CACAGT-3′, two elements), MYB1 (5′-WAACCA-3′), and MYB2 (5′YAACKG-3′) were identified at −710 and 178, −341, and −288 respectively. Furthermore, a core sequence for the Dc3 promoter binding factor (5′-ACACNNG-3′) identified in Carrot Dc3 gene which is involved in an ABA response and embryo specification was identified at position −407 (Kim et al. 1997). Two copies of a GT-1 (5′- GRWAAW-3′) consensus sequence which has been previously considered as a light regulatory element (Terzaghi and Cashmore 1995) were also found in the promoter region at positions −533 and −555. Previous studies have reported that the ARF site is a cis-acting elements associated with auxin response genes (Goda et al. 2004; Ulmasov et al. 1999). A CCTCGTGTCTC
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Fig. 3 Bar graphs showing relative levels of CnANT accumulation in different tissue types determined by RT-qPCR analysis. CnANT transcripts were normalized with respect to the elongation factor gene. Values are shown as mean±standard error (n=3). Lf: leaf, Rt: root, Sm: shoot meristem, Ii: immature inflorescence, Ec: embryogenic callus, Nec: nonembryogenic callus, Se: somatic embryo, Mde: microspore derived embryo
Fig. 2 Nucleotide sequence of the CnANT promoter and putative cisacting elements detected in the promoter region. The 828-bp upstream of the CnANT promoter from the ATG start codon and 42 bp of the protein coding region are shown. Numbering starts at the transcription start site (+1) as determined by 5′RACE. The 5′ UTR is in italics. The translation start codon is indicated in a box. The putative TATA box, CAAT, and other cis-acting elements are colored in gray and labeled below the sequence
sequence found in Glycine max GH3 gene promoter D1 element has shown related activity with the TGTCTC ARF element (Ulmasov et al. 1999). A similar sequence was identified in the CnANT promoter at −418. Several other previously identified cis-regulatory elements in other plant species such as Dof DNA binding protein core sites (5′-AAAG-3′) (Yanagisawa and Schmidt 1999), GATA boxes which bind with ASF-2 and thought to be involved in tissue specific expression (Lam and Chua 1989), were also found in the putative CnANT promoter. Expression analysis by real-time RT-PCR RNA was extracted from leaves, roots, immature inflorescence, shoot meristem, embryogenic callus, nonembryogenic callus, somatic embryos, and microspore-derived somatic embryo. During RT-qPCR, the PCR efficiencies of elongation factor and CnANT genes were approximately equal and the coefficients of correlation (R2) which was used to evaluate the quality of the standard curve for both reference and target genes were between 0.93 and 0.99. Analysis using PCR showed a greater accumulation of CnANT in tissues grown in vitro than in other vegetative tissues (Fig. 3). Results of
real-time PCR showed CnANT expression in multiple plant tissues: shoot meristem, root, and immature inflorescence, but it was not detected in leaves (Fig. 3). Among the vegetative tissues tested, CnANT transcripts were higher in roots compared to other tissues. Identification of the gene structure of CnANT To determine the gene organization of the CnANT gene, good quality DNA was isolated from spear leaf from the “Typica Tall” variety of coconut. Primers which were previously designed to amplify the cDNA sequence of CnANT were applied to the genomic DNA to isolate the genomic sequence of CnANT. Primers ANTpro1, 3R and 2 F, 4R gave the correct size bands, and only one amplicon was visualized when run on a gel. Assembled sequence gave a fragment nearly 1,150 bp in length in which five introns were detected. A fragment of approximately 600 bp was obtained for the primer pair ANT6F and CnANTR1, and no splice sites were detected in this sequence confirming a long exon towards the 3′ end of the gene. The primer pair 7 F and 8R was designed to obtain the middle portion of the gene which connects the above two partial sequences. To be more specific, one of the primers of this pair was designed from an intron sequence. This gave a fragment of approximately 670 bp. Acquired sequences were assembled, and a fulllength genomic sequence was identified for the Tall variety. Intron/exon junctions were determined by comparing gene sequence with the CnANT cDNA sequence. The CnANT gene (Fig. 4) consists of nine exons which are interrupted by eight introns of different phases (Whamond and Thornton 2006), and all splice junctions have the conserved GT-AG motifs (Breathnach and Chambon 1981). The intron types in the A. thaliana BBM
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Fig. 4 Gene structure of CnANT. Exons are colored in dark gray and introns are in light gray. Exon/intron splice sites are marked in black. Nucleotides coding the two conserved AP2 domains and the linker region are underlined. Nucleotide numbers are given on the right-hand side
gene were checked to determine whether a similar trend of intron diversity could be found in similar genes of the same subfamily. It revealed that A. thaliana BBM also has the same intron distribution. As per in CnANT, the fifth intron of the A. thaliana BBM gene is a phase 0 intron. Introns 1, 3, 4, and 8 are of phase 1, while 2, 6 and 7 are of phase 2 type. More interestingly, introns 2, 3, 4, 6, and 7 split exactly the same amino acid residue at the same position. The gene structural organization of CnANT was compared with functionally known AP2/EREBP from Arabidopsis, rice, and Brassica. The exon/intron structure
of ANT subgroup genes appears to be well conserved by having nine exons and eight introns. The lengths of exons one, two, and nine varied between different species while exon three to eight share the same number of nucleotides (Fig. 5). However, in all species, the longest exon was the ninth and the second longest was the second exon. In contrast, exon/intron splicing within the AP2 domain and linker region is well conserved in all members by having identical lengths encoding exactly the same amino acid regions of the proteins for exons 3–8. Intron lengths however varied considerably between different members of ANT genes.
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Fig. 5 Structural organization of CnANT gene and its comparison with related ANT subgroup genes. Boxes represent the exons and are drawn to scale. Lines represent the introns (not drawn to scale). Actual length of each intron is given in bp numbers
Comparison of CnANT gene structure in different C. nucifera varieties from Sri Lanka
Distinguishing Tall and Dwarf varieties using the simple sequence repeat in the CnANT gene
The CnANT sequence was compared in different coconut varieties from Sri Lanka. These included eight Tall varieties, four Dwarf, and two intermediate varieties. Sequence analysis predicted identical exons and introns in all varieties analyzed. Exon sequences were almost identical in Tall and Dwarf varieties with an exception of a single nucleotide polymorphism (SNP) located in the first exon, 88 bp downstream of translation start site (Fig. 6). This nucleotide was a cytosine in all Tall varieties and a guanine in all Dwarf varieties. This mutation changed the encoded amino acid from proline to alanine in Tall varieties and Dwarf varieties, respectively (Fig. 6). Interestingly, a 12 nucleotide insertion/deletion (Indel) was noted in the first intron for Tall and Dwarf varieties, and this could be used to differentiate Tall and Dwarf coconut varieties. In contrast, all Tall varieties have five CCTC repeats, while Dwarf varieties have only two repeats (Fig. 6). Apart from these two major characters observed between the two major groups Tall and Dwarf, few other SNPs and one indel were detected in certain coconut varieties. Dwarf Red variety was different from other varieties by having a seven nucleotide indel in intron 3 at 731 bp downstream of the translation start site. Two nucleotide polymorphisms at 793 and 838 positions located in the third intron differentiated Kamandala, San Ramon, and “King Coconut” varieties from rest of the varieties. These three varieties have cytosine in above two positions while others have thiamine. However, all these differences were found in intron sequences and thus do not interfere with the coding sequence of the gene.
The separation of the PCR amplification products of Tall and Dwarf varieties from Sri Lanka is shown in Fig. 7. The primer pair has produced unique markers for two coconut groups as predicted by the sequence. All Tall samples were distinguished by a single band “a” which appeared above the 100-bp band corresponding to the molecular weight marker, and Dwarfs were distinguished by a single band “b” appeared just below the 100-bp band corresponding to the molecular weight marker. PCR amplification was further applied to two hybrid varieties and distinguished the products along with their parents. The results indicated that the primer pair ANTdelF and ANTdelR can be successfully used to distinguish two hybrid coconuts D x T and Kapruwana from their parents Typica Tall and Dwarf Yellow of D x T and San Ramon and Dwarf Green of Kapruwana. The presence of “a” and “b” markers in the heterozygous state confirms the Typica Tall x Dwarf hybrids (Fig. 8).
Discussion Successful isolation of an AP2 subfamily gene in coconut (CnANT) allowed a comparison with other AP2 family genes in a range of species. Since the identification of the Arabidopsis BBM gene, efforts have been made to characterize the Arabidopsis BBM homologue ANT subgroup genes in a range of plant species during embryogenesis (Morcillo et al. 2007; Ouakfaoui et al. 2010; Srinivasan et
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Fig. 6 Sequence alignment of first exon [(starting from translation start site), colored in dark gray] and first intron (colored in light gray) in different coconut varieties from Sri Lanka. The SNP in the first exon
is indicated in white. Exon/intron splice sites are marked in black. The twelve nucleotide indel in the first intron of Dwarf varieties is marked as a box
al. 2007). The putative amino acid sequence of CnANT consists of two AP2/EREBP DNA binding domains and thus belongs to AP2/EREBP family. Four conserved motifs previously described (Kim et al. 2006) in ANT subgroup proteins were identified in CnANT. Identification of motifs outside the conserved DNA binding domains of transcriptional factor genes is a common phenomenon, and these motifs are thought to be functionally important and involved in transcriptional activity, protein–protein interactions, and nuclear localization (Liu et al. 1998). These are not only important in function but also identified as conserved among subgroups of large transcription gene families such as MYB, ERF, WRKY, and NAC (Kranz et al. 1998; Nakano et al. 2006; Olsen et al. 2005; Ulker and Somssich 2004) and thus have been used intensively for gene
classification. Conserved motifs have been previously identified in Arabidopsis, rice, and soybean ERF subfamily genes of the AP2/EREBP family (Nakano et al. 2006; Zhang et al. 2008). In addition, other studies suggest that conserved motifs outside the AP2 domains also play a role in gene function. In a recent publication, the BBM-1 motif which is specific to the BBM-like genes has been shown to be important in somatic embryogenesis via deletion and domain swap analysis (Ouakfaoui et al. 2010). Similarly, in Arabidopsis ANT, a serine rich region has been identified as a transcriptional activation domain which may serve as a protein phosphorylation site, thereby control AP2 activity (Hunter and Karln 1992), and therefore be of importance in the in vivo regulation of organ growth (Krizek and Sulli 2006).
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Fig. 7 Agarose gel separation of allelic distribution for the primer pair ANTdelF and ANTdelR. 1–8 are Tall varieties and 9–12 are Dwarf varieties. M: 10 bp ladder, 1: Typica Tall, 2: Nawasi, 3: Pora pol, 4: Bodiri, 5: Gon Thembili, 6: Kamandala, 7: Ran Thembili, 8: San Ramon, 9: Dwarf Yellow, 10: Dwarf Green, 11: Dwarf Brown, 12: Dwarf Red
The analysis carried out on different vegetative tissues of coconut using qRT-PCR confirmed that CnANT mRNA was present at high levels in zygotic embryos and in vitro cultured tissues. However, CnANT mRNA was also present in several vegetative organs such as root, immature inflorescence, and shoot meristem, though at very low levels. This is particularly in contrast with the findings of Nole-Wilson et al. (2005) and Tsuwamoto et al. (2010) on Arabidopsis
Fig. 8 Allelic distribution pattern of two hybrids D x T and Kapruwana and their male and female parents with ANTdelF and ANTdelR primers. M: 10 bp ladder, 1: Typica Tall, 2: Yellow Dwarf, 3: D x T hybrid, 4: San Ramon, 5: Green Dwarf, 6: Kapruwana hybrid
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AIL5 which is expressed in actively dividing tissues but not in mature tissues like leaves or stems. A weak signal was observed in roots which is a common feature amongst AIL genes (Feng et al. 2005; Nole-Wilson et al. 2005). Oil palm EgAP2-1, the closest relative of CnANT, showed no transcript accumulation in most of the vegetative tissues but was shown to be expressed throughout embryo development (Morcillo et al. 2007). The expression of CnANT at higher levels in in vitro cultured tissues and in zygotic embryo developmental stages is consistent with the suggestion that CnANT is associated with cell proliferation in coconut. A similar conclusion has been reached in a recent study of related ANT genes in apple (Dash and Malladt 2012). The suggestion from the present study that CnANT may play a role during the embryo development phase is also supported by the details of the promoter of this gene which is predicted to have several ABA-responsive and dehydration/stress-induced cis-acting elements. Abscisic acid (ABA) plays an important role during seed maturation and is thought to function in the response to stresses caused by dehydration (Galau et al. 1986). The late embryogenesis abundant gene family, one of a group of genes predominantly expressed during late stages of seed development, has been demonstrated to respond to ABA. Detailed analysis of their promoters has revealed conserved motifs referred to as ABREs (Busk and Pages 1998; Luo et al. 2008). These elements contain a CACGTC motif with the ACGT G-box core element (Izawa et al. 1993; Zhu 2002) and have been identified in many ABA inducible promoters in a range of species (Abe et al. 2003; Guiltinan et al. 1990; Izawa et al. 1993; Luo et al. 2008; Torres-Schumann et al. 1992). In the present study, analysis using the PLACE program predicted one ABRE motifs located in the CnANT promoter at positions −495. Furthermore, MYB and MYC recognition sites have been identified in dehydration/stress responsive gene promoters which act as cis-acting elements involved in ABA-responsive signal transduction (Abe et al. 1997; Luo et al. 2008). Arabidopsis RD22, a dehydration-responsive gene mediated by ABA, contains a MYC recognition site (CANNTG) and a MYB recognition site (C/TAACNA/G) for transcription factors AtMYC2 and AtMYB2, respectively, in the promoter and jointly activate gene expression (Abe et al. 1997, 2003). In the CnANT promoter, MYC and MYB cis-acting elements were detected by PLACE analysis providing more evidence for its putative activity at late stages of embryogenesis. However, further experiments such as beta-glucuronidase (GUS) reporter gene constructs analysis of a series of deletion and orientation mutants of the CnANT promoter are needed to confirm the detailed functions of the cis-acting elements present in the coconut CnANT gene. The analysis of genomic organization of CnANT and its comparison with similar ANT genes revealed striking similarities. ANT subfamily genes from different species have
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equal number of exons and introns. It was revealed that not only the amino acid sequence but also the gene structure is conserved within the two AP2 domains and linker region with highly conserved intron positions despite having varied intron size. Highly conserved exon structure in DNA binding domains has been reported previously. For example, exon/intron organization of Arabidopsis MYB DNA binding domain is highly conserved allowing little or no R2R3 domain variation (Matus et al. 2008). Sequence comparison of the genomic sequence of CnANT in different coconut varieties revealed one SNP and one indel in the first exon and first intron, respectively, which differentiate the Tall group from Dwarfs (Fig. 6). This nonsynonymous SNP encodes an amino acid change. However, this substitution occurred in the poorly conserved pre-domain region which is considered to have little importance to the function of the ANT genes. Functionally conserved elements of AP2 subfamily genes include the YRD and RAYD elements present in the two conserved domains; in particular, the RAYD element is thought to be involved in DNA binding through its amphipathic α-helix formed by the conserved central core of 18 amino acids (Okamuro et al. 1997). Four other conserved motifs, one located within the first AP2 domain and the rest of them located predomain are also considered functionally important as transcription activators (Kim et al. 2006). Since the above nucleotide substitution of Dwarf varieties does not affect any of these conserved motifs, the functional differences of CnANT between Tall and Dwarf coconut varieties are expected to be small. However, additional functional analysis such as transformation, and gene knockout experiments are needed to confirm this. The few other SNPs recognized in some of the individual verities were always located in introns without affecting the polypeptide sequence. It should be stressed that these results do not imply a causal relationship between the sequence variations and the difference in height, but probably denote a shared origin for the Tall and Dwarf forms, respectively. The indel sequence occurring in the first exon can be considered a simple sequence repeats (SSR) marker and was successfully used to distinguish Tall and Dwarf major groups as well as to develop a marker system in order to identify parental coconut varieties used in coconut breeding program in Sri Lanka and their hybrids. According to a systematic classification carried out for Sri Lankan coconut varieties, Tall (Typica), Dwarf (Nana), and Intermediate (Aurantiaca; intermediate between Tall and Dwarfs) varieties can be distinguished (Liyanage 1958). Indigenous coconut varieties from Sri Lanka have been classified into these varieties according to their morphology and breeding habits (Liyanage 1958). Under this classification, “Typica Tall,” “Bodiri,” “Pora Pol,” “Nawasi,” “Gon Thembili,” “Kamandala,” and “Ran Thembili” are grouped as Tall
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varieties; “Dwarf Yellow,” “Dwarf Green,” “Dwarf Brown,” and “Dwarf Red” are grouped as Dwarf varieties, and Intermediate varieties include King Coconut and Rathran Thembili. Studies carried out using amplified fragment length polymorphism profiling (Perera et al. 1998) and microsatellite DNA markers (Perera et al. 2004) have generated evidence for the above grouping. Even though the Aurantiaca group accession was considered to be intermediate between Tall and Dwarf accessions, the above two studies mentioned above provided evidence that Aurantiaca is more similar to dwarfs than Talls. However, in the present study, the intermediate varieties showed more similarity to Tall varieties by sharing a “C” at its SNP site in the first exon as well as lacking the 12 nucleotide indel which was observed in Dwarf varieties in their first intron. The results provided here indicate that the primers based on the presence of the indel in the first intron of CnANT can be successfully used to distinguish Tall, Dwarf Yellow, San Ramon, and Dwarf Green and their resultant hybrids Dwarf Yellow x Tall (D x T) and Dwarf Green x San Ramon (Kapruwana). In the case of the two hybrids, two bands detected corresponded to the two alleles contributed by the respective Tall and Dwarf parents. Confirming the identity of parental varieties and their hybrids has significant practical importance. For example, maintaining a high degree of genetic purity during hybrid production is very important. Mixing of seed lots during harvesting and transportation and mislabeling of seed beds are possible when handling large number of seeds from different cultivars. The primer pair described here is a simple and easy PCR-based method to eliminate some of the problems encountered during hybrid production process by testing a few individuals from the lot in doubt. Microsatellite markers have been successfully used previously to identify parents in the Sri Lanka coconut breeding program and the resulting hybrids (Perera et al. 2004). In another study, markers specific to the San Ramon variety for identification of noncontaminated materials for multiplication of this variety in seed gardens have been determined (Bandaranayake et al. 2005). However, polyacrylamide gels and silver staining have been used for the above experiments. In the present study, agarose gels at higher concentration gave good resolution for band separation. Hence, this is a simple and easy method for hybrid testing, in particular if it is necessary to check whether a palm is a Tall, Dwarf, or a hybrid. The potential drawback of this method, in that it is not possible to distinguish members within the Tall group or Dwarf group, limits its application when handling several varieties at the same time. In conclusion, the above data support the designation of CnANT as an AIL5 homologue based on the gene sequence, conserved motifs at N terminal, and C terminal and genomic sequence. We demonstrate that CnANT transcripts accumulate
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in zygotic embryos and tissues grown in vitro while no or low transcript levels are detected in organs such as leaf, root, immature inflorescence, and shoot meristem. Involvement of CnANT during embryogenesis was emphasized by the structure of the promoter by having several ABA-responsive elements. Furthermore, we describe here how the SSR primers developed from CnANT can be successfully used for the detection of Tall and Dwarf varieties within the Sri Lanka germplasm and that it is a simple and easy PCR-based method to eliminate some of the practical problems encountered during hybrid production process by testing a few individuals from any material in doubt. Acknowledgments The authors are grateful to the Tissue Culture Division of the Coconut Research Institute of Sri Lanka for providing coconut plant material. This project was supported by the Commonwealth Commission of the United Kingdom and the Association of Commonwealth Universities, through the British Council.
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