DOI : http://dx.doi.org/10.9787/KJBS.2012.44.4.470
Phylogenetic Analysis of Common Millet (Panicum miliaceum L.) using NTS of 5S Ribosomal DNA
Chung1*,
1Dept. of Genetic Engineering, Dong-A University, College of Natural Resources and Life Science,
2Dept. of Functional Crop, National Institute of Crop Science, RDA, 3National Crop Experiment Station, Rural Development Administration
Received on September 18, 2012. Revised on October 21, 2012. Accepted on October 31, 2012
Abstract
Twenty two common millet (Panicum miliaceum L.) varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences with a hope to provide the basic information on their exact origin. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS. Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragments, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region. Amplified DNAs of 400 bp fragment were subcloned and used for further investigation. The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. And critically different areas with insert or deletion were also confirmed. Those sequence difference seems to be used for discrimination of cultivars from different origin and use as molecular markers for origin identification. In phylogenic tree construction, the clear classification was shown where the genotypes from China and Russia is positioned together and stay away from domestic genotypes.
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INTRODUCTION

Common millet (Panicum miliaceum L.) is mainly cultivated in Korea, China, Japan, India and Russia. It is an annual herbaceous plant and its genome contains 2n = 18 or 20 chromosomes. It has been believed that common millet is cultivating more than several thousand years in China and its northeast and northwest region (Hu et al. 2009). Moreover, in recent discovery, ancient domestication of common millet extended to 10, 000 years ago (Lu et al. 2009). Comparison and contrast of the world’s oldest millet remains, found in the Early Neolithic site of Cishan (northern China), by using microfossil morphology and biomolecular components revealed that common millet was earlier agricultural crop than foxtail millet. Both common millet (Panicum miliaceum L.) and foxtail millet (Setaria italica) were the most important ancient crops in the region. 

Not only in East Asia, common millet was also the oldest cereal crop in Europe due to its short growing season, resistance against various abiotic stress (salt, alkali and drought) and its adaptability to many environments including high altitude (Wang et al. 2004). It was the one of main crop for some part of Europe and Eat Asia until the reach of potato and rice cultivation. After a long period of its usage as a feeding crop for pets, ignoring as a food material in developing country, recent highlight on common millet is based on many health benefits of this crop for human dietary. Compared to its total protein content to wheat, millet contained higher ratio of essential amino acids such as leucine, isoleucine and methionine (J Kalinova & J Moudry 2006). Especially, lysine content was very high, which is well known limiting amino acid. 

5S rRNA genes (5S rDNA) are well-known for the most easily identifiable genome organization where they formed the cluster of tandem repeats and contained thousand copies of repeat units (Baum and Johnson 1994). So far, various sizes of 5S rDNA repeats have been reported from plant species (Fulnecek et al. 2006). The basic organization of repeat in a plant species consists of highly conserved coding regions and variable size of non-transcribed spacers (NTSs) or nontranscribed intergenic spacer (IGS) region. The highly conserved transcribed region can be used for the alignment of many sequences from different organisms whether they are closely related or not. Nonetheless, more information on phylogeny of close relatives must be acquired from non-transcribed spacer because of its diversity from relatively rapid evolution (Baker et al. 2000; Baum et al. 2004; Kitamura et al. 2005; Baum and Johnson 2007). 

In Korea, a few studies have been reported on agronomical characteristics of common millet (Park et al. 1999). Recently, many health benefits of common millet as a food resource was highlighted and widely spread over public. Because of public interest growing over health problem and public preference on health-beneficiary food, such a crop like common millet will be cultivated in wider area and the increasing price of the crop will stimulate more cultivation every year. Most of the common millet growing in Korea has unclear origin of the resource. In this study, 22 common millet varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences. The analysis was carried out with the reasons. First, we hope to provide the basic information of the agricultural product on their putative origin when consumer tries to purchase common millet in the market. Second, identification of their exact origin will let us classify them into genetic resources in breeding program. 

MATERIALS AND METHODS

Plant materials

Twenty two common millet (Panicum miliaceum L.) cultivars were provided by the National Institute of Crop Science, Milyang, Gyeongsangnamdo, Korea (Table 1). Fully expanded plant leaves were cut into approximately 3-cm-long segment, and use immediately for DNA isolation or stores at -80℃. 

ebb3f3f72fcd04c97e7bb62e95aa197d.JPG.resized.JPGTable 1. List of common millets (Panicum miliaceum L.) used for study and their origin.

DNA extraction and amplification of 5S rRNA gene

Nucleic acids were extracted from leaves of common millet using a modification of the CTAB (cetyltrimethylammonium bromide) protocol (Rogers and Bendich 1985). After ground completely, powder of plant sample was resuspended and incubated in a mix of 500 μl 2X CTAB buffer (2% [w/v] CTAB, 100 mM Tris–HCl pH 8.0, 1.4 M NaCl, 20 mM 0.02 M EDTA pH, 1.4M NaC1, 1% PVP (polyvinylpyrrolidone) MW 40 000, and 5 μl β-mercaptoethanol) at 60℃ for 1 h. One volume of chloroform/isoamyl alcohol (24:1) was then added and the mixture was vortexed thoroughly. Following 10-min centrifugation at 13,000 rpm in a benchtop centrifuge, the supernatant was transferred into a clean tube, and the chloroform/isoamyl alcohol clean-up stage was repeated. Two volume of ice-cold isopropanol was added to the supernatant. Precipitated DNA was pelleted by 3-min centrifugation at 13,000 rpm in a benchtop centrifuge, and pellets were washed in 500 μl 70% (v/v) ethanol. Following a final centrifugation as above, the ethanol supernatant was discarded and pellets air dried prior to resuspension in 100 μl water. 

Two primers were used for PCR amplification of 5S rRNA gene and spacer regions. Primer 5s-rRNA1 (GTG CTT GGG CGA GAG TAG TAC) anneals to nucleotides 58–34 of the 5S rRNA gene, and primer 5s-rRNA2 (AGT TCT GAT GGG ATC CGG TGC) is complementary to nucleotides 81–105 of the 5S rRNA gene (Fig. 1). PCRs were 25μl in volume and cotained 200nM of each primer; 0.4mM each dNTP, 2X buffer (TOYOBO, Japan), 10ng template DNA, and 0.5 units of KOD FX DNA polymerase (TOYOBO, Japan). Cycling condition were as follows: 9 4℃ 5 min, followed by 35 cycles of denaturing at 94℃ for 1 min, annealing 60℃ for 20 sec, extension at 72℃ for 30 sec and 1 min and final extension 72℃ for 10 min. The resulting PCR product was cloned to TOPclonerTM blunt kit (Enzynomics, Korea) and sequenced (Cosmogenetech, Korea). 

ccba0c3173be6e36888563f73af20d43.JPG.resized.JPGFig. 1. Organization of the 5s rDNA repeat consisting of 5S rRNA gene and the nontranscribed spacer. Diagrammatic representation of the positions of two primers, 5srRNA1 and 5s-rRNA2, used to amplify the non-transcribed spacer (NTS) region.

Analysis of 5s ribosomal NTS DNA sequence

Searches for information and homology of nucleotide and amino acid sequence were performed using homology search with BLAST against DNA sequences in the Genbank and EMBL DNA database. Multiple sequence alignment was made by CLUSTALX (Thompson et al. 1997), and annotated using GeneDoc version 2.5.006 (Nicholas et al. 1997). The molecular evolutionary genetic tree through the Neighbor-joining method was constructed using MEGA4 from the web site (http://www.megasoftware. net/index.html). The phylogenic tree was tested using bootstrap (1500 replicates; seed = 64238). Pairwise deletion was selected for gaps/missing date (Tamura et al. 2007).

RESULTS AND DISCUSSION

Totally, 22 cultivars were investigated for their sequence of 5S rDNA to confirm their sequence differences by origin of location. Among 22 cultivars, 12 cultivars were from Korea, 7 from China, 2 from Russia and 1 from North Korea. Most of Korean cultivars were local collection and their exact information on genetic background or pedigree could not be obtained. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS sequence and a portion of the adjacent transcribed region (Fig. 1). The highly conserved coding region is not chosen for comparison due to limited variability. The amplification of NTSs or IGS in the basic organization of repeat in a plant species provides specific information on phylogeny of close relatives. In terms of evolution, the sequences of NTSs have been varied revealing higher genetic diversity in a plant species. 

Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragment, from most of plant materials analyzed, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region (Fig. 2). It is well known from other researches that two distinguish 5S rDNA variants, named as long and short repeat were detected almost every time regardless of origin of the plants (Baker et al. 2000; Baum et al. 2004; Kitamura et al. 2005; Baum and Johnson 2007). Amplified DNAs of 400 bp fragment were subcloned and 20 colonies for each transformation were screened to verify the presence of the insert. For every genotype, five clones for each type of fragment were sequenced and compared. Sequence analysis revealed that all inserts contained the 5S rDNA sequences, which included the full length of the NTS region. According to sequence analysis on transcribed region, nearly complete sequence homology was detected in those areas. These sequences were also highly homologous to the corresponding regions of the 5S rRNA genes of many other plant species as we expected. 

6353886b3f1e836233e00a69b1a87c76.JPG.resized.JPGFig. 2. Gel electrophoresis separation of PCR products obtained with the 5s-rRNA1 and 5s-rRNA2 primers. Lanes 1-9, 20-22, Korean cultivar; lanes 10-11, 15-19, Chinese cultivar; lane 12-13, Russian cultivar; lane 14, North Korean cultivar; lane M, 1kb Plus DNA ladder (SolGentTM, Korea)

The long repeats found in the plant materials were also examined for their sequences. Analysis of the fragments showed that they comprised a 700bp of NTS region flanked by the terminal 40 bp and initial 58 bp of the 5S rRNA gene. Not like transcribed region, intergenic spacer sequences exhibited relatively high variability in nucleotide. There are some base substitutions and also insertions /deletions (indels) of some nucleotides among 22 samples. The identity between spacers isolated from the same genotype ranged from 90% to 97%, while the identity among all spacers was 85%. The big sequence differences in a genotype indicated that various size of long NTS repeats were existed over the common millet genome. The sequences of long repeat have been evolved more rapidly over short repeat of NTSs (data not shown).

Due to its sequence similarity within a genotype the short repeats were analyzed to address phylogenetic relationship among the plant materials in this study. The subcloned inserts were sequenced and compared each other (Fig. 3). The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. We demonstrate the sequences by the degree of homology where primary shading (black) corresponds to 100% sequence similarity, secondary shading (dark gray) corresponds to 80% sequence similarity and tertiary shading (light gray) corresponds to 50% sequence similarity. And we also pointed critically different area with insert or deletion with boxes. As indicated in boxed sequences, most of plant materials with foreign origin such as China and Russia can be distinguish from those sequences in boxes. From 4 boxed areas, 7 common millet genotypes originated from China have 2 insertions and 5 deletions. Instead, 2 genotypes originated from Russia have 1 insertion and 3 deletions out of 4 boxes. Interestingly, one genotype from North Korea doesn’t contain different sequences compared to Korean genotypes. The result here indicated that those sequence difference in boxed areas in NTS region have potential to discriminate cultivars from different origin and use as molecular markers for origin identification. Recently, it is very important public concern in Korean seed market whether some agricultural products in market were really produced domestically or imported from China or foreign country.

103f762ff9ed3adbe7a9775047ec1398.JPG.resized.JPGFig. 3. Fig. 3. CLUSTALW multiple aligment of 5S rDNA NTS sequences amplified from the different common millet cultivars. Boxes indicate largely different region of the sequence. Numbers on the right indicate sequence length and (-) denote gaps. Primary shading (black) corresponds to 100% sequence similarity, secondary shading (dark gray) corresponds to 80% sequence similarity and tertiary shading (light gray) corresponds to 50% sequence similarity.

Finally, phylogenic tree was constructed using MEGA4 program (http://www.megasoftware.net/index.html) with obtained sequences of NTSs (Fig. 4). Even though the amount of sequences is limited to some degree, the clear classification was shown from the analysis. Most of genotypes originated from China and Russia is positioned together; we set a putative box in the figure. The grouping was anticipated from the sequence analysis in several critical area in NTSs. This result once again provides convincing information on the idea whether we can use short repeat sequence of 5S rDNA as molecular marker for origin identification. More widely, specific indels can be applied to discriminate species and even breeding lines in specific breeding program. According to previous reports (Zanke et al. 1995; Menke et al. 1996), a synthetic specific sequence from NTS of 5S rDNA was used as a hybridization probe and confirm the presence of the parental S. pinnatisectum genome in somatic hybrids obtained by protoplast fusion of this species with the breeding line S. tuberosum. 

9fef785842d1a417cdfbf25b8c58b609.JPG.resized.JPGFig. 4. Phylogenetic analysis of 5S ribosomal DNA NTS region sequences. The branch length indicates the percentage of sequence similarity.

In this study, we could not include many common millet genotypes from various origins all over the world as plant materials. It is because of difficulty to collect various genetic resources from other countries over the protection and barrier built by researchers or government. Considering the substantial years of the cultivation of this crop by human being, there must be wide and large variants existed among the countries from East Asia to Europe and Africa. More accurate validation of common millet with various genetic resources should be carried with a consensus of researchers working in same field. 

ACKNOWLDEMENTS

This work was supported a grant from Agenda Program, Rural Development Administration, Republic of Korea. PJ006638032012). 

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