sign in sign up
<<
Home , Chickpea, Phaseolus vulgaris

BIHAREAN BIOLOGIST 9 (1): 35-39 ©Biharean Biologist, Oradea, Romania, 2015 Article No.: 141128 http://biozoojournals.ro/bihbiol/index.html

Studying conserved regions of tufm gene between common ( vulgaris), ( arietinum) and (Pisum sativum)

Maryam GHORBANI1, Kianoosh CHEGHAMIRZA1, 2,* and Alireza ZEBARJADI1,2

1. Agronomy and Breeding Department, Razi University, Kermanshah, 2. Biotechnology Research Department for Drought Resistance, Razi University, Kermanshah, Iran *Corresponding author, K. Cheghamirza, E-mail: [email protected], Tel/ Fax: +98 8338323731

Received: 26. July 2014 / Accepted: 15. September 2014 / Available online: 09. April 2015 / Printed: June 2015

Abstract. As a new science, genomics explores the sequencing of genomes in specific organisms. One type of genomics is comparative genomics which can be regarded as a valuable tool for develop molecular resources for crops and identify major genes in . Increasingly, the accessibility of sequence data for genomes of multiple species allows us to compute their evolutionary pathway on a genome-wide basis. This in turn allows a better understanding of the homologous relationships between genes. The present study has made an attempt to identify and compare conserved areas for tufm gene in common bean (Goli and AND1007), chickpea (Bivanij and ILC482) and pea (Flagman and native Gorgan) genomes using STS markers. Evaluation of protected areas in these plants showed that the differences between the tufm gene have emerged. For this purpose after amplification and observation of polymorphism, the isolated fragments of each sample were sequenced. The NCBI database and DNAStar software provided valuable information on the genomes of the crops including the conserved areas. The highest and lowest similarity was belonging to Bivanij and ILC-482, and Flagman and Goli, respectively. ILC-482 and Flagman has highest difference, and ILC-482 and Bivanij has lowest difference among all cultivars. This could be as the result of the mutations in evolution process. Except for chickpea, it was also revealed that intraspecies sequences are far greater than interspecies.

Key words: comparative genomics, genomic conservation, , STS marker, tufm.

Introduction been successfully developed and used for selection in vari- ous crops such as , common bean, and pea Genomics is a field of genome studies that includes intensive (Wang et al. 2011). Due to the significant differences in the efforts to determine the sequence of DNA and RNA using genome, reproducibility, degree of polymorphism, locus high-throughput sequencing strategies (Kahrizi 2014). Ap- specificity and technical demand, there are different types of plication of genomic technology in all areas of biology has molecular markers (Agarwal et al. 2008). lowered the barriers that once separated the plant, animal, Among the many molecular markers, sequence tagged microbial research communities (Varma & Shrivastava 2008). sites (STS) developed based on gene sequences are largely One type of genomics is comparative genomics which has genome specific and thus have the advantages of identifying been a valuable tool for the development of molecular re- cultivars/species from mixture of or plant materials sources for crops and the identification of key genes for crop especially when they are morphologically similar (Nagaich improvement. It is also an indispensable tool to study evolu- et al. 2009). The STS primers due to their co-dominant inheri- tion of genomes and gene families (Kumpatla et al. 2012). tance, analytical easiness, and high reproducibility for ge- Legumes are one of the largest crops and are considered as netic map construction have been used as bridge markers having a key role in the biology of the cycle (Choi et (Jones et al. 2002). The mitochondrial translation factor Tu al. 2004). (TuFM) delivers aminoacylated tRNAs to the mitochondrial Among legumes, chickpea (Cicer arietinum L.) ranks third ribosome as part of a mitochondrial translation elongation worldwide (Singh et al. 2008). Information about the rela- complex 35. Because of its central role in mitochondrial tionships among elite breeding populations and the genetic translation, increased or decreased levels of TuFM have di- diversity in available germplasm is requisite for designing rect effect on the synthesis of mtDNA-encoded and establishing new chickpea breeding programs (Bhagy- (Nordgaard et al. 2008). The purpose of the present study is awant & Srivastava 2008). The pea as another legume is one to identify conserved areas for TuFM locus (EST-STS of the world's most important legumes and serve as a marker) in common bean, chickpea and and find simi- valuable source in the diet of humans and animals larities and differences between the obtained sequences. (Ayeh et al. 2009). The common bean ( L.) is one of the most important legume crops in the world. Due to its high protein and low content, it has become a princi- Materials and Methods pal source of dietary protein and fiber in many developing The name and the origin of plant materials used in the current study countries (Broughton et al. 2003). are given in Table 1. DNA was extracted from tissues of two The molecular markers such as proteins (Kakaei et al. week old seedlings of pea (Pisumsaivum), chickpea (Cicer arietinum), 2010, Kakaei & Kahrizi 2011, Rostami-Ahmadvandi et al. and common bean (Phaseolus vulgaris) cultivars using Dellaporta 2011, Masoumi et al. 2012) and DNA (Rostami-Ahmadvandi method (Dellaporta et al. 1983). For EST-STS marker (TuFM) analy- et al. 2013) have been applied for evaluation of genetic di- sis, PCR amplifications were performed in 25 μl reactions containing versity in crop plants. The DNA molecular markers are PCR Buffer 10 x, 0.2 mM dNTPs, 0.44 μM of the forward (5´- based on differences in DNA sequence and, as such, are not GATGGACCWATGCCTCAAAC-3´) and reverse (5´- ACCCTCATCCTTTGTGAGTAC-3´) primers 1 U of Taq DNA po- subject to environmental influence (Rostami-Ahmadvandi et lymerase (BioFlux), 3.2 mM MgCl2 and 30 ng/μl of genomic DNA. al. 2013). Currently, various kinds of DNA markers have The cycle conditions were initial denaturation step at 94 ˚C for 4 36 M. Ghorbani et al. minutes followed by 5 cycles of 92 ˚C for 30 s, annealing at optimum annealing temperature (Ta) for 2 minutes, extension at 72 ˚C for 1.5 minutes followed by 30 cycles of 92 ˚C for 15 s and then extension at 72˚C for 1.5 minutes with a final extension at 72 ˚C for 8 min. The amplified products were electrophoresed on 2% agarose gel stained with ethidiumbromide.

Table 1. The name and the origin of the studied plants in current research.

Number Plant Name Scientific Name Origin 1 Goli Phaseolus vulgaris var. Mesoamerican 2 AND1007 Phaseolus vulgaris var. Andean 3 ILC482 Cicer arietinum var. 4 Bivanij Cicer arietinum var. Iran 5 Flagman Pisum sativum var. 6 native Gorgan Pisum sativum var. Iran

Figure 1. The amplified fragment with specific primers of TuFM

marker in common bean, pea and chickpea, respectively (1: Goli, After observation the fragments, they extracted from the gel, 2: AND1007, 3: ILC482, 4: Bivanij, 5: Flagman and 6: native Gor- then prepared for sequencing. In order to find the similarities and gan. M: size marker, 1 kb). differences between interspecies, intra-species as well as between genera online and offline data bases were used.

NCBI BLAST was used for similarity search. Further analysis of the DNA sequence was done using DNASTAR software. and 13/8% respectively. The conserve region between cultivars of Flagman and native Gorgan showed 7.8% similarity and 86% difference Results (Fig.2-c).

In this study the researchers are interested in comparative Interspecific comparisons mapping of family and in the development of STS Cultivars of common bean and pea, common bean and markers that potentially can be applied across different gen- chickpea, pea and chickpea were separately compared for era. conserved regions of tufm locus. The differences were ob- TuFM marker did not exhibit polymorphism between served in the genus level. This comparison showed the high- pea and chickpea cultivars. A 1007 bp fragment was pro- est divergence and similarity between native Gorgan and duced for both plants. Length polymorphisms were ob- Flagman (pea cultivars) (139.5) and between Goli (bean cul- served between common bean cultivars. A 2500 bp and 2100 tivar) and native Gorgan (pea ) (48), respectively bp fragment was produced for cultivar AND1007 and culti- (Fig.2-d). Cluster analysis grouped common bean and pea var Goli, respectively. The results confirm the findings of cultivars into 2 clusters. Cultivars AND1007 and Flagman Aghaee nejad (2009) and (Moffet 2006). The segment related were placed in a cluster and Goli and native Gorgan were to the marker has a role in elongation phase of protein syn- grouped in the other cluster (Fig.3-a). Cultivars Bivanij and thesis (Moffet 2006). ILC-482 showed the highest value of similarity (85.1) and cultivars of Goli and AND1007 revealed the highest diver- Sequencing results gence (152.4) (Fig.2-e). Phylogenetic tree were cut based on In order to find conserved regions and also to detect the minimum deference among cultivars and three groups were level of similarities and differences in the studied locus (am- obtained; the first group includes two subgroups (Fig.3-b). plified regions between two primers) amplified fragments Chickpea's cultivars (Bivanij and ILC-482) are placed in the were used for direct sequencing. MegAlign software pack- first subgroup and Goli is located in the second subgroup. age (Windows version 3.12e; DNASTAR, Madison, Wis.) Only Bean cultivar (AND1007) was placed in the third was used to align the sequences for intraspecies and be- group. tween genus comparisons. The highest amount of similarity (85.1) was observed be- tween Bivanij, ILC-482 and cultivars of native Gorgan. Be- Intraspecific comparisons sides, ILC-482 showed highest divergence (152.2) compared Comparing AND1007 and Goli cultivars of common bean to to pea and chickpea cultivars (Fig.2-f). Two cultivars of pea the extent of common conserved areas showed differences in and chickpea were separately placed in two groups (Fig.3-c). nucleotide level (Fig.2-a) and values of and similarity were Amount of similarity and divergence between 6 cultivars of 99.7% and 2.4% respectively. The large difference between pea, chickpea and common bean for TuFM locus were two cultivars of was contrary to the expectation, shown in Table 2. The highest and lowest values of similar- which could be as a result of the non-uniform size and exis- ity were between Bivanij and ILC-482 (85.1) and Flagman tence of gap in sequences. and Goli (2.2), respectively. ILC-482 and Flagman has high- Chickpea’s cultivars, ILC-482 and Bivanij showed simi- est difference with 219.3, and ILC-482 and Bivanij with 13.8 larities in many regions and only in few differences were ob- has lowest difference amount among all cultivars (Fig.2-g). served (Fig.2-b). For ILC-482 and Bivanij cultivars, the After comparing all studied cultivars, six cultivars were amount of obtained similarities and differences were 85/1% grouped into three clusters. The result showed that cultivars

Studying conserved regions of tufm gene between common bean, chickpea & pea 37

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure. 2. Comparison of conserved regions between different cultivars using TuFM locus. (a) common bean cultivars, (b) chickpea cultivars, (c) pea cultivars, (d) common bean and pea, (e) common bean and chickpea, (f) pea and chickpea, (g) common bean, pea and chickpea, and (h) all cultivars whit three other plant.

from different genus were located in the same group, this and O. sativa (86.2) and O. sativa and AND1007 (1.9). may be because of common family, similarity in sequence of studied marker, and unequal in obtained sequence length. AND 1007, Flagman, Goli, native Gorgan, Bivanij and ILC- Discussion 482 were placed in cluster 1, 2 and 3 respectively (Fig.3-d). Sequences of TuFM locus for these plants were selected from Based on the results, fragment of 1700 bp was conserved in NCBI database and relationship of this data with obtained pea and chickpea cultivars and it seems that, no changes has data of the current study was evaluated (Fig.2-h). Diver- occurred over time in this region; however, the amplified gence and similarity analysis revealed the highest diver- fragment with specific primers of TuFM locus in common gence for Flagman and Z. mays (303.7) and the lowest diver- bean was different for two studied cultivars. As Table 1 gence for ILC-482 and Bivanij(13.8) (Table 3). The highest showed, origin of Goli cultivar is Mesoamerican and origin and lowest similarity values were observed between Z. mays of AND1007 cultivar is Andean, so one reason for this differ-

38 M. Ghorbani et al.

(a) (b)

(c) (d)

(e)

Figure. 3. Phylogenetic tree of different cultivars based on conserved region for TuFM marker. (a) cultivars of common bean and pea, (b) cultivars of common bean and chickpea, (c) cultivars of pea and chickpea, (d) cultivars of common bean, pea and chickpea, and (e) cul- tivars of common bean, pea and chickpea with three other plant.

Table 2. Amount of similarity (upper off-diagonal matrix) and difference (Lower off-diagonal matrix) between six cultivars from pea, chickpea and common bean for TuFM locus.

AND 1007 Bivanij Flagman Goli ILC-482 native Gorgan AND 1007 3.7 42.5 2.4 2.8 2.7 Bivanij 192.1 4.6 5.3 85.1 2.8 Flagman 63.3 198.2 2.2 3.5 7.8 Goli 175.6 154.7 149.4 5.2 48 ILC-482 204.4 13.8 219.3 161.5 2.9 Gorgan 181 154.7 149.8 67.1 155.1

Table 3. Similarity (upper off-diagonal matrix) and dissimilarity (Lower off-diagonal matrix) values for pea cultivars, chickpea and common bean and three other plants that obtained from NCBI for TuFM locus.

Arabidopsis native Oryza Zea AND1007 Bivanij Flagman Goli ILC-482 thaliana Gorgan sativa mays AND1007 2.9 3.7 42.5 2.4 2.8 2.7 1.9 2 Arabidopsis thaliana 200.5 4.3 4.9 4.6 3.3 4 68.1 67.5 Bivanij 224.9 139.4 4.6 5.3 85.1 3.8 2.5 5.3 Flagman 63.3 230.1 190.1 2.2 3.5 7.8 4.7 2.5 Goli 218.1 155.1 171.5 244.6 2.2 48 4.3 5.5 ILC-482 257.3 136.1 13.8 219.7 187.4 2.9 2 4.3 native Gorgan 206.3 152.8 206.1 209.6 67.1 214.7 3.6 3.6 Oryza sativa 244.1 31.6 138.3 289.4 139.9 144.6 145.9 86.2 Zea mays 254.1 33.4 135.2 303.7 144.2 132.5 153.3 15.2

ence could be the different origin for the plants. Based on ple species, increasingly allows us to compute their evolu- natural theory of evolution, suggested that most changes at tionary trajectories on a genome-wide basis. This in turn al- the molecular level were the result of mutation and genetic lows for a better understanding of the homologous relation- drift, rather than selection (Kimura 1968). It was also pro- ships between genes. (Koonin 2005). Boutin et al. (1995) re- posed that selection (selective removal of deleterious alleles) ported that considerable conserved synteny existed between plays a bigger role than positive selection. Therefore, the ob- the linkage maps of Phaseolus and Vigna species and that served differences in band sizes for common bean, pea and some gene order conservation also existed between the gen- chickpea cultivars (Fig.1) could be due to changes result era Glycine and Phaseolus. In a similar study, by map com- from mutation, deletion or addition of plant genomes. Ori- parisons between these genera and Arabidopsis, Lee et al. gin of Bivanij cultivar is Iran and ILC-482 cultivar come from (2001) also found out that there is a synteny among them. Turkey. Iran and Turkey have geographical shared border, Since Zea mays belong to cereal family and Flagman belongs therefore the low distance between the origins of this two to legume family such a difference was somehow predict- cultivars can be one of the reasons for this high similarity. able but ILC-482 and Bivanij belong to the same family and The availability of sequence data for genomes of multi- same genus. ILC-482 and Bivanij showed similarities in Studying conserved regions of tufm gene between common bean, chickpea & pea 39 many regions and differences were observed only in few ar- Bhagyawant, S., Srivastava, N. (2008): Genetic fingerprinting of chickpea (Cicer eas in intraspecific comparisons (Fig.2-b). Z. mays and O. sa- arietinum L.) germplasm using ISSR markers and their relationships. African Journal of Biotechnology 7: 4428-4431. tiva belong to the same family, but O. sativa and AND1007 Boutin, S., Young, N., Olson, T., Yu, Z.H., Vallejos, C., Shoemaker, R. (1995): belonged to different family. It appears that sequence of this Genome conservation among three legume genera detected with DNA region in the genome has fewer changes in genus which be- markers. Genome 38: 928-937. Broughton, W., Hernandez, G., Blair, M., Beebe, S., Gepts, P., Vanderleyden, J. long to one family. The phylogenetic tree confirmed this re- (2003): Beans (Phaseolus spp.) model food legumes. Plant and Soil 252: 55- sult. 128. According to dendrogram all of studied plants in this re- Choi, H.K., Mun, J.H., Kim, D.J., Zhu, H., Baek, J.M., Mudge, J., Roe, B., Ellis, N., Doyle, J., Kiss, G.B. (2004): Estimating genome conservation between search and the plants used in same other works were de- crop and model legume species. Proceedings of the National Academy of rived from three groups. The first group including two sub- Sciences of the of America 101: 15289-15294. clusters, O. sativa, Z. mays and Arabidopsis thaliana were Dellaporta, S.L., Wood, J., Hicks, J.B. (1983): A plant DNA minipreparation: version II. Plant Molecular Biology Reporter 1: 19-21. placed in subcluster 1. Bivanij and ILC-482 were located in Fitch, W.M. (1970): Distinguishing homologous from analogous proteins. subcluster 2. Cluster two and three were similar to the pre- Systematic Biology 19: 99-113. vious subcluster (Fig.3-e). The concept of homology is cen- Jones, E.S., Mahoney, N.L., Hayward, M.D., Armstead, I.P., Jones, J.G., tral to comparative genomics. Homology is a general con- Humphreys, M.O., King, I.P., Kishida, T., Yamada, T., Balfourier, F. (2002): An enhanced molecular marker based genetic map of perennial ryegrass cept for genes that are related through common ancestry. (Lolium perenne) reveals comparative relationships with other Poaceae These homologs may be further classified as either paralogs genomes. Genome 45: 282-295. (genes that arose as a result of duplication of a single gene) Kakaei, M., Kahrizi, D. (2011): Evaluation of storage protein patterns of ten wheat varieties using SDS-PAGE. Biharean Biologist 5: 116-118. or orthologs (genes that arose from a single gene as a result Kakaei, M., Kahrizi, D., Mostafaie, A. (2010): Study on powdery mildew disease of speciation) (Patel 2011). Orthologous genes arise due to related proteins expression in winter wheat cultivars via SDS-PAGE. speciation events (Fitch 1970). Biharean Biologist 4: 169-171. Kahrizi, D. (2014): Reduction of EPSP synthase in transgenic wild turnip Paal, Enolase, and TuFM were first identified in the (Brassica rapa) weed via suppression of aroA. Molecular Biology Reports syntenic area and allowed identification of fin/det, one of 41(12): 8177-8184. several TFL genes already mapped in bean, as another Kimura, M. (1968): Evolutionary rate at the molecular level. Nature 217: 624- 626. orthologous loci between pea and bean. Gene Product of Koonin, E.V. (2005): Orthologs, paralogs, and evolutionary genomics 1. Annual tufm gene in genome is mitochondrial elongation factor Review of 39: 309-338. (Moffet 2006). Considering that the tufmgene has an impor- Kumpatla, S., Buyyarapu, R., Abdurakhmonov, I., Mammadov, J. (2012): st tant function in plant genome, it seems that this genome ori- Genomics-assisted plant breeding in the 21 century: technological advances and progress. pp. 131-184. In: Abdurakhmonov, I.Y. (ed.), Plant Breeding. gin has changed less over time. Intechopen, Croatia. Evaluation of protected areas in these plants showed that Lee, J., Grant, D., Vallejos, C.E., Shoemaker, R. (2001): Genome organization in the differences between cultivars for the tufm gene have dicots. II. Arabidopsis as a’bridging species’ to resolve genome evolution events among legumes. Theoretical and Applied Genetics 103: 765-773. emerged. These differences can occur due to mutation or Masoumi, S.M., Kahrizi, D., Rostami-Ahmadvandi, H., Soorni, J., Kiani, S., natural divergence during evolution. Overall, in this study it Mostafaie, A., Yari, K. (2012): Genetic diversity study of some medicinal was found that the tufm gene is largely conserved between plant accessions belong to Apiaceae family based on seed storage proteins patterns. Molecular Biology Reports 39: 10361-10365. plants in a genus, especially chickpea cultivars and in lower Moffet, M.D. (2006): Identifying regions of conserved synteny between pea level among genera of family. Although this region of the (Pisum spp.), (Lens spp.), and bean (Phaseolus spp.). M.Sc. Thesis, genome may exist in different organisms, it can change due Montana State University-Bozeman, College of .,100p. Nagaich, D., Kumar, S., Chandra, A. (2009): Genome-specific STS markers-a to several factors such as mutations, transposons, deletions, tool for species testing of important forage range legume Stylo. National additions and recombination. We wish, the result will be Academy Science Letters 32: 327-331. useful for breeding programs and for further research stud- Nordgaard, C.L., Karunadharma, P.P., Feng, X., Olsen, T.W., Ferrington, D.A. ies such as studding of genetic polymorphism, marker as- (2008): Mitochondrial proteomics of the retinal pigment epithelium at progressive stages of age-related macular degeneration. Investigative sisted selection, phylogenetic relatively, and genetic and mo- Ophthalmology and Visual Science 49: 2848-2855. lecular mapping. Patel, R. (2011): Orthologous Gene Identification in Plant Species. M.Sc. Thesis, University of Toronto, 111p. Rostami-Ahmadvandi, H., Cheghamirza, K., Kahrizi, D., Bahraminejad, S.

(2013): Comparison of morpho-agronomic traits versus RAPD and ISSR markers in order to evaluate genetic diversity among Cuminum cyminum L. Acknowledgments. The author's acknowledge the technical accessions. Australian Journal of Crop Science 7: 361-367. experience provided by Dr. Leila Zarei. Razi University has Rostami-Ahmadvandi, H., Kahrizi, D., Zebarjadi, A., Mostafaie, A., Sohrabi- financially supported this research Project. Thanks to Zagros Babahadi, F., Kiani, S. (2011): Study of Peganum harmala genetic diversity Biotechnology, Razi University Incubator for all Supports. based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) analysis. Journal of Agricultural Science and Technology A1: 1300- 1302.

Singh, R., Sharma, P., Varshney, R.K., Sharma, S., Singh, N. (2008): Chickpea improvement: role of wild species and genetic markers. Biotechnology and References Genetic Engineering Reviews 25: 267-314. Varma, A., Shrivastava, N. (2008): The role of plant genomics in biotechnology. Agarwal, M., Shrivastava, N., Padh, H. (2008): Advances in molecular marker Encyclopedia of Life Supports. In: Biotechnology, Vol 8. Encyclopedia of Life techniques and their applications in plant sciences. Plant Cell Reports 27: Support Systems (EOLSS). 617-631. Wang, H., Yang, T., Guan, J., Ma, Y., Sun, X., Zong, X. (2011): Development and Aghaee nejad, M. (2009): Evaluation of conserved regions and similar genomic characterization of 20 novel polymorphic STS markers in (fava in legumes using STS Marker. M.Sc. Thesis, Razi University, Compose of bean). American Journal of Botany 98: 189-191. Agriculture and Natural Resources. Ayeh, K.O., Lee, Y., Ambrose, M.J., Hvoslef-Eide, A.K. (2009): Characterization and structural analysis of wild type and a non-abscission mutant at the development funiculus (Def) locus in Pisum sativum L. BioMed Centralplant Biology 9: 76.