The evaluation of morphological differences is a traditional method of evolutionary and pedigree relationship
determination. It was particularly useful in maize, where phenotypic differences occur (e.g. color, kernel type and kernel
size). However, only molecular markers provide information that is independent of environmental influences or a plant
development phase. Therefore, techniques of DNA analysis have become more and more important. Methods based on
polymerase chain reaction -PCR- are used widely in research. Ones of the most used methods are the RAPD (Randomly
Amplified Polymorphic DNA) metho

Leonte, Constantin

Murariu, Danela

Murariu, Marius

Simioniuc, Dănuţ Petru

Simioniuc, Dănuț-Petru

ALSE Repository

Lucrări Stiintifice – vol. 55 (2) /2012, seria Agronomie  215  GENETIC CHARACTERIZATION OF SOME LOCAL MAIZE LANDRACES COMING FROM ROMANIA BY RAPD METHOD    Marius MURARIU, Danela MURARIU, Constantin LEONTE, Dănuţ Petru SIMIONIUC  e-mail: dmurariu@suceava.astral.ro   Abstract The evaluation of morphological differences is a traditional method of evolutionary and pedigree relationship determination. It was particularly useful in maize, where phenotypic differences occur (e.g. color, kernel type and kernel size). However, only molecular markers provide information that is independent of environmental influences or a plant development phase. Therefore, techniques of DNA analysis have become more and more important. Methods based on polymerase chain reaction -PCR- are used widely in research. Ones of the most used methods are the RAPD (Randomly Amplified Polymorphic DNA) method Key words: : maize local landraces, cold test index, RAPD method   Reconsidering of the evaluation work, documentation and use of maize genetic resources represented by old local landraces, no studied or inadequately studied, represents an actual necessity, at the national and international level. Not incidentally, the work report of the ECPGR Maize Working Group Meeting Rome, Italy (1996) has noted two major needs for collaboration on maize genetic resources:  Identify of old local populations,  valuables  for their agronomic characters;  Establish joint prebreeding programs. The maize local landraces are distinguished by a high capacity for adaptation and physiological characteristics specific to certain areas, as well as high yield capacity and the its quality attributes (Moşneagă and collab., 1957; Cristea,  2006;  Căbulea and collab., 1975;  Hallauer and Miranda, 1981; Murariu and collab  1998, 2010). The Romanian maize local landraces are very different as the ecological conditions in our country under the influence of which were formed and over which were superimposed the effects of empirical selection made by thousands of growers, each in its own way. Although, the maize landraces are very heterogeneous, they are grouped into distinct races, each occupying a certain area (Cristea, 2006). The fundamental aim of this research is the evaluation of genetic diversity of some Romanian maize landraces stored at the Suceava Genebank. It makes possible a quick examination of genetic material in a large number of samples at a relatively low cost. In a research conducted in the Agriculture Faculty of Iasi, the modified RAPD technique with a system of primers containing additional DNA sequences partly complementary to the semi-conservative sequences of intron – exon junctions proved to be very useful in a variety of plant species.  These primers, known also as semi-random primers, were with success used by Weining and Langridge  to target diverse regions of genome in cereals.  This method is used especially for genetic diversity identification and philogenetic studies (Bagheri A. and all, 1995, Hoey B.K. and all, 1996, Iqbal N.J. and all, 1997, Samec and all, 1998,  Tinker and all, 1993).  The objective of this research was to determine the genetic variability existing among maize landraces coming from Romania, very resistant to cold temperatures. We hoped to obtain information on the level of intervarietal divergence which is essential for plant breeders. The additional practical goal was to eliminate possible duplicates.   MATERIALS AND METHODS  Sixty one maize landraces taken to the preliminary analysis are maintained in the Suceava Genebank. The selected Maize landraces are very resistant to cold temperatures and coming from different area of Romania (table 1).  All molecular analyses were carrying out in the Agriculture Faculty of Iasi, in frame of molecular markers laboratory. For genetic characterization of maize germplasm by RAPD method, first, was necessary extracting genomic DNA. Of each variant were taken leaves  Universitatea de Ştiinţe Agricole şi Medicină Veterinară Iaşi  216 Table 1  Local maize landraces coming from Romania, very resistant to cold temperatures,  which  were characterized from genetic point of view through RAPD method (USAMV, 2011)       Table 2 RAPD primers used  Nr crt. Primer Secvenţa  (5’-3’) 1. ROTH A15 TTC CGA ACC C 2. ROTH A16 AGC CAG GCA A 3. ROTH A17 GAC CGC TTG T 4. ROTH B02 TGA TCC CTG G 5. ROTH B08 GTC CAC ACT C 6. ROTH B13 TTC CCC CGC T 7. ROTH B14 TCC GCT CTG G 8. ROTH B16 TTT GCC CGG A     Fig.1. The obtained images with support of B13 primers and analyzed with RFLP Scan 2.1, to those 61 maize local landraces. Accession number Accession name Origin Accession number Accession name Origin SVGB-8012 METES 1 AB SVGB-8026 ZLATNA 4 AB SVGB-8022 ZLATNA AB SVGB-1790 PETROSANI 2 HD SVGB-9800 CIUNGANI HD SVGB-3599 MAGURA ILVEI 1 BN SVGB-9807 CERBAL 2 HD SVGB-1806 CN-21-84C CJ SVGB-845 B-173 SM SVGB-8043 RACOVITENI 3 BZ SVGB-7750 VLADESTI AG SVGB-9966 SANTMARTIN HG SVGB-5880 GLODENI 6 GJ SVGB-911 DUMBRAVA CJ SVGB-5483 POLOVRAGI 4 GJ SVGB-4813 CALAFAT 2 DJ SVGB-7900 PADES 2 GJ SVGB-7820 SARADIS 2 CJ SVGB-7282 PRUNDU BARGAULUI 13 {A} BN SVGB-1423 LUDESTI 2 HD SVGB-5219 STARCHIOJD 6 PH SVGB-3764 NEREJU 12 VN SVGB-3971 CARBUNESTI 480 GJ SVGB-1357 JITIA DE JOS 3 VN SVGB-3973 BALCESTI 1 CJ SVGB-3722 RODNA 10 BN SVGB-7811 BOLDUT 2 CJ SVGB-11575 CRISTINESTI BT SVGB-7745 DRAGHICI AG SVGB-5226 IZVOARELE 3 PH SVGB-5168 TEREGOVA 5 CS SVGB-7812 VALEA LUI CATI CJ SVGB-11231 POIANA TEIULUI 5 NT SVGB-7645 PONOARELE 6 MH SVGB-9591 SATU MARE B 139-84 HR SVGB-4005 PLENITA II DJ SVGB-1399 BALSA 9 HD SVGB-7624 BEZDEAD 1 DB SVGB-8865 BRUSTUROASA 10 BC SVGB-4023 ALMAJ  1 DJ SVGB-5874 RUNCU 3 GJ SVGB-9577 SATU MARE B 172-84 HR SVGB-981 B-159 SM SVGB-16145 DINTE CAL DE FRASINET CL SVGB-499 ILVA MICA 43 BN SVGB-9887 BAISOARA 5 CJ SVGB-595 CEAHLAU 154 NT SVGB-4019 TURNU SEVERIN 1 MH SVGB-7701 STOENESTI 3 VL SVGB-1015 CHICHIS CV SVGB-952 B-181 HR SVGB-1179 MIERCUREA MS SVGB-5557 PIETRARI VL SVGB-1244 ZOLTAN CV SVGB-9920 BLAJ CIUGUD AB SVGB-1640 GEOAGIU 28 HD SVGB-5172 TEREGOVA 6 CS SVGB-11584 GEORGE ENESCU 4 BT SVGB-7754 VALEA SILISTII AG SVGB-14153 JELNA 1 BN SVGB-9919 BLAJEL SB    Lucrări Stiintifice – vol. 55 (2) /2012, seria Agronomie  217 of young maize plants (14 days after emergence), which were placed in plastic tubes (Eppendorf tubes) and immediately frozen in liquid nitrogen. The maize samples were kept in a freezer at-700C. For DNA extraction CTAB  method was used (hexadecyltrimethilammonium bromide) as amended by Doyle and Doyle in 1987. The steps were:  plant material (100-200 mg) of each variant was grinded  in liquid nitrogen in a mini tube (1.5 ml)  and introduced again   in liquid nitrogen   at each mini tubes with prepared plant material were added  700 ml of extraction D & D* solution, after that,  the mixture was incubated for 20-30 minutes at 6500C  *D & D solution - containing 100 mm Tris-HCl (pH 8.0), 20 mm EDTA (pH 8.0), 1.4 M NaCl, 2% CTAB, 1% Na2S2O5, H2O, all were  autoclaved and then 0.2% mercaptoethanol was added.  700 ml CIA* was added and stirred  5 minutes; * CIA solution (24:1) contains 24 parts chloroform and one part isoamilalcool.  It was centrifuged at 10,000 rpm (4 0C) for 10 minutes;  the liquid phase was extracted,  was added 600μl CIA and stirred 5 minutes;  it was centrifuged at 10,000 rpm (4 0C) for 10 minutes;  the liquid phase was transferred in new mini tubes adding  50 ml NH4OAc (10 M) and 60μl NaOaC (3M, pH 5.5) and was stirred gently;  500μl 2-propanol was added and stirred gently;  It was centrifuged at 4000 rpm, 4 minutes;  the liquid was removed using a pipette;  it added (for washing for 10 minutes) 70% ethanol solution 10mM NH4OAc;  it was centrifuged at 4000 rpm, for 4 minutes;  the liquid was removed  with a pipette;  the mini tubes were dried (open) in a thermostat at 37°C until complete evaporation of alcohol;  in each mini tube was added 100 ml TE solution, containing 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0);  the mini tubes were kept in a freezer at – 2000C. For establishing DNA concentration fluoro spectofotometer NanoDrop 2200 type, was  performed. The appropriate dilutions for PCR mixture (5 ng / ml) were performed with TE solution. In order to determine the genetic diversity of 61 maize landraces, 8 decameric RAPD primers (Table 2.) were selected, after making an initial screening with 20 primers. It was chose only those primers that generated polymorphic fragments. For PCR mixture in a volume of 20μl was performed, that were pipetted: 5 ngADN genomic, 10 mm of dNTP, 25 mM MgCl2, 5pmol / ml decamer primer (ROTH), 0.1 units Taq DNA polymerase (Go Taq Polymerase - Promega) and 10x buffer. Amplification with an Eppendorf termocicler was performed. The conditions in which amplification was performed were: initial denaturing for 3 min at 950C after which followed a number of 45 cycles of amplification, each amplification, having  the following steps:  Distortion: 1 min at 930C;  Primers attaching: 1 min at 340C;  Extension: 1 min at 720C. The last phase was the final extension, 10 min at 720C. Electrophoresis separation of the amplification products was carried out in agarose gel with a concentration of 2%. The fragments visualizing by staining with ethidium bromide was achieved, at a concentration of 0.5 ml/ml.  RESULTS AND DISCUSSIONS  The genetic classification on related genetic groups was performed using similarity coefficient Lei, Ni and UPGMA (unweighted pair-group method arithmetic average). In order to determine the similarity of 61 maize populations was used RAPD (Random Amplified Polymorphic DNA), resulting in a total of 91 bands with sizes between 74 and 1687 bp, of which 86 were polymorphic. The lowest number of amplified fragments was 6 (A15) and the highest number was 17 (ROTH B13 and B14). As shown in table 3, the polymorphic bands level, in the 8 primers used for RAPD analysis, ranged between 83% (ROTH A15) and 100% (ROTH A16, A17 and B08). Table 3.  The amplified fragment numbers, polymorphic bands numbers and polymorphism percentage for each used primer to the RAPD analyses  With support of SPSS software program, it calculated genetic similarity between genotypes analyzed, resulting dendrogram in Figure 2. and Primer Număr fragmente amplificate Fragmente polimorfice Mărime  fragmente (bp) Procent polimorfism (%) ROTH A15 6 5 373-972 83% ROTH A16 10 10 376-1058 100% ROTH A17 7 7 204-873 100% ROTH B02 10 9 253-964 90% ROTH B08 14 14 74-1281 100% ROTH B13 17 16 413-1678 90% ROTH B14 17 16 283-1397 94% ROTH B16 10 9 351-1207 90% Universitatea de Ştiinţe Agricole şi Medicină Veterinară Iaşi  218 DNA bands frequency chart in Figure 3. In the first chart is noted that from point of similarity degree view, the samples were grouped into three clusters (C1-.. C3), and in the second graph (Fig. 3), we see that there are not duplicates in the 61 maize local populations analyzed; the matrix values are very close but not similar.  CONCLUZIONS 1. The sixty one maize local landraces, coming from Romania, very resistant to cold temperature from point genetic molecular of view, by RAPD method  were analyzed,  resulting a total of 91 bands with sizes between 74 and 1687 bp, of which 86 were polymorphic. 2. There are not duplicates in those 61 analyzed maize local populations; the matrix values are very close but not similar.  BIBLIOGRAPHY  1. Bagheri A, Paull JG, Langridge P, Rathjen AJ, 1995. Genetic distance detected with RAPD markers among selected Australian commercial varieties and boron-tolerant exotic germplasm of pea (Pisum sativum L.). Mol. Breeding 2:193-197; 2. Căbulea I.şi colab 1975, Germoplasma locală de porumb din Transilvania şi utilizarea ei în lucrările de ameliorare. Probleme de  Genetică teoretică şi aplicată, vol. VII, nr. 1 57-90; 3. Cristea, M., 2006 - Monografia porumbului, Edit. ASAS Bucuresti;. 4. Hallauer A.R. şi Miranda J.B., 1981 Quantitative Genetics in Maize Breeding Iowa State University. Press Amer; 5. Hoey BK, Crowe KR, Jones VM, Polans NO, 1996. A phylogenetic analysis of Pisum based on morphological characters, and allozyme and RAPD markers. Theor. Appl. Genet. 94: 860-864; 6. Iqbal NJ, Saeed Aziz, Zafar Y, Malik KA, 1997. Genetic diversity evaluation of some elite cotton varieties by RAPD analysis. Theor. Appl. Genet. 94 :139-144; 7. Moșneagă V., Velican, V., Priadcencu Al., 1957, Ameliorarea porumbului, Porumbul- Studiul monografic, Editura Academiei RPR; 8. Murariu Danela, Cătălina Buzdugan, M. Murariu, (1998). The aspects concerning the precocity and the resistance to low temperatures to some maize landraces conserved in Suceava Genebank, Simpozionul Stiinţific de Agronomie, Iaşi 25-26 octombrie , 1998, vol. 41, pag. 95-100 9. Murariu Marius,  Murariu Danela, Has Voichita, 2010  Maize Local Landraces Used Like Prebreeding Material For Simultaneous Improvment Of Main Agronomic Trait, Lucrări ştiinţifice - vol. 53, Nr. 2/2010, seria Agronomie 10. Samec P, Posvec Z, Stejskal J, Nasinec V, Griga M, 1998. Cultivar identification and relationships in Pisum sativum L. based on RAPD and isozymes. Biologica Plantarum 41: 39-48; 11. Tinker NA, Fortin MG, Mather DE, 1993. Random amplified polymorphic DNA and pedigree relationships in spring barley. Theor. Appl. Genet. 85: 976-984                                                    Lucrări Stiintifice – vol. 55 (2) /2012, seria Agronomie  219                                               C1 C2 C3 Fig. 2. Relationships among  the investigated maize landraces using RAPD data (Jaccard distance, UPGMA clustering) Universitatea de Ştiinţe Agricole şi Medicină Veterinară Iaşi  220                                  Fig. 3. The DNA bands frequency to those 61 maize local landraces  

Genetic characterization of some local maize landraces coming from Romania by RAPD method

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Genetic characterization of some local maize landraces coming from Romania by RAPD method

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