Microsoft Word - BSN RAPD rice_Joshi_Final two column.docx Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   16 ORIGINAL RESEARCH ARTICLE Genetic  Relationship  among  Nepalese  Rice  Landraces  and  Cultivars  based  on   RAPD  Markers Bal K. Joshi*, Hari P. Bimb, David Kansakar and Ekta Ghimire Biotechnology Unit, NARC, PO Box 1136 Kathmandu, Nepal * Corresponding author: Email: joshibalak@rediffmail.com Abstract Genetic information of any genotype is necessary to manage and utilize them in conservation and breeding program. A total of 28 RAPD markers were used to relate the genetic structure among 50 Nepalese rice genotypes consisting of 29 landraces, 12 breeding lines and 9 released cultivars. Some of them are aromatic and blast resistance. Only four primers (P41, P60, P109 and P141) amplified the DNA of these genotypes with scorable bands. Primer 60 produced the highest number of bands (8). The highest number of present bands (6) was shown by primer 41 in 10 rice genotypes. Grouping of these genotypes based on the adaptation to agro-climatic zone was not observed, probably due to low percentage coverage of genome by four primers. Most of the genotypes grouped in two clusters. Kali Marsi and IR-24 formed separate individual cluster. Mansara and Jarneli were the most similar landraces (0.96). Churenodhan and Pranpyuri were the most closely related with Masuli. Only one genotype NR-285-18 has fallen in the first quadrant by principal component (PC) analysis and the fourth quadrant was empty. The highest contribution in PC1 was from the second band of primer 41. This RAPD information can be used for selecting lines and for blast resistance breeding. Key words: Genetic distance, rice, RAPD Introduction Nepal is rich in rice genetic resources [1, 2]. Knowledge on genetic diversity contributes significantly for the better management and utilization of these resources. Diversity analysis with the help of molecular markers provides reliable information which can be utilized for breeding purposes. RAPD (Randomly Amplified Polymorphic DNA) [3] markers though dominant markers, provides fast, reliable and cost effective determination of genetic diversity in plant varieties, breeding lines and accessions [4-6]. In RAPD, a single random primer is added to the template DNA and subjected to polymerase chain reaction (PCR). This simple but effective method of revealing polymorphism is cheap and Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   17 universally applicable [7, 8]. The indica and japonica cultivars are classified into separate groups by cluster analysis using RAPD [5]. We studied the genetic diversity of rice particularly adapted to mid and high hills using RAPD markers to support for effective management and utilization of rice genetic resources. Materials and methods a. Plant materials and plant DNA extraction The rice genotypes analyzed are given in Table 1. A total of 50 rice samples consisting of landraces, breeding lines and released cultivars were used. DNA was extracted employing the Modified CTAB method of [9]. b. DNA amplification For RAPD analysis 28 decamer primers were tested (Table 2). Amplification was carried out in a 10 µl reaction volumes consisting of 10mM Tris-HC1 pH 8.3, 2mM MgC12, 0.2mM dNTPs, 1mM primer, 0.35 unit of Taq DNA polymerase and 1 ng of total DNA as template. The amplification reaction was carried out in PTC-100 thermocycler (MJ Research, USA). The first cycle consisted of denaturation of template DNA at 93.5oC for 1 min, primer annealing at 36oC for 2 min and primer extension at 72oC for 3 min. In the next 44 cycles, the three steps of first cycle were repeated. In the last cycle it is hold at 72oC for 7 min and then at 4oC for 3 min. PCR products were separated on a 1.8% agarose gel using TAE buffer. The gels were run for 2.5-3 hr at 70 V and stained with ethidium bromide. DNA fragments were visualized under UV light and photographed using Gel Doc system. Only the four primers amplified the DNA of test lines. Polymorphisms were scored for the presence or absence of bands on a 1/0 basis and data analyzed using the NTSYS-pc software [10]. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   18 Table 1. Rice landraces, cultivars and breeding lines used in this study. S.N. Genotype Collection site Altitude, m Collection year Remarks 1 Krishnabhog Achham 1000 1985 Landrace 2 Thapachini Bajura 1768 1995 Landrace 3 Tauli Bhojpur 1219 1987 Landrace 4 Tunde dhan Dailekh 1400 1995 Landrace 5 Rato dhan Dadeldhura 1585 1995 Landrace 6 Hansraj Dadeldhura 1128 1995 Landrace 7 Mansara Dadeldhura 1128 1995 Landrace 8 Chureno dhan Dang 2120 1985 Landrace 9 Anpjhutte Gorkha 1981 1988 Landrace 10 Jarneli Gulmi 2000 1998 Landrace 11 Bhuwa dhan Humla 1970 1985 Landrace 12 Jhul dhan Humla 1350 1985 Landrace 13 Pahele Kaski 1075 1998 Landrace 14 Radha-7 Kaski 1040 1998 Released 15 Pakhe Lamjung 1920 1988 Landrace 16 Pranpyuri Lamjuing 1996 1988 Landrace 17 Madise Lamjung 1524 1988 Landrace 18 Kali marsi Mugu 2600 1985 Landrace 19 Ghaiya dhan Mugu 2380 1985 Landrace 20 Dhokro Mugu 2350 1985 Landrace 21 Maine pokhreli Mustang 1400 1985 Landrace 22 Lekali dhan Myagdi 1800 1985 Landrace 23 Hanse Sallyan 1200 1992 Landrace 24 Pale dhan Sindupalchok 1500 1985 Landrace 25 Bageri dhan Solukhumbu 1707 1989 Landrace 26 Jethobor Tanahun 1250 1988 Landrace 27 Pokhara masino Tanahun 1250 1988 Landrace 28 Chananchur Udaypur 1829 1989 Landrace 29 Lalshar Udaypur 1829 1989 Landrace 30 NR10315-145 ABD, Khumaltar Breeding line 31 NR10286-6 ABD, Khumaltar Breeding line 32 Manjushree-2 ABD, Khumaltar Released 33 NR10375-20 ABD, Khumaltar Breeding line 34 Khumal-11 ABD, Khumaltar Released 35 NR10353-8 ABD, Khumaltar Breeding line 36 NR285-18 ABD, Khumaltar Breeding line 37 NR10276-15 ABD, Khumaltar Breeding line 38 NR10414-25 ABD, Khumaltar Breeding line 39 NR10414-34 ABD, Khumaltar Breeding line 40 Taichung-176 ABD, Khumaltar Released 41 Jumli White ABD, Khumaltar Landrace 42 Chandan nath-1 ABD, Khumaltar Released 43 Chandan nath-3 ABD, Khumaltar Released 44 NR10276-9 ABD, Khumaltar Breeding line 45 NR10285-29 ABD, Khumaltar Breeding line Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   19 S.N. Genotype Collection site Altitude, m Collection year Remarks 46 Sabitri NRRP, Hardinath Released, BR 47 IR-24 NRRP, Hardinath Released, BR 48 A57-115-8 NRRP, Hardinath Breeding line, BDI (3 gene pyramid) 49 CO39 NRRP, Hardinath Breeding line, BS 50 Masuli NRRP, Hardinath Released, BS Note: ABD , Agriculture Botany Division. NRRP, National Rice Research Program. B, Blast. R, Resistant. S, Susceptible. DI, Differential line. Table 2. Details of RAPD primers used in this study. S.N. Primer Sequence Band scored Remarks 1 P36 GGGGGTCGTT - 2 P40 GGCGGACTGT - 3 P41 GAGTGCGCAG 6 Rice genome 4 P42 CCGGACTGAG - 5 P48 GAAGGCGCGT - 6 P52 GGCACCACCA - 7 P60 CATCGGCCCT 8 8 P109 TGGCCACTGA 3 9 P141 GTGATCGCAG 7 Operon Tech 10 P142 CAATCGCCGT - 11 P144 CAGCACCCAC - 12 P165 CTGACGTCAC - 13 P169 AGTCGACGCC - 14 P181 ACGGACGTCA - 15 P189 TGGGTCCCTC - Operon Tech 16 P191 CTGCGCTGGA - 17 P194 AGGCCCGATG - 18 P197 GACCCCGGCA - 19 P198 GCCTGGTTAC - 20 P202 CGCAGACTTG - TAG 91:65-667.’95. Lentil 21 P205 GCCGTGAAGT - TAG 91:65-667.’95. Lentil 22 P209 GGCGTCGGGG - TAG 91:65-667.’95. Lentil 23 P217 GGGTTGCCGT - TAG 85:937-95.’93.Vicia faba 24 P222 GTCACCCGGA - TAG 85:937-95.’93.Vicia faba 25 P225 AGTGGTCGCG - 26 P232 GCGCATTAGA - Bio/Tec 10:686-690. Conifer 27 P270 AGCCAGTTTC - TAG 85:190-196.’92. Brassica 28 P292 CAAACGGCAC - TAG 86:788-794.’95. Alfalfa Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   20 Results and discussion a. Primers and genetic similarity Among the 28 RAPD primers, only four primers (P41, P60, P109 and P141) amplified the genomic DNA of test lines (Table 2). The percentage of primers that amplified the DNA was very low. These four primers showed polymorphism. We considered only those primers that could amplify the DNA of all samples with scorable bands (Figure 1, 2). Most of the primers did not work probably due to the old or not related to rice genome or poor quality of template DNA. Polymorphism percentage of the tested RAPD primers are 90.0 in the study of [11] and 67 in [12]. In their study, with selected primers, sufficient polymorphism is detected to allow identification of individual varieties. RAPD analyses offer the greatest chance of detecting small genetic differences, since a larger component of the genome can be scanned than in other systems [8, 13]. Primer 60 produced the highest number of bands (8). The highest number of present bands (6) was shown by primer 41 in 10 rice genotypes. The genetic similarity ranged from 0.00 to 0.96. Mansara and Jarneli were the most similar landraces (0.96). The second most similar landraces were Tunde dhan and Krishnabhog. IR-24 showed the zero similarity coefficients with all genotypes. The zero similarity coefficient of Kali Marshi with Thapachini, Krishnabhog and Tauli indicates the most genetic dissimilarity. The similarity index between Chandannath-1 and Lalshar was also zero. A57-115-8 showed the zero similarity index with Chandannath-3. Two blast susceptible varieties, Mansuli and CO-39 have mostly the similar coefficients with all tested genotypes. Fig.1. RAPD polymorphism of different rice genotypes with primer 141 M            1            2          3          4          5            6          7          8          9        10      11      12        13      14        15      16        17   A B Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   21 (M, marker; Sample A: 1, Kali Marshi; 2, Ghaiya dhan; 3, Dhokro dhan; 4, Maine Pokhreli; 5, Lekali dhan; 6, Hanse; 7, Pale dhan; 8, Bageri dhan; 9, Jethobor; 10, Pokhara Masino; 11, Chananchur; 12, Lalshar; 13, NR10315-145-2-3; 14, NR10286-6-3-2-2; 15, Manjushree-2 ; 16, NR10375-20-1-2; 17, Khumal 11. Sample B: 1, NR10353-8-2-1; 2, NR28518-3-2-3-1; 3, NR10276-15-2-3-3-2; 4, NR10414-25-2; 5, NR10414- 34-2-3; 6, Taichung-176; 7, Jumli White; 8, Chandhannath-1; 9, Chandhannath-3; 10, NR10276-9-3-3-3-2; 11, NR10285-29-3-1; 12, Sabitri; 13, IR-24; 14, A57-115-8; 15, CO 39; 16, Masuli;17, Check3 from Jumla, 2 from Humla and 3 Mugu). Fig.2. RAPD polymorphism of different rice genotypes with primer 41 (M, marker; Sample A: 1, *Krishnabhog; 2, *Thapachini; 3,Tauli; 4,Tunde dhan; 5,Rato dhan; 6, *Hansraj; 7, Mansara; 8,Chureno dhan ; 9, Anpjhutte; 10,Jarneli ; 11, Bhuwa dhan; 12, Jhuldhan; 13, *Pahele; 14,Radha-7; 15,Pakhe ; 16, Pranpyuri; 17, Madise. Sample B: 1, Kali Marshi; 2, Ghaiya dhan; 3, Dhokro dhan; 4, Maine Pokhreli; 5, Lekali dhan; 6, Hanse; 7, Pale dhan; 8, Bageri dhan; 9, *Jethobor; 10, *Pokhara Masino; 11, Chananchur; 12, Lalshar; 13, NR10315-145-2-3; 14, NR10286-6-3-2-2; 15, Manjushree-2 ; 16, NR10375-20-1-2; 17, Khumal 11. Sample C:1, NR10353-8-2-1; 2, NR28518-3-2-3-1; 3, NR10276-15-2-3-3-2; 4, NR10414-25-2; 5, NR10414-34-2-3; 6, Taichung- 176; 7, Jumli White; 8, Chandhannath-1; 9, Chandhannath-3; 10, NR10276-9-3-3-3-2; 11, NR10285-29-3-1; 12, Sabitri; 13, IR-24; 14, A57-115- 8; 15, CO 39; 16, Masuli;17, Check3 from Jumla, 2 from Humla and 3 Mugu). * Aromatic rice. b. Cluster analysis The dendrogram generated by the RAPD analysis showed four distinct groups (Figure 3). IR-24 and Kali Marsi formed the separate individual cluster. Most of the genotypes fell in two clusters. Grouping of these genotypes based on the adaptation to agro-climatic zone was not observed, probably due to low percentage coverage of genome by four primers. Mansara and Jarneli were the most similar landraces followed by NR-10276-9 and NR-10285-20. Churenodhan and Pranpyuri were the most M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 A B C Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   22 closely related with Masuli. The three blast resistance genes pyramided rice genotype, A57-115-8 was genetically near with Anpjutte, Tauli and Thapachini. Fig.3. Clustering of 50 rice genotypes based on RAPD markers. c. Principal component analysis A scatter plot was drawn based on the similarity coefficients among the 50 rice genotypes (Figure 4). All genotypes except NR-285-18 fell in the second and third quadrant. Only one genotype NR-285-18 has fallen in the first quadrant by principal component analysis and the fourth quadrant was empty. The highest contribution in PC1 was from the second band of primer 41 (Table 3). Considerable overlapping among the various samples is evident, which suggests that genetic variation among them is rather narrow. Nevertheless, some rice samples appeared separate from the overlapping ones e.g. aromatic rice like Pahele, Jethobor, Maine Pokhreli, Pokhara Masino, and Hansraj. The level of Coefficient 0.00 0.24 0.48 0.72 0.96 Krishnabhog Tundedhan JumliWhite PokharaMasino Jhuldhan ChandanNath-1 NR10276-15 Sabitri Hansraj Mansara Jarneli Bhuwadhan NR10315-145 Pahele NR10414-25 Taichung-176 RatoDhan NR10276-9 NR10285-29 ChandanNath-3 Radha-7 Madise Pakhe Ghaiyadhan MainePokhreli Paledhan Dhokrodhan Hanse Chananchur Lekalidhan Jethobor Bageridhan Manjshree-2 NR10353-8 NR10414-34 Thapachini Tauli Anpjhutte A57-115-8 Churenodhan Pranpyuri Masuli Lalshar NR10286-6 NR10375-20 Khumal-11 NR285-18 CO39 KaliMarsi IR-24 Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   23 distinctness versus overlapping was in good concordance with that of the cluster. This preliminary genetic information could supplement for breeding and conservation works based on morphological markers. For increasing the value of genetic information derived from RAPD markers, number of primers should be increased. Choudhury et al. [14] suggest that a set of 10 primers can be employed for an initial assessment of genetic diversity in a large number of collections. Because of multilocus nature of RAPD, its use is considered more suitable for fingerprinting and genetic diversity measurement. Table 3. Eigen vectors of RAPD primers based on 50 rice genotypes. Primer Band PC1 PC2 PC3 P41 1 -0.363 0.203 -0.123 2 -0.374 0.217 0.019 3 -0.300 0.370 0.046 4 -0.371 0.247 -0.012 5 -0.299 0.240 0.078 6 -0.148 0.174 0.536 P60 1 -0.225 -0.284 -0.022 2 -0.252 -0.357 -0.071 3 -0.167 -0.200 -0.327 4 -0.096 -0.053 -0.172 5 -0.097 -0.153 0.029 6 0.197 0.268 -0.034 7 0.124 0.071 0.083 8 0.065 0.063 -0.037 P141 1 0.085 0.124 -0.060 2 -0.157 -0.257 0.472 3 -0.112 -0.321 0.372 4 -0.083 -0.143 0.198 5 0.068 0.016 0.048 6 -0.045 -0.090 0.059 7 -0.009 0.003 0.032 P109 1 -0.202 -0.097 -0.221 2 -0.199 -0.135 -0.230 3 -0.180 -0.175 -0.157 Eigenvalue 1.062 0.629 0.396 Proportion 0.243 0.144 0.090 Cumulative 0.243 0.386 0.477 Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   24 Fig.4. Scatter plotting of 50 rice genotypes based on four RAPD markers. 0-1-2-3-4 2 1 0 -1 -2 PC I P C II Masu liCO 3 9A 5 7 -1 1 5 -8 IR-2 4 Sab itri N R1 0 2 8 5 -2 9 N R1 0 2 7 6 -9 Ch an d an N ath -3 Ch an d an N ath -1Ju mliWh ite Taich u n g -1 7 6 N R1 0 4 1 4 -3 4 N R1 0 4 1 4 -2 5 N R1 0 2 7 6 -1 5 N R2 8 5 -1 8 N R1 0 3 5 3 -8 K h u mal- 1 1 N R1 0 3 7 5 -2 0 Man ju sh ree-2 N R1 0 2 8 6 -6 N R1 0 3 1 5 -1 4 5 Lalsh ar Ch an an ch u r Po k h araMasin o Jeth o b o r Bag erid h an Paled h an H an se Lek alid h an Main ePo k h reli D h o k ro d h an G h aiy ad h an K aliMarsi Mad ise Pran p y u riPak h e Rad h a-7 Pah ele Jh u ld h an Bh u w ad h an Jarn eli A n p jh u tte Ch u ren o d h an Man sara H an sraj Rato D h an Tu n d ed h an Tau li Th ap ach in i K rish n ab h o g Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  16  –  25                                                                                                                      Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   25 References 1. 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