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Philippine Journal of Science136 (1): 45-56, June 2007ISSN 0031 - 7683

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Merlyn S. Mendioro*, Sharon L. Madriñan,Jeneylyne F. Colcol and Felipe S. Dela Cruz, Jr.

Key Words: table-type bananas, isozymes, malate dehydrogenase, phosphogluconate dehydrogenase, phosphoglucoisomerase, zymogram

*Corresponding author: [email protected]

Isozyme Polymorphism of Selected Cultivars of Table-and Cooking-type Banana (Musa sp.) in the Philippinesand Genome Identification of the Table-type Cultivars

INTRODUCTIONBananas are among the most common fruits known to man and are among the most dominant crops in Philippine agriculture (Yorobe 1999). They can be found in almost all parts of the world (Molina and Valmayor 1999) and are primarily cultivated in many

Isozyme profiles of 20 table-type and 6 cooking-type banana (Musa sp.) cultivars were studied. The different isozymes used include malate dehydrogenase (MDH), 6-phosphogluco dehydrogenase (PGD), phosphoglucoisomerase (PGI), and phosphoglucomutase (PGM). Different banding patterns were observed. Banding patterns (BP) of the different cultivars with known genomes (18) were compared with newly collected cultivars (5 table-types; 3 cooking-type) with unknown genome (UK) to determine the genome identity of the latter. The 4 isozymes were not useful in identifying the genome of the 3 cooking-type cultivars namely, Balatay (BLT), Bataan (BAT), and Dumanese (DUM). To determine the genetic relationships of the different table-type cultivars, dendrogram was created using Numerical Taxonomy and Multivariate Analysis System (NTSYS). Clusters were identified based on the results of Unweighted Pair Group Method Using Averages (UPGMA). Using similarity coefficient of 0.85, the different table-type cultivars were grouped into 6 clusters. The cultivars with similar genomes were not found in the same cluster. In the first cluster, cultivars with AAB (HHG, CAN), AAA (BWE) and AA (LAK) genomes were together. MCS (UK) was found in this cluster. The same thing is true for the second cluster; AAB (LTL and MLA), AAA (TDK), and AA (IBL). KMY (UK) belonged to this group. MPD (UK) clustered with LAT (AAB). MNF (UK) formed a cluster with the rest of the cultivars with AAA genome (BUN, SBG, DCV, PST, and TMK). The clustering of cultivars with AAA and AAB genome may indicate common genomic origin.

countries because of their great socio-economic importance. They generate national export revenues for countries involved in the industry and help in the establishment of employment (Cull 1985; Avise et al. 1986; Valmayor 1986). Bananas are basically part of the diet, which provide nutrition to millions of people and valuable economic benefits from production activities.

Genetics and Molecular Biology Division, Institute of Biological Sciences,College of Arts and Sciences and Institute of Plant Breeding, Crop Science Cluster,

College of Agriculture, University of the Philippines Los Baños,College, Laguna Philippines

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Mendioro et al.: Isozyme Polymorphism of Selected Cultivars of Philippine Banana

Philippine Journal of ScienceVol. 136 No. 1, June 2007

Table 1. Cultivar names, their designated abbreviations, genomes, and origin of Banana (Musa sp.) maintained at field genebank of the National Plant Genetic Resources Laboratory IPB, UPLB used in the studyCultivar Name (Designation) Genome OriginTable-type

Inarnibal (IBL) AA Dolores, QuezonLakatan (LAK) AA Sta. Cruz, MarinduquePastilan (PST) AAA Torrijos, MarinduqueSuay Baguio (SBG) AAA Abuyog, LeyteTudok (TDK) AAA Guinobatan, AlbayTumok (TMK) AAA Diffun, QuirinoBinalatong (BLT) AAA Bago Oshiro Davao CityBungulan (BUN) AAA Torrijos, MarinduqueDwarf Cavendish (DCV) AAA Diffun, QuirinoBinawe (BWE) AAA Bago Oshiro, Davao CityLatundan Tall (LTL) AAB Diffun, QuirinoManila (MNL) AAB Rizal, CagayanHilao-Hinog (HHG) AAB Bago Oshiro, Davao CityCantong (CAN) AAB Atok, BenguetLatundan (LAT) AAB Guinobatan, AlbayLatundan Puti (LPT) Unknown Catanauan, QuezonManang Costa (MCS) Unknown Pinabacdao, SamarKinalamay (KMY) Unknown Abuyog, LeyteManifun (MNF) Unknown Rizal, CagayanMangipod (MPD) Unknown Torrijos, Marinduque

Cooking-TypeSaba (SAB) BBB Los BañosMatavia (MTV) ABB Sta. Cruz, MarinduqueTiparot (TPT) ABBB Bago Oshiro, Davao CityBalatay (BAL) Unknown San Felipe, ZambalesBataan (BAT) Unknown Limay, BataanDumanese (DUM) Unknown Diffun, Quirino

Genomic identification of established cultivars was based on Valmayor et al. 2000.

Banana nomenclature has already been established. Following the International Code of Nomenclature for Cultivated Plants, bananas are classified based on the scientific names of their wild parents (Valmayor et al. 2000). However, confusion still exists and is unavoidable because of the occurrence of many vernacular names for single variety. Furthermore, morphological characterization is time consuming and environmental influence may lead to inconclusive observations (Bhat et al. 1992a; 1992 b). Thus, the use of biochemical markers like isozymes are encouraged because the process is more reliable and provides easier way for identifying and classifying cultivars.

This study therefore was conducted to determine the isozyme profile of 20 table-type and 6 cooking-type banana cultivars maintained at the field gene bank of the National Plant Genetic Resources Laboratory of the Institute of Plant Breeding (IPB), College of Agriculture University of the Philippines Los Baños,

College, Laguna. The different isozymes considered were malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (PGD), phosphoglucoisomerase (PGI), and phosphoglucomutase (PGM). These isozymes were also considered by other researchers in banana (Bhat et al. 1992b; Jarret and Litz 1986; Espino and Pimentel 1990). The isozyme profiles of the table-type cultivars with known genomes were compared with the isozyme profiles of the newly collected table-type cultivars to determine their genome identity.

MATERIALS AND METHODS

Name and Genome IdentificationThe cultivar names, designated abbreviations, and the genomes for the table- and cooking-type banana cultivars are presented in Table 1. The table-type cultivars are

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HHG, LTL, MNL, CAN, and LAT with AAB genome while, BWE, BLT, BUN, DCV, PST, SBG, TDK and TMK with AAA genome; LAK and IBL are diploids with AA; genome; MTV with ABB; SAB with BBB while TPT with ABBB (Valmayor et al. 2002). The genomes of other 5 table-type and 3 cooking-type cultivars are unknown. Both table-type and cooking-type cultivars were then subjected to isozyme analysis to determine their isozyme profile.

Isozyme AnalysisOne gram of leaf tissue taken from the central region of the leaf blade of the second unfolded leaf from the top was ground in refrigerated ice mortar and pestle placed in a basin containing crushed ice to maintain cool temperature. and 1 mL chilled extraction buffer (20 mL of extraction buffer with the following composition: 175 mL 0.1 M Tris-HCl buffer pH 7.5, 31.5 mg mM DL-dithiothreitol, 2 mL 10% (v/v) glycerol, 2 g 10% (w/v), PVP40, 1 mL 0.5% Triton X-100) was added. Thirty mL of leaf- extract and the tracking dye were loaded into a 13% starch gel and placed into the electrode vessel of Advance Mupid 2 electrophoresis apparatus. The set up was filled with TRIS-HIS buffer (0.009M Tris-0.005M Histidine). Electrophoretic runs lasted for 4 h at 50 volts. Gels were stained for the presence of MDH, PGD, PGI and PGM. Isozyme banding patterns were observed by placing the stained gels over a light box. Zymograms were constructed for each enzyme system. Bands were scored as SS (slow moving bands), FF (fast moving bands) and FS (combination of slow and fast bands).

Data Collection and AnalysisStained gels were placed in a light box to determine their isozyme banding patterns (BP). The number of bands was recorded and their relative mobility (Rf) was obtained using the formula:

RF= Distance travelled by the band

distance traveled by the tracking dye

Zymograms were prepared to depict the isozyme profiles. Multivariate analysis of the isozyme profiles was done (Lebot et al. 1993) and clustering was based on the results of Unweighted Pair Group Method Using Averages (UPGMA) cluster analysis performed on the Jaccard’s similarity coefficient matrices. Dendrograms presenting the genetic relationships of the different cultivars were constructed using the Numerical Taxonomy and Multivariate Analysis System (NTSYS).

RESULTS AND DISCUSSION

Isozymes of Table-type BananasMalate dehydrogenase (MDH): The 2 zones of activity observed were designated as MDH1 and MDH2. MDH1 has an average RF range of 0.02-0.24 while MDH2 has 0.34-0.48. Jarret and Litz (1986), Espino and Pimentel (1990) and Lebot et al. (1993) also observed 2 zones of activity for MDH. Figure 1 shows the photograph, zymogram and designated genotypes for MDH in selected table-type bananas. Four BPs were noted (Figure 2). BP1 was observed in HHG, LTL, MLA, CAN, and LAT. Three genotypes were observed for MDH1, SS, FS, and FF and only 2 for MDH2, FF and FS. These cultivars have AAB genome. The 3 unknown cultivars LPT, MCS, and KMY showed BP1. This same BP was also exhibited by cultivars with AA genome (LAK and IBL). BP3 was noted in BLT, BUN. DCV, PST, and TDK. The said cultivars have AAA genome. The BP of MPD is similar to BP3.

Phosphogluconate dehydrogenase (PGD): For PGD, 2 zones of activity were observed for PGD. Similar observation was noted by Jarret and Litz (1986). Two presumptive loci were observed, PGD1 and PGD2, with RF range of 0.07-0.10 and 0.19-0.28, respectively (Figure 3). Only SS genotype was noted. No distinct BP was observed for cultivars with AAB, AA or AAA genomes (Figure 4). BP1 was observed in HHG, LTL, and MLA. These are cultivars with AAB genome. BP1 was also noted in LAK and IBL with AA genome and BLT, BUN, SBG, and TDK with AAA genome. Three accessions with unknown genome (LPT, KMY and MNF) showed BP1 while MCS and MPD showed BP 2.

Phosphoglucoisomerase (PGI): For PGI, the 2 zones of activity (Figure 5) were designated as PGI1 (Rf range of 0.02-0.19) and PGI 2 (0.23-0.40). Only 1 genotype (SS) was noted for PGI1 but two for PGI2, SS and FF. HHG, LTL, MLA, and LAT showed BP 1 (Figure 6). The 3 unknown cultivars (LPT, MCS, and KMY) also showed BP1. The BP of the 2 unknowns (MNF and MPD) is similar to the cultivars with AAA genome (BLT, BUN, DCV, PST, SBG, TDK, and TMK).

Phosphoglucomutase (PGM): Two zones of activity were noted for PGM (Figure 7) designated as PGM1 and PGM2. The genotype for the 2 zones was SS. This is different from the results of Lebot et al. (1993) for they observed 3 zones in banana. Three types of BP were observed (Figure 8) for PGM. Two BP were noted for AAB genomes, BP1 (HHG and CAN) and BP2 (LTL, MLA, and LAT). BWE with AAA genome showed BP1. BP2 was noted for IBL with AA and SBG, TDK, and TMK with AAA genome. BLT, BUN, DCV, and PST known to

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Figure 2. Zymogram, banding pattern and designated genotypes for malate dehydrogenase of table-type banana (Musa sp.)

Legend

Cultivars used in the study

BLT - Binalatong MPD - Mangipod

BUN - Bungulan SBG - Suay Baguio

DCV - Dwarf Cavendis TDK - Tudok

MNF - Manifun TMK - Tumok

PST - Pastilan CRL - ControlPNK - Pisang Nangka (introduced variety)

Figure 1. Photograph of the gel, zymogram and designated genotypes of malate dehydrogenase (MGH) of table-type bananas

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Legend


BLT - Binalatong MPD - Mangipod

BUN - Bungulan SBG - Suay Baguio

DCV - Dwarf Cavendis TDK - Tudok

MNF - Manifun TMK - Tumok

PST - Pastilan CRL - ControlPNK - Pisang Nangka (introduced variety)

Figure 3. Photograph of the gel, zymogram and genotypes for 6-phosphogluco dehydrogenase (PGD) in table-type bananas

Figure 4. Zymogram, banding pattern and designated genotypes for 6 phosphogluconate dehydrogenase

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Legend


LAT - Latundan BWE - Binawe

IBL - Inarnibal MLA - Manila

LAK - Lakatan LTL - Latundan Tall

CAN - Cantong LPT - Latundan

KMY - Kinalamay HHG - Hilao-Hinog

MCS - Manang Costa

Figure 6. Zymogram, banding pattern, and designated genotypes of the table-type bananas (Musa sp.)

Figure 5. Photograph of the gel, zymogram and genotypes for phosphoglucoisomerase (PGI) in table-type bananas

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Legend


HHG - Hilao-Hinog KMY - Kinalamay

LPT - Latundan Puti CAN - Cantong

LTL - Latundan Tall LAK - Lakatan

MLA - Manila IBL - Inarnibal

BWE - Binawe LAT - Latundan

MCS - Manang Costa

Figure 7. Photograph of the gel, zymogram and genotypes for phosphoglucomutase (PGM) in table-type bananas

Figure 8. Zymogram, banding pattern, and designated genotypes for phosphogluconate of the table-type bananas (Musa sp.)

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Legend:


MTV - Matavia

SAB - Saba

TPT - Tiparot

BAL - Balatay

BAT - Kinalamay

DUM - Dumanese

have AAA genome showed BP3. The 2 unknowns, MNF and MPD showed BP3.

Isozymes of Cooking-type BananasMalate dehydrogenase (MDH): Two zones of activity were noted and designated as MDH1 and MDH2. The genotype noted was only SS. Average RF range for MDH1 is 0.11-0.13 while for MDH2 is 0.26-0.37. A representative photograph and its zymogram are presented in Figure 9. One type of BP was observed. MTV, SAB, TPT, BAL and DUM exhibited the same BP. The genomes of the 3 unknowns (BAL, BAT, and DUM) cannot be determined since the BP of cultivars with ABB or BBB genomes are the same for MDH.

Phosphoglucodehydrogenase (PGD): One zone of activity was again noted. PGD1 has RF range of 0.19-0.27 (Figure 10). The genotype was designated as FF. All cultivars exhibited the same BP, hence, PGD is not useful in establishing the genome of the 3 unknown cultivars.

Phosphoglucoisomerase (PGI): PGI showed 2 zones of activity. They were labeled as PGI1 and PGI2 (Figure 11). PGI1 has Rf range of 0.32-0.35 while PGI2 has 0.66-0.72. FF genotype was noted both for PGI1 and PGI2 . MTV, SAB, TPT, and DUM have only PGI1 while BAL and BAT have both PGI1 and PGI2. This may imply the presence of inherent variation in banana. Since banana is asexually propagated, occurrence of any new mutation may persist in the individual. Since no BP that is specific for a particular genome was noted, the genomes of the 3 unknowns cannot again be determined.

Phosphoglucomutase (PGM): For PGM, 3 zones of activity were noted (Figure 12). Average Rf range for PGM1, PGM2, and PGM3 were 0.13-0.18, 0.32-0.42, and 0.47-0.53, respectively. FF and FS genotypes were observed in PGM1, SS and FF for PGM2 and only SS for PGM3. Four types of BP were noted. MTV (with ABB genome) showed BP1. This is similar to the BP of DUM, the cultivar with unknown genome. TPT and BAT showed BP2 while BAL showed BP3. SAB showed BP4.

Figure 9. Photograph of the gel, zymogram and genotypes for malate dehydrogenase (MDH) in cooking-type bananas

Legend:


MTV - Matavia

SAB - Saba

TPT - Tiparot

BAL - Balatay

BAT - Kinalamay

DUM - Dumanese

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Legend


MTV - Matavia

SAB - Saba

TPT - Tiparot

BAL - Balatay

BAT - Kinalamay

DUM - Dumanese

Legend


MTV - Matavia

SAB - Saba

TPT - Tiparot

BAL - Balatay

BAT - Kinalamay

DUM - Dumanese

Figure 11. Zymogram for phosphoglucoisomerase (PGI) in cooking-type bananas

Figure 10. Photograph of the gel, zymogram and genotypes for 6-phosphogluco dehydrogenase (PGD) in cooking-type bananas

Legend


MTV - Matavia

SAB - Saba

TPT - Tiparot

BAL - Balatay

BAT - Kinalamay

DUM - Dumanese

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Legend


DUM - Dumanese

TPT - Tiparot

BAT - Kinalamay

BAL - Balatay

SAB - Saba

MTV - Matavia

Figure 12. Photograph of the gel, zymogram and genotypes for phosphoglucomutase (PGM in cooking-type bananas

The 4 isozymes were not useful in determining the genomes of the 3 cooking type cultivars. No specific BP was obtained to which the isozyme profile of the unknown cultivars can be compared. According to Espino and Pascua (1992), aside from MDH, peroxidase and polyphenol oxidase can be used to delineate the cultivars belonging to BB/BBB from those of AAB and ABB cultivars.

Genetic Relationships of the Table-type Banana CultivarsUsing the Numerical Taxonomy and Multivariate Analysis System (NTSYS) program, similarity indices among the table-type cultivars were derived. Using a similarity index of 0.85, the different cultivars were grouped into 6 clusters (Figure 13) . The first cluster is composed of 2 cultivars with AAB genome (HHG and CAN). But BWE with AAA genome is also found in this cluster. LAK which is a diploid (AA) is separated from the rest. MCS (with unknown genome) is included in this cluster. The second cluster includes LTL, MLA and the 2 cultivars with unknown genome, LPT and KMY. Their genome is AAB. IBL (AA), TDK (AAA) and BLT (AAA) were also found in this cluster. LAT (AAB) and MPD (AAA)

were included in the third group. The fourth, fifth, and sixth group include varieties with AAA genome namely BUN, SBG, DCV, PST, and TMK. MNF (with unknown genome) is included in this cluster.

SUMMARY AND CONCLUSIONTwenty table-type and 6 cooking-type cultivars of banana (Musa sp.) were used in the study. The different table-type cultivars have different genomes. Cultivars with AAB genome include Hilao-Hinog (HHG), Latundan Tall (LTL), Manila (MNL), and Cantong (CAN). Binawe (BWE), Binalatong (BLT), Bungulan (BUN), Dwarf Cavendish (DCV), Pastilan (PST), Suay Baguio (SBG), Tudok (TDK), and Tumok (TMK) have AAA genome. Five cultivars have unknown genomes namely; Latundan Puti (LPT), Manang Costa (MCS), Kinalamay (KMY), Manifun (MNF), and Mangipod (MPD). For the cooking-type Matavia (MTV), Saba (SAB), and Tiparot (TPT) have genomes ABB, BBB and ABBB, respectively. The genomes of Balatay (BAL), Bataan (BAT) and Dumanese (DUM) were unknown. These cultivars were subjected to isozyme studies to establish their isozyme profiles.

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Figure 13. Dendogram showing the genetic relationships among table-type cultivars of banana based on similarity index of four isozyme profile

Isozymes studied include malate dehydrogenase (MDH), 6 phosphodehydrogenase (PGD), phosphoglucoisomerase (PGI), and phosphoglucomutase (PGM). The isozyme profiles of the cultivars with known genomes were compared with the profile of the newly collected cultivars to determine their genome identity.

The 4 isozymes were not useful in identifying the genome of the 3 cooking-type. No specific BP was obtained to which the isozyme profile of the unknown cultivars can be compared. The genetic relationships of the 20 table-type cultivars were established by constructing a dendrogram using NTSYS. Six clusters were identified using 0.85 similarity coefficient. Cultivars with AAB, AAA and AA clustered together. The clustering together of cultivars with AAA, and AAB would simply indicate that their genomes are of the same origin.

The 4 isozymes considered failed to cluster the different table-type cultivars based on their genome composition. No clear clustering was noted for the cultivars with AAB, AAA, and AA genomes. More isozymes need to be considered so that the genome of MCS, LPT, KMY,MPD, and MNF be determined or better yet use DNA markers.

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