Genetic Variability of Local Corn Cultivars from Kisar Island Southwest Maluku Regency using Microsatellite Molecular Marker

This research aim was to reveal the genetic variability of local corn cultivars from Kisar Island using microsatellite molecular marker. The use of microsatellite markers is based on one reasons that the genetic information is polymorphic, and will be better to identify genetic diversity of plants including corn. Corn cultivars used were obtained from farmer in Kisar Island, and three reference varieties as outgroup were obtained from Institute of Cereals in Maros South Sulawesi. Polymerase chain reaction was conducted following the protocols from Fast Start kit (Qiagen-USA). The data obtained then analyzed with MVSP 3.1A Software for dendogram construction. The results showed that the appearance of DNA bands was vary (monomorphic and polymorphic) for local cultivars as well as reference varieties, with one or two bands on each primer. Based on the dendogram, there are three main clusters with similarity index ranged from 20% -100%. It can be concluded that there is very low similarity and distant kinship of local cultivars. The novelty of this research is knowing kinship relationship between local corn in Kisar Island which is not known yet. This results is expected to provide benefits regarding the breeding program, and for instance, it can be an important information regarding the development of local corn as a source in assembly the superior corn cultivars.

the high genetic diversity of local corn can also be used to reduce subjection of corn hybrids derived from multinational companies which cause minor attention to the local cultivars or landraces (Pabendon, 2010).
Research on genetic diversity can be done by utilizing a system of genetic markers for both morphological, and molecular markers (Farooq & Azam, 2002;Bhat et al., 2010).Molecular markers is an effective technique in genetic analysis and has been widely applied in the breeding program especially in plants.According to Farooq & Azam (2002) some genetic marker-based on polymerase chain reaction (PCR) that has been widely used is randomly amplified polymorphic DNA (RAPD), Sequence Characterized amplified regions (Scars), sequence tagged sites (STS), single strand conformational polymorphism (SSCP), single nucleotide polymorphism (SNPs) and microsatellite or short tandem repeats/short sequence repeats (STRs/SSRs).Some of these genetic markers are relatively simple, easy to use, can be automatically tabulated, and sometimes codominant (Farooq & Azam, 2002;Bhat et al., 2010;Fibriana & Hadiyanti, 2016).One of the genetic markers have also been developed extensively is microsatellites markers.The advantages of microsatellite markers is easy to implement, and the degree of information about the number of alleles per locus is very high.Microsatellite markers can be used to detect genetic differences between species in many eukaryotic organisms.The use of microsatellite markers to analyze the genetic diversity of corn have been also reported (Smith et al., 1997;Senior et al., 1998;Gethie et al., 2002;Lie et al ,. 2004;Pabendon et al., 2006;Pabendon et al., 2008;Zhang et al., 2012).
Research on the genetic diversity of corn in Indonesia using microsatellite markers have been reported by Pabendon, et al. (2003), andPabendon (2006), or a combination of morphological markers and microsatellite (Pabendon, et al., 2008, andFate, et al., 2009).Especially for local corn on Kisar Island Southwest Maluku Regency, there is no research had never done to determine the genetic diversity of these cultivars.The purpose of this study was to determine the genetic diversity of local corn cultivars in Kisar Island SouthWest Maluku Regency based on molecular characterization using microsatellite molecular markers.
The novelty value of this research is knowing kinship relationship between local corn cultivars in Kisar Island of Southwest Maluku Regency which is not known yet.The results of this study are expected to provide benefits to the

INTRODUCTION
Corn is a plant species that has a very large genetic diversity, widely distributed, and one of perspective species in world plants utilization (Wijayanto, 2007;Saputro et al., 2016).This high genetic diversity was caused by the natural traits of corn as cross-pollinated plants, can lead variation in genetic composition of one varieties with another (Sudjana et al., 1991).According to Iriany et al. (2008) nowadays, corn varieties is estimated about 50.000 varieties.This including local varieties, as well as breeding of improved varieties.
In Indonesia, the genetic diversity of corn is the highest in Asia (Pabendon, 2010), this is due to in many area in Indonesia, there are many local corn cultivars with different characteristics.Local cultivar is formed through a process of isolation of genotypes that are changing and adaptation to specific agroclimate (Iriany et al., 2008) such as the diversity of geographical conditions, and islands, as well as bulk fluctuate, causing the diversity of climatic and soil conditions from one region to another (Wijayanto, 2007).
Southwest Maluku Regency, is one area in Maluku which has high diversity of corn germplasm (Pesireron, et al., 2013a).In this area also, corn be the main commodity that already been cultivated since many years ago in any farming activities and in all growing season (Pesireron, et al., 2013a).Formerely, exploration and documentation of corn germplasm has been done by Alfons et al. (2003), and found that in Kisar Island Southwest Maluku, there are seven local corn cultivars specific to this island.These cultivars consist of: Merah Delima Tongkol Cokelat (MDTC), Merah Delima Tongkol Putih (MDTP), Merah Darah (MD), Putih (PTH), Pulut (PLT), Kuning Genjah (KG) and Kuning Dalam (KD).
A very striking nature of those local cultivars is the diversity of seed color.The presence of local corn cultivars with specific characteristic and high ability in adaptation to specific environmental condition is the main genetic resources that must be conserved to maintain the specific traits that can be used in the construction of superior varieties.According to Juhriah et al. (2011) germ plasm is the source of gene that can be used to increase plant diversity.This is usefull in improving some traits of population, and to make corn varieties.Previously, Pabendon et al. (2003) stated that germ plasm and the relationship between breeding material is very important to know and understand regarding to the planning program for producing better hybrid.In addition, breeding of local corn cultivars and the protection and conservation of corn germplasm in South west Maluku Regency.

DNA Extraction
Leaves of corn used as sample were taken from the second leaf from the tip at 10 days after planting.DNA isolation was done following the protocol of ilustra phytopure kit.As much as 0.1 g of freshleaf was taken, and gently crushed with a mortal and pestle.Furthermore, 500 mL of ilustra phytopure I kit reagent were added, and placed into eppendorf tubes.Then 150 mL reagent of phytopure II were added and shaken gently and incubated at 65ºC for 10 minutes in waterbath.Subsequently the samples were placed in ice for 20 minutes, added with 400 mL of cold chloroform and 20 mL of resin phytopure into the sample carefully and centrifuged at 3000 rpm for 10 minutes to obtain a supernatant.
The supernatant was taken and placed in another tube.After that, cold isopropanol with the same volume as the supernatant was added through the tube, and centrifuged at 10.000 rpm for 10 minutes to obtain a DNA pellet.The supernatant was discarded and the tubes containing pellets dried on tissue then added 100 mL of 70% ethanol and again centrifuged at 10.000 rpm for 5 minutes.

DNA Amplification and Electrophoresis
DNA obtained was used for PCR analysis using 10 types of primer SSR (phi 026, phi 046, phi 047, phi 058, phi 068, phi 069, phi 074, phi 079, phi 116, and phi 120) (Smith et al., 1997;Senior et al., 1998;Qi-Lun et al., 2008), using Fastart PCR kit.DNA that has amplificated from PCR reaction, then ran in gel agarose electrophoresis, and documented with gel doc to see the appearance of DNA banding pattern.

Data Analysis
Molecular data such as DNA band was scored with the value of one (1) for the band presence and zero (0) for unpresence of DNA band.The data then processed to construct the dendogram using MVSP 3.1a Software.

RESULT AND DISCUSSION
The result showed that performance of the DNA bands varies (monomorphic and polymorphic) both on local corn cultivars (Figure 1-7) as well as reference varieties Lamuru, Anoman, and Srikandi (Figure 8-10).
Total amount of DNA varies between one to two bands on each primer.For Pulut cultivar (Figure 3) there were only one DNA bands in 9 primers while in the phi 068 primer, the appearances DNA banding pattern not so obvious.For the Kuning Genjah cultivar (Figure 4) there were polymorphic DNA bands with 1-2 bands for all primers.In Kuning Dalam cultivar (Figure 5) there     According to Stuessy (1990), kinship is defined as a pattern of relationships or total similarity between groups of plants based on their nature or specific character of each group of plants.The distance of the kinship is affected by the similarities and differences between the characteristics of the local corn cultivars indicated by the similarity index.The value of the similarity between corn cultivars tested, ranged between 20% -100%.The high index shows that the similarity between corn plants tend to high, otherwise when the similarity index is small, the similarity is tend to small.The fenetic approach, if the similarity index reaches a 85%, the similarity between the operational ta-xonomical unit (OTU) is considered as a species, 65% for genus, and 45% for family.
Merah Delima Tongkol Putih, Merah Delima Tongkol Cokelat, and Kuning Genjah cultivars were clustered into same cluster (Cluster B) which means that these cultivars have high similarity index (70-80%).The value of the similarity between Merah Delima Tongkol Cokelat, and Kuning Genjah cultivars as much as 80%, Merah Delima Tongkol Putih and Merah Delima Tongkol Cokelat as much as 70%, while between Merah Delima Tongkol Putih and Kuning Genjah only about 50%.This result showed that in cluster B even though these cultivars are in same cluster, but Merah Delima Tongkol Putih cultivars were separated from Merah Delima Tongkol Cokelat, and Kuning Genjah cultivars.
Srikandi, Lamuru, and Anoman varieties as a reference (outgroup) looks to be on the same cluster (cluster C).However, in this cluster it involves the local cultivar Merah Darah (MD), and Putih.The similarity value of C cluster reaches 100%, indicating that reference varieties and local cultivars Merah Darah and Putih had a very high similarity.In cluster C also showed that the Pulut cultivar and Anoman varieties were separate from the Srikandi, Lamuru and local cultivars Putih, and Merah Darah.
The similarity value among Anoman, Srikandi, Putih, Merah Darah, and Lamuru as much as 80%, while the similarity value between Anoman varieties with Pulut cultivars as much as 70%.This result indicated that the Anoman varieties was closely related to Srikandi varieties, and Lamuru, also the local cultivars Putih, and Merah Darah, or in other words Srikandi varieties, and Lamuru, as well as the local cultivar Putih, and Merah Darah is more similar to Anoman varieties than the Pulut cultivar.
Based on the dendogram, it can be seen that Kuning Dalam (KD) cultivar were separate from other cultivars.This mean was that Kuning Dalam cultivar had a low level of similarity index with other cultivars.The similarity index of Kuning Dalam cultivar with other cultivars range between 20% -50%.The similarity index below 65% indicated that there are a very long or very different genetic variation.Overall, based on the fenetic analysis with SSR markers it can be known that most cultivars have similarities below 85%.This suggests that the similarity between local corn cultivars with one another is not too high.These results are quite provide an overview and information on the phylogenetic relationship between local corn cultivars in Kisar Island South west Maluku.It is alleged that local cultivars have close genetic relationship because it is at the same location.But apparently based on the results of the analysis with the molecular characterization of microsatellite markers, it is known that the local corn cultivars has kinship distant and very quite different.These results indicate that although these cultivars were grown in the same environment, but they are not similar in their genetic material.This is consistent with the statement of Jose et al., (2005) that the genotype in the same area do not always shows similarity in their genetic arrange.This is allegedly caused by the genotype has a different arrangement of genetic material, resulting in the appearance of different phenotypes, even if they were grown in the same environment.
It is known that the molecular characterization of an investigation to complement and reinforce the allegations obtained from the morphological characterization.Thus, the results of morphological characterization show much variety and at least a great similarity between local corn cultivars, strengthened by the results of molecular data were indicate local corn cultivars in Kisar Island SouthwestMaluku were genetically different, resulting in the appearance of phenotypically different, despite being on a the same neighborhood.
Kinship distant of local corn cultivars in Kisar Island means that there is a high genetic variation and this is a very important information regarding to the breeding program.Corn cultivars can be used as a source of genes for corn germplasm conservation in the future.According to Sukartini (2007), accessions ties distant resemblance is good for plant breeding activities.Instead accessions has very close resemblance relationship is not good for plant breeding activities for at least the possibility of genetic variation within the species.
Nevertheless, all of cultivars with distant kinship, the Kuning Dalam cultivar seems to be more difference than the other local cultivars.Morphologically, these cultivars have very little in common with other local cultivars.For example, in the female flower characters.Other six local cultivars shows purple color when appears, except for Kuning Dalam, it has pink color of female flower when appear.The differences of Kuning Dalam cultivars with other six local cultivars also shown in fruit characters.All of six cultivars have a large category of cob diameter with small category, while the Kuning Dalam cultivar has a very large cob diameter with the diameter and medium category.These results then confirmed to the result of molecular characterization that placed this Cultivar in to one cluster alone with a very low similarity value, which means that this cultivar has a far kinship relationship with other local cultivars.
The results of Sinay & Karuwal (2013) studies based on the trials of drought resistance at the early vegetative phase of six local corn cultivars from Kisar Island Southwest Maluku Regency under greenhouse condition, also found that the Kuning Dalam cultivar had higher proline and total soluble sugars content than the other local cultivars, as well as reference varieties Srikandi, Lamuru and Anoman.
Another result also reported by Sinay et al., (2015) about the proline content and yield of local corn cultivars on natural conditions in Kisar Island, which indicates that the Kuning Dalam cultivar has the highest proline content (in line with the results of greenhouse experiments, according to Sinay & Karuwal, 2014), as well as high yields such as the number of seeds per cob, the number of rows of seeds, the cob weight, the cob length, and the cob diameter.
Based on these observation result in the field or in their natural habitat in Kisar Island, shows that Kuning Dalam cultivar is the most widely grown and consumed by the people.Also the result of an experiment under greenhouse condition, and under natural condition, give an information that Kuning Dalam has a better or higher response than other local cultivars.Therefore this cultivar can be recommended as a source of germplasm that is urgently needed in its preservation and development as a superior corn cultivar in the future, although other aspects must continue to be made regarding its potential development as a candidate for superior corn cultivars, mainly through multilocation testing.
The study concluded that local corn cultivars of Kisar Island Southwest Maluku Regency have low similarity and distant kinship.The novelty value of this research is knowing kinship relationship between local corn cultivars in Kisar Island of Southwest Maluku Regency which is not known yet.The results of this study are expected to provide benefits regarding the breeding program of local corn cultivars and for instance, this result can be an important information regarding the development of local corn cultivars as a source in assembly the superior corn cultivars.

CONCLUSION
Based on these findings, it was concluded that the results of the molecular characterization using microsatellite markers grouping local corn cultivars from Kisar Island into three main groups with similarity index ranged from 20%-100%.Molecular characterization results indicate that low similarity or distant kinship between local corn cultivars in Kisar Island Southwest Maluku Regency.

Figure 11 .
Figure 11.Dendogram of local corn cultivars and reference varieties based on UPGMA algorithme with Simple Matching Coefficient Method Based on the molecular characterization, the local corn cultivars and reference varieties were devide into three main clusters (cluster A, B, and C).Cluster A consists of only one cultivar that was Kuning Dalam (KD) cultivar.Cluster B consists of three local cultivars Merah Delima Tongkol Putih (MDTP), Merah Delima Tongkol Cokelat (MDTC), and Kuning Genjah (KG), while the cluster C consists of local cultivars Pulut (PLT), and Merah Darah (MD), and also reference varieties Srikandi (S), Lamuru (L), and Anoman (A).According toStuessy (1990), kinship is defined as a pattern of relationships or total similarity between groups of plants based on their nature or specific character of each group of plants.The distance of the kinship is affected by the similarities and differences between the characteristics of the local corn cultivars indicated by the similarity index.The value of the similarity between corn cultivars tested, ranged between 20% -100%.The high index shows that the similarity between corn plants tend to high, otherwise when the similarity index is small, the similarity is tend to small.The fenetic approach, if the similarity index reaches a 85%, the similarity between the operational ta-