SSR diversity on rice landraces collected from Yogyakarta Province

In Indonesia, rice is the most common food, and many areas have a lot of rice landraces. Therefore, characterization is very important to be done to identify potential gene sources to be used for the breeding program. The current study aims to learn more about the genetic diversity of rice landrace accessions collected from Yogyakarta province using simple sequence repeat (SSR) markers. A total of 56 accessions of rice landraces originated from Yogyakarta province, and 7 accessions of standard varieties, were used in the study. The DNA sample was extracted from young leaves and isolated for further molecular analysis by using eleven SSR markers. Data analysis was conducted using Power Marker v3.25 for genetic analysis, and DARwin v6.0.021 for cluster analysis and dendrogram visualization. The results of the genetic analysis showed that the average values of major allele frequency, number of alleles, gene diversity, heterozygosity, and polymorphic information content (PIC) were 0.27, 10.1, 0.83, 0.15, and 0.80, respectively. All eleven SSR markers were highly informative (PIC>0.50), as their PIC values ranged from 0.69 to 0.85. These markers have great potential to be used for marker-assisted selection of the germplasm collections. The result of cluster analysis showed that all the rice landrace accessions were separated into three different groups, each consisting of 29, 28, and 6 accessions.


Introduction
Rice is an important commodity as a staple food for most of Indonesia's population.According to Statistics Indonesia [1], rice production for food consumption reached 31.54 million tons in 2022, an increase of 184.50 thousand tons or 0.59 percent as compared to rice production in 2021 of 31.36 million tons.Meanwhile, rice consumption was 30.2 million tonnes, an increase compared to 2021, which was 30.04.However, this still encourages the government to continue increasing production given the high demand for rice and the increasing population growth rate.Based on data from the Central Statistics Agency (BPS) [2], Indonesia's population was 275.77 million people in 2022.This number has increased by 1.13% compared to 2021 of 272.68 million people.As predicted [3], Indonesia's population and rice consumption in 2023 will increase by as much as 280.13 million people and 30.5 million tonnes, respectively.The efforts to increase agricultural crop production, need to be supported by the availability of a collection of genetic resources.
Characterization is an important activity to identify the characteristics of a collection of genetic resources, so that they are more ready to be utilized.As a result, determining genetic diversity is crucial to the development of breeding programs, conservation strategies, and utilization plans [4].Appropriate 1255 (2023) 012048 IOP Publishing doi:10.1088/1755-1315/1255/1/012048 2 characterization methods are needed to accurately identify the materials [5,6] as well as to determine the relationships among accessions [7].
One of the most widely used molecular characteristic methods today is using microsatellite markers (SSR, or simple sequence repeats).This marker was chosen because it is co-dominant, spread throughout the plant genome, highly reproducible, and easy to score [8].SSR markers are reported to be able to produce a better spectrum of genetic diversity due to their being multi-allelic, highly informative, and transferable among related species [9].
Yogyakarta province is in the middle-southern part of Java Island.It is about 0-2,911 m above sea level and has an area of 3,185.80 km 2 [23], with a diverse type of typology stretching along the areas.These conditions contribute significantly to the high diversity of genetic resources in this area.As inventory and collection activities have been carried out, many rice landraces can be found and are still cultivated by farmers, in particular in the districts of Sleman, Kulon Progo, Bantul, and Gunung Kidul [24].Therefore, this research aimed to study the genetic diversity of rice landraces collected from Yogyakarta province and determine the relationships among the accessions.The information derived from present study is very important for the development of appropriate strategies in the conservation of rice landraces.Furthermore, it would also help breeders identify and select individual parents to be used for the breeding program.

Samples preparation
A total of 56 accessions of rice landraces collected from Yogyakarta province were used in the study.In addition, 7 accessions were also included as a standard variety.Seeds of these accessions are maintained in Agricultural Genebank, BBPSI Biogen, Ministry of Agriculture, Bogor.A complete list of the accessions is presented in Table 1.Seeds of the rice accessions were sown in pots for further reared in the greenhouse.A sampling of plant material was taken on plantations that were about 1 month old (3-5 weeks).Sampling was carried out by taking part of the young leaves from 10 different plants, then the leaf samples were mixed (bulk).Plantings were then maintained until seed harvest for observation of the main morpho-agronomic characters.

DNA extraction and isolation
DNA was extracted and isolated from the young leaves of several sample plant accessions (3-5 weeks after seedling) using the Genomic DNA Mini Kit (GenAll GenEx Plant Sx).DNA extraction and isolation were carried out at the Molecular Biology Laboratory, BBPSI Biogen Bogor.
Detailed protocol for DNA extraction and isolation are as follows (for a single sample): A total of 100mg of finely chopped young leaf samples was put into a 1.5 ml Eppendorf tube.The amount of 500µl of PL buffer and 3µl of RNase A solution were added to the Eppendorf tube, then mixed by placing it in the Vortex machine.Incubated at 65 o C for 15 minutes.Centrifuged at 14,000xg for 30 seconds, then about 400µl of the supernatant was transferred to a new Eppendorf flask.Add 140µl PP buffer, then vortex it for 15 seconds.Incubate the tube on ice for 5 minutes, then centrifuge at 14,000xg for 5 minutes.Transfer the 400µl supernatant using a pipette into a new Eppendorf tube containing 300µl isopropanol (room temperature), then the solution was mixed by gently inverting the tube until white DNA threads appear in the solution.
Centrifuge the solution at 14,000xg for 1 minute, then discard the supernatant and add 300µl of 70% ethanol (room temperature).Mix by gently inverting the tube to wash the DNA pellet and the side wall of the tube.Centrifuge at 14,000xg for 1 minute, then remove the ethanol by pipetting.Place the Eppendorf tube upside down on the absorbent paper and dry the DNA pellet for 5-10 minutes.Add 100µl of RE buffer (or distilled water), then rehydrate the DNA by incubating at 65 o C for 20 minutes (or at room temperature for 1 hour).The DNA sample solution was then stored at -20ºC for further use.

DNA quantification
The DNA samples were then measured for concentration and purity quantitatively by using a NanoDrop ND-1000 full-spectrum spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).Electrophoresis on 0.8%-1% agarose gel was used to check the quality of the DNA samples as well.The DNA sample was then diluted with a final concentration of 20-40 ng/µl for the PCR amplification reaction.

PCR analysis
A total of eleven SSR markers (Table 2), which are designed for rice [25] were used in this study.Each sample was amplified in a total reaction of 20µl containing 20ng of template DNA; 10 µl Kapa2G Fast ReadyMix (containing dNTP mix (0.2 mM of each 1x dNTP, buffer, 1x MgCl2 (1.5 mM), DNA Polymerase (0.5 U per 25µl reaction), and 2x loading dye) (Kapa Biosystems, USA); each primers F and R were 10µM/µl; and distilled water.The PCR reaction was carried out in a PCR Thermocycler machine (Biometra, Germany) with the following PCR conditions: an initial denaturation was carried out at 95 o C for 3 minutes, followed by a total of 35 cycles of the denaturation process at a temperature of 95 o C for 15 seconds, annealing (primer attachment stage) at 55 o C for 15 seconds, and elongation at 72 o C for 1 minute.The PCR reaction ended with a final extension cycle (last stage of base extension) at 72 o C for 1 minute.
To ensure the presence of amplicons, electrophoresis with 1% agarose gel containing SYBR Gold dye was used to check the PCR product.After all the rice accessions were amplified perfectly, the next process was separation using 8% non-denatured polyacrylamide gel in 1xTBE buffer solution.The gel electrophoresis results were then stained with an ethidium bromide solution, and a gel documentation system was used to see them on a UV transilluminator.

Electrophoresis
The PCR product was then electrophoresed on 2% agarose gel using 0.5 TAE buffer.After the gel was stained with ethidium bromide, the Bio-Rad Geldoc Documentation System was used to see the DNA bands.Data was manually scored based on the size of the DNA bands, using a standard DNA ladder as a reference.

Data analysis
Several statistical parameters (frequency of major alleles, gene diversity, heterozygosity, and polymorphic information content) were calculated using PowerMarker v3.2.5 [26] to estimate the level of diversity of the SSR markers used.Allelic data resulting from the scoring of DNA bands were analyzed using DARwin v6.0.021 (Dissimilarity Analysis and Representation for Windows) [27] to perform cluster analysis based on simple matching index, and to generate a dendrogram using unweighted Neighbour-Joining method.The relationship between accessions then can be visually determined by observing the dendrogram pattern.

Results and discussion
The amplification results showed that eleven primers used in the study produced clear and scoreable profiles of polymorphic DNA fragments.Figure 1 shows an example of visualizing DNA fragments from amplified samples of rice accessions using the primers RM19, RM144 and RM474.High genetic diversity was detected in rice accessions used in this study.The average major allele frequency was 27.29% with the lowest value being 22.86% (RM11) and the highest value being 35.21% (RM19) (Table 3).The population's richness is indicated by the number of alleles.One of the most widely used measures of population genetic diversity is allelic richness [28].SSR markers are short tandem repeats, therefore good markers typically produce 2 to 7 alleles per locus [29].The average number of alleles from the amplified SSR markers in the present study was 10.09 alleles per marker with a range of 6-13 alleles per locus.This result is greatly higher than has been reported in Malaysian aromatic rice [29] and coloured upland rice [30].
The average value of gene diversity is 0.83.The highest gene diversity was indicated by the RM11, which was 0.86, while the lowest gene diversity value was indicated by the RM105, which was 0.73.In this study, the degree of polymorphism produced by markers is reflected in the index of gene diversity.Three markers, namely RM474, RM215, and RM11 demonstrated heterozygosity at values of 0.18, 0.65, and 0.81, respectively.
The ability of markers to produce a polymorphic allele is indicated by the PIC (Polymorphism Information Content) values.The PIC values ranged from 0.69 to 0.85 with an average value of 0.80.This result is quite similar as reported in 36 accessions of Indian rice landraces having different therapeutic values [31].The lower genetic diversity was reported in 40 Pakistan rice accessions, with PIC value of 0.38 and an average of 2.75 alleles/locus [32].The lowest PIC value (0.69) was generated by RM105, and the highest value (0.85) was generated by RM11.For genetic diversity study, high-PIC molecular markers are a good choice.SSR markers that have PIC values greater than 0.50 are regarded as effective [33].The results showed that all SSR markers used in the study had a PIC>0.5, this indicate that all markers are informative and are very useful for distinguishing rice accessions.The informative SSR markers have great potential to be developed as The results of the cluster analysis showed that the 63 accessions of rice landraces used in this study could be clustered into three groups, each consisting of 29, 28 and 6 accessions (Figure 2).The dendrogram shows that the grouping pattern does not reflect to provenance, as well as the grain characteristics of the accessions (grain type and colour).
The accuracy and usefulness of the SSR markers for tracing the phylogeny or pedigree of a germplasm and breeding materials can be examined through dendrogram visualization.Accessions that are grouped together are thought to have a high genetic similarity, whereas those that are grouped far apart are thought to be divergent.
It can be clearly noticed that in the first group there are two accessions showing high similarity, which indicates that the two accessions are duplicates.The two accessions are Saodah Merah and Andel Merah, both are red rice from Bantul and Kulon Progo districts.On the other hand, all the seven standard varieties are placed in the second group, in the same sub-group together with three black rice accessions that are Pari Ireng, Beras Hitam Bantul, and Cempo Hitam Cemani.
A high level of genetic differentiation is indicated by the population's high variability.High variability within population will tend to further strengthen population divergence.In order to produce the desired heterotic groups in breeding populations, high genetic differentiation in the germplasm is also crucial [34].

Conclusion
All SSR markers used in the study had a PIC>0.5, indicating that they are informative markers and are very useful for distinguishing rice accessions.Further investigation is needed to explore their potential as a functional marker linked certain to interested traits.The result of the cluster analysis showed that the 63 accessions of rice landraces used in this study could be clustered into three groups, each consisting of 29, 28 and 6 accessions.Saodah Merah and Andel Merah are considered as duplicate accessions.There was no correlation between grouping pattern and provenance, as well as grain characteristics of the rice accessions.

Figure 1 .
Figure 1.The visualization results of three gel electrophoresis show the amplified DNA fragments using the RM19 (top), RM144 (middle) and RM474 (bottom) markers.(Notes: Far left and right are standard DNA ladders; each gel electrophoresis contains 48 DNA samples of the accessions).

Table 1 .
List of rice accessions used in the study.

Table 2 .
SSR markers for rice used in the study.

Table 3 .
Summary statistics derived from 11 SSR markers used in the study.