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KEPROK (Citrus reticulata) GENETIC DIVERSITY ASSESSMENT BASED ON MORPHOMETRIC STUDIES IN INDONESIA Amelia Tridiptasari, Jamilatus Sa’diyah, Nurfitri Arfani, Nia Kurniawan Ecology Laboratory of Mathematics and Natural Science Brawijaya University, Malang Email: [email protected]

ABSTRAK Indonesia is a tropical country that is known as a producer of citrus. Some areas in Indonesia was known as a producer of citrus, especially keprok, namely in Kalimantan, Sumatra, and Madura. The number keprok producing areas in Indonesia with different geographical conditions can influence the diversity of the keprok. This study aims to determine the genetic diversity of keprok (Citrus reticulata) from several regions in Indonesia based on morphometric characteristics and the influence of the environment. Keprok used from Situbondo, Jember, Madura, and other three areas from Bengkulu, Borneo, Pacitan were sampled from Balitjestro, Batu, East Java. The result shows two groups of keprok based morphometric proximity genetic basis, i.e. keprok group I of Pacitan, Situbondo, and Madura and group II of Bengkulu, Jember, and Kalimantan. Keywords: Citrus reticulata, Morphometric, Diversity, Environment

INTRODUCTION Keprok orange (Citrus reticulata) included in the family Rutaceae (Etebu & Nwauzoma, 2014) is a horticultural crop most traded in the world (Turner & Betty, 2013). This plant has an important role in the world market and within the country, either in the form of fresh or processed. The development of citrus cultivation in Indonesia has reached about 70-80% annually. Development opportunities citrus cultivation Indonesia at the ASEAN level is quite good considering Indonesia become Country with the largest harvested area and production for citrus in ASEAN (the Ministry of Agriculture, 2015). Almost the entire territory of the 33 provinces in Indonesia has developed a citrus. There are five areas that dominate the production of oranges (70%) of the overall production Indonesia, among others: North Sumatra, East Java, South Sumatra, Sulawesi Selatan, and West Kalimantan (Morey, 2007). Based on information from the Ministry of

Agriculture (2015) note that this type of local oranges are cultivated in various regions in Indonesia covers keprok (Citrus reticulata / Nobilis L.), tangerine (C. microcarpa L. and C.sinensis L.) consisting of Siam Pontianak, Siam Garut, Siam Lumajang, as well as a large citrus (C. maxima Herr.) consisting of orange Nambangan-Madison and Bali. Citrus crop diversity in Indonesia is influenced by various factors. These factors include the different geographic circumstances in various parts of Indonesia, local citrus harvest unbalance with needs of the domestic market throughout the year, causing the entry of imported oranges, and genetic factors that plants (Hanif, 2015). All these factors caused changes in the diversity of citrus spread in Indonesia. In addition, the identification of citrus plant taxonomy is still difficult because of the many crosses spontaneously and commercial (Turner, 2013).

Identification of the citrus crop diversity can basically be done in various ways. One way is to use morphometric analysis. Morphometric analysis is a way to assess the phenotypic variation that is due to biotic and abiotic factors. A morphometric method performed by using the size and the ratio of the size of an organism to measure variations of a biological specimen, and comparing the genetic variations, environmental and phenotype (Paris et.al, 2016). This method has also been applied in plants and plant organs for years (Cope, et al, 2012). Morphometric methods, in general, has been widely used to determine the kinship. METHODS Study Area The citrus sample used in this study was from six locations in Indonesia. These locations include Bengkulu, West Kalimantan, Pacitan, Jember, Situbondo,

of a particular species, the differentiation of the various species, to determine the variation of species, and for the identification of a species (Soladoye et al, 2010). Based on the foregoing, the research on "Keprok (Citrus reticulata) Assessment Based On Genetic Diversity Studies morphometric In Indonesia" needs to be implemented. The purpose of this study was to determine the genetic diversity of citrus fruits based morphometric and to explain the influence of the environment on the genetic diversity of citrus fruits based morphometric.

and Madura. Keprok Bengkulu, Pacitan, and West Kalimantan were used in this study were taken from the Balitjestro, Batu, East Java. Map location of the origin of citrus fruits can be seen in Figure 1.

2

1 6 3

5 4

Figure 1. Origin site of C. reticulata sampled (1) Bengkulu, (2) Kalimantan Barat, (3) Pacitan, (4) Jember, (5) Situbondo, (6) Madura

The study was conducted in OctoberDecember 2016. The samples of citrus fruit Bengkulu, Pacitan, and Kalimantan conducted at the Research Institute for Citrus and Subtropical Fruit (Balitjestro),

Batu, East Java. Keprok Jember, Situbondo, and Madura were taken from the area of origin of citrus fruits. Geographical conditions of the area of origin of citrus presented in Table 1.

Table 1. Geographically characteristics of C. reticulata No Region Elevation Altitude (mdpl) 1 2 3 4 5 6

Bengkulu Pacitan West Kalimantan Jember Situbondo Maduta

High Low Low Low Low Low

800-1000 700 < 500 100-500 250 200-500

Measurement Data Morphometric Citrus Fruits This study uses descriptive exploratory with quantitative and qualitative approaches. Characters measurement morphometric includes fruit width (LB), fruit length (PB) (Bercu, 2016), the amount of flesh (SDB), the width of flesh (A), the width rind (mesocarp / albedo) (B), broad beans (LBJ), and the number of seeds (SBJ). Part of morphometric measured citrus fruit is shown in Figure 2.

Air temperate (oC)

Rainfall (mm/year)

Fertility

19 22 22 23 24,7 28

2.500 1000 2.800 1.969 994 1.328

Thriving Clay sanding Lightly thriving Arid Arid Arid

a

c b

Figure 2. (a) Length, (b) width, (c) mesocarp / albedo

Abiotic factors data used in this study include altitude, temperature, and rainfall at each sampling locations keprok. Measurement morphometric performed using calipers and gauges while data from literature abiotic Ministry of Agriculture Agricultural Research and Development Agency, 2015. The results of morphometric measurements keprok in millimeters (mm). Data analysis Citrus fruit morphometric measurement results were tabulated and compiled using Microsoft Excel. Furthermore, the data used to determine the kinship keprok by using software PAST 3 with Principal Component Analysis (PCA) includes the analysis with dendrogram and Biplot analysis. PCA analysis with dendrogram with BrayCurtis similarity scale can determine kinship with Biplot keprok while analysis to determine the character that supports the grouping of keprok. RESULT AND DISCUSSION Comparison of morphometric characters in this study was determined by Principal Component Analysis (PCA) to see morphometric characters that have relevance to other characters. PCA aims to simplify the observed variables by

reducing its dimensions. This is done by eliminating the correlation between independent variables through the transformation of the independent variable origin to new variables that are not correlated at all. Based on the analysis of the PCA, obtained a major component that is able to retain most of the information which was measured using the total diversity by using a bit of the main components only. In this study,

the PCA is done by using biplot analysis and dendrogram analysis. Dendrogram Analysis Dendrogram analysis is a multivariate analysis that aims to classify the objects of the data based on similar characteristics examined its (Mattjik, 2011). The similarity of these characteristics is usually measured using the size of the proximity between objects that can be a similarity measure or unsimilarity. One technique of grouping is engineering hierarchy, that began the grouping of two or more objects with

similarities closest, and so on until it forms a kind of trees where there is a hierarchy (levels) which clearly between objects, from the most similar to least similar (Santoso, 2004). Based on the dendrogram analysis obtained measurement results morphometric (Citrus reticulata) were get from different regions (Figure 3), which showed that the index of similarity BrayCurtis, there are two clusters with similarity of 85% where cluster 1 consists of keprok from Pacitan, Situbondo, and Madura while the second cluster consists of Kalimantan, Bengkulu, and Jember.

Figure 3. Dendrogram analysis of Citrus reticulata (1,2,3: Bengkulu; 4,5,6: Pacitan; 7,8,9:Kalimantan; 10,11,12: Madura; 13,14,15: Situbondo; 16,17,18: Jember)

In cluster 1, showed that keprok Pacitan and Situbondo have 97% similarity. Both of this keprok have similar characteristics between the flesh of the fruit, altitude, and also rainfall. In addition, in cluster 2, between Kalimantan and Bengkulu has 93% similarity with the character of measurement similar to the albedo and altitude.

Biplot Analysis Grouping formed on dendrogram analysis can be seen in two dimensions using biplot analysis. Biplot analysis is used to describe the line (object) and columns (variables) that exist in the data matrix together in a low-dimensional graph (usually two or three) (Ariawan, et al., 2013). This depiction includes diversity and correlation between variables, as well as the proximity between

objects that will be able to identify the object grouping. Biplot analysis is a statistical technique that can descriptive multiple dimensions simultaneously serving a cluster object of observation and variables in a graph on a plane two dimension so

that the characteristics of the variable and the object of observation and the variables can be analyzed (Diyarti, 2003). A group of keprok in various areas based on biplot analysis in this study showed variations of different groups in various areas studied (Figure 4).

Figure 4. Biplot analysis of Citrus reticulata

Keprok from Madura and Situbondo has character temperatures were positively correlated. While the individual variation of keprok in Madura look bigger than the individual variation in the area of Situbondo. Madura and Situbondo are an area with an average temperature of 2428oC. The difference in temperature at the source of the keprok used in the study essentially influence the differences in skin color and flesh color areas that have differences in the temperature range even if the fruit of the same age (Figure 5), it is due to the presence of pigment in the skin and flesh that changes with changes in temperature. Iglesias (2007) states that changes chloroplasts to chromoplast led to a decline of chlorophyll and carotenoid pigments additions and changes the color of skin and flesh from green to orange. This happens because the ambient temperature is low, the availability of

carbohydrates, the hormone ethylene, causing greening occurrence. While the high environmental temperature, nitrogen availability, and hormone gibberellin cause regreening. Character heights in this study correlated inversely with the temperature factor, ie the higher the temperature of an area in the lower area. Discoloration of the skin and flesh of the fruit in each region in this study had a parallel correlation. It supported the statement Alquezar et al. (2008) that there is a correlation parallels between changes in skin color and flesh color keprok is caused by the difference in carotenoid pigments that are specific to the skin and fruit, on citrus fruits are ripe, the carotenoid content β, β-xanthophylls can reach 98% of total carotenoids in flavedo and albedo (Rodrigo in Alquezar et al., 2008).

Figure 5. (a,c) peel color and flesh of Keprok from Madura (280C), (b,d) peel color and flesh of Keprok from Bengkulu (190C). source: private documentation

Pacitan keprok characterized by mesocarp (albedo), altitude, fruit length and fruit width contrasting with Jember keprok. Jember keprok characterized by the amount of fruit flesh contrasting with Pacitan. In this study note that the most conservative character is the amount of flesh. Character fruit length, fruit width, and the amount of flesh on each region may be associated with nutritional imbalance citrus that affects the size of a keprok. Kays research results (1999) states that the lack of intake of nitrogen, potassium, zinc, and iron in citrus caused the small size of keprok. Bengkulu keprok seeds are characterized by wide characters. This characterization can be seen from the length of the vector of the character that is longer than the vector formed by other groups. In addition to differences in seed width characters, different characters skin texture and grain of each region in the observation were also observed. This occurs because the character differences

citrus fruit maturation process that led to the decline in the production of ethylene, sensitivity, and respiration which gradually led to changes in skin texture and grapefruit (Iglesias, 2007). Environmental factors that affect the quality of the next keprok soil as a growing medium and provider of plant nutrients. Kalimantan and Bengkulu have the character of rainfall with rainfall 25002800 with fertile soil conditions. Iglesias (2007) explains that a good ground for the development of citrus is soil water content and nutrients is always fulfilled all the time with the availability of abundant humus. With these findings, it can be seen various characters from different areas of keprok in Indonesia which can then be used for the development of local keprok in Indonesia. CONCLUSION Based on morphometric measurements can be seen that there is a genetic diversity of keprok (Citrus reticulata) from various regions in Indonesia with biplot analysis and dendrogram analysis. In this study, we obtained two clusters with 85% similarity index where the first cluster consists of Pacitan, Situbondo, and Madura while the second cluster consists of Borneo, Madura, and Bengkulu. Environmental influences that cause the genetic diversity of citrus fruits in various areas including air temperature, altitude, and rainfall.

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