Metoda Parentage analysis Analisis Kebenaran Tetua Dan Keragaman Genetik Populasi Kelapa Sawit (Elaeis Guineensis Jacq) Asal Kamerun Menggunakan Marka Ssr (Simple Sequence Repeat)
14 Beberapa penulis mendefinisikan mikrosatelit sebagai pengulangan dari 2
sampai 8 bp, atau pengulangan dari1sampai 6 bp atau bahkan 1 sampai 5 bp. Mikrosatelit dihasilkanberasal dari daerah yang merupakan ragam sederhana dari
motif berulang sekuen DNA yang telah terwakili. Mekanisme mutasi dominan pada mikrosatelit merupakan fenomena tergelincirnya proses perpasangan untai
DNA. Proses terjadi ketika sintesa DNA dalam array mikrosatelit mengalami slipped strand mispairing, yang berakibat pada bertambah atau berkurang satu,
atau lebih sekuens unit berulang, bergantung pada apakah yang keluar adalah dari rantai DNA loop yang baru disintesis atau rantai template-nya.
Reaksi PCR untuk SSR dijalankan dengan primer forward dan reverse yang menempel pada ujung 5 `dan 3` dari masing-masing template DNA.
Fragmen PCR biasanya dipisahkan pada gel poliakrilamida yang dikombinasikan dengan pewarnaan AgNO3, autoradiografi atau sistem deteksi fluoresens. Agarose
gel biasanya 3 dengan EtBr juga dapat digunakan ketika perbedaan ukuran alel antara sampel lebih besar dari 10 bp. Namun penggunaan SSR dibatasi oleh
lambatnya data yang dihasilkan dan terbataskan pasangan marka serta dibutuhkannya biaya yang tinggi untuk melakukan pengembangan marka. Namun
demikian, perbaikan dibidang teknologi molekular terus dikembangkan seperti adanya metoda multiplex PCR untuk amplifikasi DNA.
Multipleks PCR yaitu metoda PCR yang menggunakan beberapa marka dalam satu reaksi. Metoda ini bertujuan meningkatkan jumlah informasi dari yang
diperoleh per reaksi, dan untuk mengurangi pemakaian bahan dan biaya tenaga kerja. Kombinasi penggunaan bahan yang berbasis fluorescen berpendar mampu
secara otomasis mendeteksi DNA dan ukuran fragment sehingga mampu mempercepat proses, hasil yang lebih akurat dan perolehan data dengan biaya
yang lebih murah Mitchell et al.1997. Namun pengembangan multiplex PCR pada tanaman seringkali mengalami kesulitan karena besarnya ukuran genom dan
polyploidy. Namun saat ini telah dikembangkan metoda baru yang dikenal dengan metoda “Multiplex ready PCR” yang dikembangkan oleh Hayden et al. 2008
yang menggabungkan teknologi berbasis fluorescens dengan penggunaan primer M-13 . Metoda ini menggunakan deteksi SSR yang berpendar dan analiser
fragmen DNA otomatis untuk mengukur ukuran alel yang merupakan metoda analisis genotipe SSR yang paling cepat dan akurat Ziegle et al. 1992. Prosedur
ini berdasarkan pemisahan SSR yang berlabel oleh gel elektroforesis. Keuntungkan dari SSR yang berpendar adalah SSR dapat dipisahkan dalam gel
kapiler berdasarkan ukuran fragmennya tanpa tumpang tindih satu sama lain. Pada kasus saat SSR memiliki ukuran alel yang sama akan terjadi tumpang tindih,
maka pemisahan dapat dilakukan dengan melabel produk SSR dengan dye berpendar yang memiliki emisi panjang gelombang yang berbeda. Sehingga
keuntungan dari multiplex ready PCR yaitu kemampuannya menggunakan banyak marka dalam satu reaksi dan dapat dibedakan secara akurat melalui pendaran yang
memiliki panjang gelombang yang berbeda.
15
3 GENETIC DIVERSITY ANALYSIS BASED ON
SSRMARKERS OF TXT CROSS AND DURA SELF OIL PALM
Elaeis guineensis Jacq. PARENTAL POPULATIONS ORIGINATEDFROM CAMEROON
Abstract
The success of oil palm breeding activities depends on the availability of diverse germplasm parental populations, especially between the Dura and the
Pisifera types. Therefore, evaluation of the potential Dura and Pisifera genetic diversity is neccessary. The objectives of this research are to analyze the T x T
crosses and Dura self oil palm Elaeis guineensis Jacq. parental populations genetic diversity and evaluate their potential value for creating hybrid progenies in
oil palm breeding program. A total of 148 individuals from three T x T crosses and three Dura self populations were evaluated. Genotyping was conducted using
16 SSR marker loci. DNA isolation and PCR amplifications for all of the SSR loci were conducted at the Biotechnology Laboratory, PT. Astra Agro Lestari Tbk.
The genotype data were analyzed using software for population genetic and genetic diversity analysis. Results of the analysis indicated the T x T male parent
populations were more diverse than the Dura self. The 16 evaluated microsatellite markers are either highly or moderately polymorphic based on their PIC values.
Hence, they could be used for further analysis for larger number of Astra Agro Lestari’s T x T and Dura Self population samples. The results of clustering and
PCA analysis showed the all populations are grouped into three groups consisting of 1 B02, 2 B57, and 3 the rest of populations B01 and the three Dura Self
populations. In the meantime, the third group is further divided into five subgroups, consisting of sub-group 1: the B01, and sub-group two to five
comprised of a mix individuals from members of at least two different Dura self populations. All the studied T x T populations could potentially be used as
improved male parents for producing future oil palm hybrid varieties. The T x T populations has a wider genetic distance than that of the D Self populations.
Moreover, member of Dura self oil palm population should not be grouped based on the family but it should be based on results of clustering analysis. The reported
data should be beneficial for aiding future oil palm breeding in Indonesia.
Keywords:
Afican oil palm, Dura, Pisifera type oil palm, Population structure,
Simple Sequence Repeat 3.1 Introduction
Palm oil is the major vegetable oil producing crop in the world. Palm oil supplies at least 32 to the total world vegetable oil demand while the rest is
from other vegetable oils, such as: soybean 29 , rapeseed 16 , sunflower 8 , nut 3 , cotton 3 , and minor vegetable oils 9 USDA, 2011. The
demand outlook for palm oil is most probably still increasing in the years to come. It is predicted that In 2050, Oil palm can fullfil all demand of world vegetable oil
to reach 240 Million tons Corley,. 2009; Barcelos et al., 2015.
16 The 2014 data from Directorate General of Plantations, Republic of
Indonesian Ditjenbun, 2014 indicated there were rapid increases in oil palm plantation areas in Indonesia. The total areas of oil palm plantations in 2004 were
only 5,284,723 ha while in 2012 were 10,956,231 ha, respectively. However, the Indonesian government through Presidential Instruction, Republic of Indonesia
No 102011, has implemented a moratorium on forest to plantation land conversion since 2011 Ditjetbun, 2013. Moreover, availability of arable land
suitable for growing oil palm has also become a limiting factor for the opening of new oil palm plantation Danielsen et al., 2009. Therefore, meeting the increasing
future demand for palm oil has to come from more productive planting materials while those better yielding planting materials should come from effective oil palm
breeding programs Barcelos et al., 2015.
One of the critical success factors in generating more productive and superior quality oil palm planting material through breeding program is the
availability of diverse oil palm breeding materials. Diverse oil palm breeding materials provide oil palm breeders raw materialsfor developing new and
improved cultivars with desirable characteristics Govindaraj et al., 2015.Therefore, understanding genetic diversity and population structure of the
breeding materials is an important aspect of oil palm breedingThongthawee et al., 2010; Barcelos et al., 2015. Breeding for superior oil palm variety is generally
conducted through reciprocal recurrent selection RRS method Purba et al., 2000. In one generation of RRS, potential female Dura type and male Pisifera
type parental lines are cross hybridized to generate D x P progenies. The D x P progenies are evaluated for their superior characters in the field. To generate
improved parental lines, the Dura types are self-pollinated and the Pisifera types are generated through crossing of two Tenera types T x T crossesNCHU et
al.,2015. The cycle is continually repeated to eventually identify improved parental lines capable of producing the new superior D x P cultivars.
In RRS method, the existence of illegitimate progeny can happen in any stage of the controlled crosses in the oil palm breeding activities, from the initial
stage of selecting and labeling parents, to the final stage of field evaluation trials.
Many factors affect the percentage of illegitimacy or contamination in oil palm breeding such as the biology of the plant flower i.e. the existence of a few
hermaphrodite flowers, human errors during pollen collection and damage of the
pollination bag for covering the flowers because of animals and the environment
factors allowing insect pollinators to cross pollinate the female flowers Hama-Ali et al., 2015. Improved parental lines in RSS oil palm breeding methods should
not contains illegitimate progenies; therefore, identification of illegitimacy in the improved parental line populations is important. Moreover, new superior variety
should only be obtained from hybridization of genetically diverse parental populations. Therefore, generating basic information about genetic diversity of
parental populations is also necessary to identify the parental line combinations having the largest potential of hybrid vigor Arias et al., 2012.
Phenotype variation and molecular markers have been used to evaluate genetic diversity of perennial crops, such as: cacao, coconuts, and oil palm
Moose Mumm, 2008; Tornincasa et al., 2010; Zulhermana et al., 2010; Nhcu et al., 2012; Solin et al., 2014; Ajijah et al., 2015; Maskromo et al., 2015. In oil
palm, genetic diversity has been evaluated using RAPD, ISSR, AFLP, and SSR