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Abstract. Pesik A, Efendi D, Novarianto H, Dinarti D, Sudarsono S. 2017. Development of SNAP markers based on nucleotide variability of WRKY genes in coconut and their validation using multiplex . Development of molecular markers benefits coconut breeding program. The availability of DNA sequences for coconuts opens the new possibility of developing molecular markers such as single nucleotide amplified polymorphism (SNAP). The study aimed to evaluate nucleotide sequence diversities of WRKY gene in the GenBank database, design gene specific primers for generating WRKY gene specific SNAP markers, optimize multiplex PCR technique and validate SNAP marker effectiveness for evaluating Kopyor coconut germplasm. Based on 35 sequences data of coconut WRKY genes that are available in the GenBank database, we have identified eight informative SNPs and used them to generate SNP-specific primers. We have designed sixteen primer pairs and validated them using singleplex PCR. Subsequently, we optimized the Ta and primer concentrations either for duplex or triplex PCR. Duplex PCR using two sets of primer pairs was more reliable for genotyping Kopyor coconut germplasm than triplex PCR. We have successfully demonstrated the duplex PCR for genotyping Banten Tall, Jember Tall, Kalianda Tall, Pati Dwarf and Sumenep Tall Kopyor coconuts. Therefore, one may use the developed SNAP markers as a simple alternative of co-dominant markers for genetic analysis of coconuts. One may use the developed SNAP markers to investigate the possible association between markers and Kopyor endosperm and other relevant phenotypes in coconuts.

Kopyor coconut is a naturally-occurring mutant having fluffy solid endosperm in stead of the normal one. Similar type of coconut has been found in several other Asian countries, with their distinct local names, such as Macapuno (Philippines), Makhrao Kathi (Thailand), Dikiri Pol (Sri Lanka), Thairu Thengai (India). It is a delicacy coconut to Indonesian and sells as much as ten times higher than normal coconut. In nature, three types of kopyor coconut exist in Indonesia: kopyor tall, kopyor dwarf, and kopyor hybrid. There are three kopyor dwarf varieties (‘Pati Kopyor Green Dwarf’, ‘Kopyor Yellow Dwarf’ and ‘Kopyor Brown Dwarf’) officially released, and one registered tall variety (‘Puan Kalianda Kopyor Tall’). In general, kopyor fruit yield under natural conditions is only < 25% of the total harvested fruits for both the tall and the dwarf types. Traditionally, Indonesian farmers harvest kopyor fruits at ten  months after pollination while normal fruits at 11 months. They use the harvested normal fruits for propagation; but cannot guarantee whether or not they would produce korpyor fruit. Adoption of kopyor seedling production through embryo culture has been done. However, the seedling production is slow, while the price is very expensive, thus unaffordable to common farmers. Indonesian Palm Research Institute and Bogor Agricultural University have collaborated to develop alternative approaches to increase kopyor fruit production through the production of seedling that would ensure to produce korpyor fruit through control pollination. Initiated since 2010, the activities successfully overcome uncertainty in kopyor seedling production. Moreover, hybridization among local superior coconut varieties and known kopyor one have been done to broaden genetic background of kopyor trait and to develop breeding population for new kopyor varieties in the future. Overview and update of research progress on korpyor in Indonesia are presented in this paper.

<p>ABSTRAK<br />Kelapa Genjah kopyor asal Pati, Jawa Tengah merupakan<br />kekayaaan hayati asli Indonesia dengan nilai ekonomi tinggi. Informasi<br />keragaman genetik kelapa kopyor masih terbatas. Data keragaman<br />morfologi dan genetik diperlukan dalam program pemuliaan kelapa<br />kopyor. Penelitian ini mempelajari keragaman tiga varietas kelapa genjah<br />kopyor asal Pati yang telah dilepas berdasarkan karakter morfologi,<br />kuantitas endosperma, dan keragaman alel marka SSR. Penelitian<br />dilakukan di Pati dan di Laboratorium Plant Molecular Biology,<br />Departemen Agronomi dan Hortikultura, IPB. Evaluasi dilakukan terhadap<br />tiga populasi kelapa Genjah kopyor (hijau, coklat, dan kuning) dengan 30<br />tanaman sampel untuk setiap populasi. Rataan data morfologi digunakan<br />untuk menyusun dendogram. Kuantitas endosperma diamati pada satu<br />buah kelapa kopyor per tanaman yang dievaluasi. Karakteristik<br />endosperma dikelompokkan sesuai kategori yang telah ditetapkan. Untuk<br />setiap populasi, analisis marka dengan lima pasang primer SSR dilakukan<br />pada 10 tanaman sampel. Data yang didapat digunakan untuk menentukan<br />keragaman genetik kelapa Genjah kopyor asal Pati. Hasil pengamatan<br />menunjukkan keragaman morfologis dan alel SSR antar tanaman dalam<br />varietasnya (keragaman intra-varietas) rendah. Sebaliknya, keragaman<br />morfologis dan alel SSR antar varietasnya tinggi. Kuantitas endosperma<br />kelapa Genjah kopyor asal Pati bervariasi antara skor 1–6. Keragaman<br />genetik yang rendah dalam varietas dan tinggi antar ketiga varietas (coklat,<br />hijau, dan kuning) memperkuat pelepasan ketiganya sebagai varietas lokal.<br />Selain itu, keragaman genetik antar tanaman dalam varietas yang rendah<br />mendukung penggunaan ketiga varietas lokal sebagai tetua dalam program<br />perakitan varietas kelapa kopyor unggul baru. Tetua yang dipilih dapat<br />diseleksi intra-varietas berdasarkan persentase buah kopyor per tandan<br />dan skor kuantitas endosperma yang tinggi.<br />Kata kunci: Keragaman morfologis, keragaman intra dan antar varietas,<br />kuantitas endosperma</p><p>ABSTRACT<br />Kopyor dwarf coconuts are mutants from Pati, Central Java having<br />high economic values. However, morphological and genetic diversities of<br />this coconut were still limited. Morphological and genetic diversity data<br />are needed for breeding program. The research objectives were to evaluate<br />intra and inter-specific diversity based on morphology, endosperm<br />quantity, and SSR alleles. Field evaluations were conducted in Pati while<br />laboratory activities were at Plant Molecular Biology Laboratory,<br />Department of Agronomy and Horticulture, IPB. Three populations of<br />kopyor dwarf varieties (brown, green, and yellow) were evaluated. Thirty<br />trees were sampled for each population. The average of morphological<br />data were used to construct cluster analysis. Endosperm quantity was<br />scored (0 – 9) based on a single nut sample. Ten palms were analyzed<br />using five SSR loci for each population and used to determine genetic<br />diversity of populations. Results of observations indicated intra-variety<br />morphological and SSR allele variations among kopyor dwarf was low.<br />However, inter-variety variations were high. The endosperm quantity<br />scores among kopyor dwarf coconut ranged from 1–6. The low intra-<br />variety and high inter-variety variations among the three kopyor dwarf<br />coconut supported their release as different local varieties. Moreover, the<br />low intra-variety phenotypic and genotypic diversities among kopyor<br />brown, green, and yellow dwarf coconut support their use as parents for<br />new and superior kopyor coconut variety development in the future. For<br />such purpose, however, it is necessary to conduct intra-variety selection to<br />identify desirable parents based on high kopyor fruit percentage per bunch<br />and for high kopyor endosperm quantity.<br />Key words: Morphological diversity, intra and inter variety diversities,<br />quantity, endosperm</p>

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