Identification of true to type and open pollinated progenies of polyembryonic Mangifera indica cv. Harumanis using microsatellite markers

The study was conducted on a polyembryonic mango, Harumanis which contains more than one embryos including one zygotic and single or more number of nucellar ones. In this paper, we used microsatellite markers to identify whether the seedlings of Harumanis were zygotic or nucellar. A total of 95 progenies of Harumanis were evaluated using 13 polymorphic microsatellite markers. The genetic profiling revealed that a total of 14 Harumanis progenies were zygotic or open pollinated seedling as their genetic profile was different with Harumanis at least at one loci. Nevertheless, 76 Harumanis progenies were true to type or nucellar seedlings as their genetic profile was similar to Harumanis. The remaining five progenies could not be identified with the call rates of the genotypic data. Identifying true to type or nucellar seedling is useful for nursery growers to determine the true to type Harumanis progenies from the seed. Meanwhile, the open pollinated seedling or zygotic seedlings are preferred by breeders as they are considered as a new variety which increases the mango genetic variability. Key word: Mango, Harumanis nucellar, zygotic, microsatellite Introduction Harumanis is considered the “King of Mangoes,” gaining popularity in Malaysia because of its deliciousness, and sweet and aromatic fragrance. Harumanis is polyemroyonic thus its seed contains more than one embryo, which may be both zygotic and nucellar or all nucellar embryos (Shukla et al., 2004; Degani et al., 1993). In mangoes, this trait is genetically controlled by a single dominant gene while the cultivar and environmental conditions influence the number of seedlings produced from a seed (Aron et al. 1998; AndradeRodríguez et al., 2005). The nucellar tissue that covers the embryo sac forms the nucellar embryos and produces genetically identical seedlings to its parent plant (Aron et al., 1998). These seedlings from nucellar embryos are true to type as their mother and are preferred by nursery growers to produce rootstocks because using them results in a more even orchard (Rao et al., 2008). On the other hand, fertilization either by self or cross-pollination forms zygotic embryo. This type of embryo will produce new open-pollinated progenies imperative for the development of new mango varieties. Unfortunately, based on morphological criteria, it is difficult or not possible to identify whether the seedlings are derived from nucellar or zygotic embryos (Desai, 2004), making molecular markers imperative for identification purpose. In general, the most vigorous seedling from each seed is used to produce rootstocks. Unfortunately, uneven orchards may occur because the nucellar seedling is not always the most vigorous (Rocha et al., 2014) which lead to impractical identification of nucellar seedlings using morphological characteristics. To differentiate zygotic and nucellar embryos, researchers used genetic markers, including isoenzymes (Degani et al., 1993), RAPD (Ochoa et al., 2012), and ISSR (Rocha et al., 2014). To our knowledge, no reports were published using microsatellite markers for identifying nucellar and zygotic types in mango seedlings. Hence, this is the first report that used microsatellite markers for nucellar and zygotic identification. Microsatellite markers are extensively used in plant genomic studies because of their highly polymorphic, abundant and multi-allelic nature, simple analytical procedure, and transferability across genotypes (Vieira et al., 2016). Therefore, they are useful to increase the efficiency of classification and identification of mango accessions, giving necessary information for future biodiversity conservation and gene bank management. Materials and methods Genomic DNA Extraction: Young leaves from 95 Harumanis progenies and their respective mother plants were collected to extract the genomic DNA. All samples were obtained from the mango plot located at the Malaysian Agriculture Research and Development Institute (MARDI), Sintok, Malaysia (6° 28’ 53’’N, 100 ° 29’ 00’’E). We collected the leaves and stored them with silica gel in an airtight plastic bag for air drying. The leaves were punched into a 96-well plate containing stainless steel beads (2.3 mm diameter) to obtain small fragments and were immediately frozen at –80°C for a minimum of one night. The frozen tissue was ground using Tissue Lyser (Qiagen, Germany). We immediately added the extraction buffer (2 % CTAB, pH8 of 100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl, 0.05 % β-mercaptoethanol) after the tissues were ground. We extracted the total genomic DNA following the protocol by Mace et al. Journal Appl Journal of Applied Horticulture ISSN: 0972-1045