Identification and utilization of informative EST-SSR markers for genetic purity testing of coconut hybrids

Coconut palms are categorized into two forms, viz ., ‘talls’ and ‘dwarfs’ which are being utilized to produce hybrids through the process of inter-varietal or intra-varietal crosses. Hybrid coconut seedlings are generally identified and selected based on morphological traits by plant breeders, which is quite difficult and requires expertise. Even minor errors in identification may adversely affect breeding programs in coconut, which is spread over many decades. In this study, we have utilized thirty EST-SSR markers, derived from existing coconut leaf transcriptome data, for screening polymorphism between eighteen coconut parental lines. The polymorphic primers capable of differentiating the parental palms were then utilized successfully for assessment of purity of hybrids derived from these parents. Thus, the current study demonstrates the utility of EST-SSR markers in determining the genetic purity of hybrids in coconut.


Introduction
Generating and testing hybrid varieties of coconut are currently a major field of research in many countries with the objectives of increasing yield of nuts, oil content and also tolerance to abiotic and biotic stresses. There are many hybrids being developed and researched upon to cater to the climate, soil conditions and needs of each individual location. The two major varieties of coconut palms are 'talls' and 'dwarfs' (Narayana and John, 1949) with dwarfs (even though fewer than 5 per cent of the world coconut population) being in higher demand for genetic studies due to their quick emission of inflorescence and early germination (Bourdeix et al., 2008). Talls take a longer time to flower (~6 years) but live much longer (~100 years) when compared to dwarfs (~60 years). Talls (var. typica) and dwarfs (var. nana) also differ in their breeding behaviour with the talls being allogamous (cross-fertilizing) and dwarfs being autogamous (self-fertilizing) (Arunachalam and Rajesh, 2008). Inter-varietal crosses between a dwarf male parent with a tall female parent (T x D) as well as tall male parent with a dwarf female parent (D x T) and intra-varietal crosses (T x T and D x D) are methodologies followed for the development of hybrids (Arunachalam and Rajesh, 2008).
Hybrid varieties that provide better resistance to various diseases and enhanced yield have been successfully developed in coconut. Kalpa Sankara, a hybrid resistant to root (wilt) disease has been derived by crossing Chowghat Green Dwarf (CGD) and West Coast Tall (WCT) (Nair et al., 1996). Hybrids developed between Vanuatu Tall (VTT) and Rennell Island Tall (RIT) have been reported to possess better resistance towards coconut foliar decay disease, which is endemic to Vanuatu in the South Pacific (Labouisse et al., 2011). Recently, Kalpa Samrudhi, a cross between Malayan Yellow Dwarf (MYD) and WCT, has been developed which provides a much higher nut yield, copra content as well as oil output when compared to its parents (Jerard et al., 2015).
Even though the development of hybrids has contributed significantly for the increased productivity of coconut, the timely production and ample supply of hybrid seedlings, which are genetically pure, to the farmers is the key factor determining the success of hybrid technology in this crop. Morphological descriptors currently used for seed purity assessment in coconut include petiole colour, days taken for germination, seedling vigour and higher collar girth . Although hybrid purity assessments based on morphology are extensively taken up, they are often affected by environment; in addition, requirement for time and resources for such an assessment is tremendous. Selection by petiole colour, which is generally utilized marker to select hybrid seedlings in nurseries, is authentic only if parents used are homozygous for red, yellow or green petiole . Some of the drawbacks of utilizing morphological traits for genetic purity testing of coconut hybrids are that they are cumbersome and subjected to environmental influences. Furthermore, many of the varieties and hybrids are phenotypically less distinct resulting in difficulty in accurate morphological evaluation.
DNA-based markers, because of their rapidity in estimation, ease of use and cost-effectiveness, have become indispensable for use in variety identification, diversity and linkage-mapping studies. Among the common molecular markers, SSR (simple sequence repeat) are generally preferred due to their abundance, co-dominant inheritance, presence over the whole genome, higher reproducibility, multi-allelic nature, hyperpolymorphism and high transferability across species/genera (Varshney et al., 2005). SSRs have been developed and utilized in coconut for genetic diversity studies (Rivera et al., 1999;Perera et al., 2000;Meerow et al., 2003;Rajesh et al., 2008 a,b).
The use of SSRs for the authentication/ differentiation of hybrids is a widely accepted procedure in many crops (Antonova et al., 2006;Sundaram et al., 2008;Naresh et al., 2009) and has been previously used in coconut too. SSR-based identification of Kalpa Sankara hybrids has been reported by Rajesh et al. (2012). In a cross between Sri Lanka Yellow Dwarf (SLYD) and Sri Lanka Tall (SLT), progenies with yellow colour were removed as selfed progenies based on visual observations (since SLYD petioles are yellow in colour), but SSR analysis later on proved that at least 11 per cent of the discarded yellow seedlings were actually hybrids (Perera, 2010).
Although genomic SSR markers have been utilized for genetic purity studies in plants traditionally, their high cost and time involved in this process have restricted their utilization. The number of SSR markers available in coconut is limited. With the exponential accumulation of data in EST databases, EST-derived SSRs (EST-SSRs) are being utilized these days for various molecular studies. EST-SSRs are also advantageous in that these SSRs might be from gene sequences that are functional, ESTs being located in the coding region of a gene. EST-SSR markers have been utilized earlier in genetic purity assessment of annual crops like safflower (Naresh et al., 2009) and castor (Pranavi et al., 2011;Gouri Shankar et al., 2013), but there are no such reports in perennial tree crops. In this study, we aim to identify novel markers that could decisively validate different coconut hybrids through the use of EST-SSRs.

Plant materials
The plant materials used for hybrid authentication using molecular markers consisted of tall and dwarf parents and their offsprings collected from the ICAR-CPCRI Farm, Kasaragod, Kerala, India. A total of 18 parental lines and 103 progenies were used for the study (Table 1).

DNA isolation
DNA was extracted from spindle leaves of parental palms and their progenies following the modified method of Rajesh et al. (2013). To check the DNA purity, it was run in 0.8 per cent agarose gel, stained with ethidium bromide and visualized in a gel documentation system.

Assessment of parental polymorphism using EST-SSR markers
Initially, all the parental palms used in hybrid seed production were screened using the 30 novel EST-SSR primers (Table 2), which were mined from leaf transcriptome data of Chowghat Green Dwarf cultivar (Rajesh et al., 2015) as per the procedure reported in Preethi et al. (2014 The amplicons were separated on 3 per cent agarose gel and photographed on a digital gel documentation and image analysis system after staining with ethidium bromide. Polymorphic primers capable of differentiating the parental palms were then utilized for hybrid purity assessment studies.

Results and discussion
Thirty novel EST-SSR primers were used to screen polymorphism among eighteen parental lines. Those primers capable of detecting polymorphism among the parental palms in a particular cross were selected (Table 1). Confirmation of the results was achieved through repeated testing. For all these markers, the alleles present in the parents were of different sizes and both parental alleles were detected in the hybrids, EST-SSRs being co-dominant markers.
The hybridity of 14 F 1 plants derived from CGD x WCT were tested through the use of CnKGDEST126 and CnKGDEST117 primers, which displayed polymorphism between the parental lines. Out of 14 F 1 progenies, a total of 11 were confirmed to be true hybrids while three were deduced to be selfed or off types using CnKGDEST117 primers (Fig. A). Out of a total of six F1 progenies tested from a cross between MYD and TPT, two offsprings were deduced to be offtypes and the other four as true hybrids using the primer CnKGDEST126 (Fig. 1B). In a cross between COD x WCT, two pure hybrids and two selfed F1 progenies were detected using the primer CnKGDEST126 (Fig. 1C). F 1 progenies of the  crosses GBGD x PHOT (Fig. 1D), GBGD x LCOT (Fig. 1E) and LCOT x CCNT (Fig. 1F) were all confirmed to be true hybrids when checked with primer CnKGDEST130. Two selfed F 1 progenies were detected out of a total of eight probable hybrids in GBGD x FJT cross using the primer CnKGDEST130 (Fig. 1G). The primer CnKGDEST117 could aid in identifying one offtype from among ten F 1 progenies with the others confirmed as true hybrids in WCT x COD (Fig. 1H). LCT x COD cross revealed three offtypes and seven pure hybrids using the primer CnKGDEST117 (Fig. 1I). Progenies of crosses between COD x CCNT ( Fig. 2A), CRD x CCNT (Fig. 2B) and MYD x CCNT (Fig. 2C) showed true hybrids in all the lanes of the F 1 progenies used for testing with the primer CnKGDEST117. In CGD x CCNT (Fig. 2D) and MYD x SNRT (Fig. 2E), out of four progenies, two pure hybrids and two offtypes were identified using the primer CnKGDEST117. The same primer, CnKGDEST117, was used for the assessment of hybrid purity in MOD x SNRT (Fig. 2F) and MGD x CCNT (Fig. 2G) which showed that out of four F1 progenies, only one was a true hybrid with the others being offtypes. Assessment of progenies of COD x SNRT with the primer CnKGDEST117 revealed that there was an offtype among the four F 1 progenies (Fig. 2H). In the cross between GBGD and SNRT, when tested with the primer CnKGDEST117, a total of three selfed progenies were detected among the four F 1 progenies tested (Fig. 2I). Identifying hybrids in an early stage is of prime importance for breeders; using morphological markers for this purpose is an unreliable method to identify a hybrid mainly due to the fact that the morphological traits are limited, display dominant expression thus reducing statistical capability, are influenced by the environment and they might change according to the development phase of the plant (Kumar et al., 2009). Despite these disadvantages, morphological traits like petiole colour, days taken for germination, seedling vigour in terms of leaf production and higher collar girth over a specific duration are still utilized for identification of hybrids in coconut . With reference to a perennial crop like coconut, it is also of utmost importance that proper hybrid identification be done at an early stage due to the long time that it takes to grow, flower and bear fruit. Commercial hybrids are hugely popular in coconut with both public and private sectors being actively invovled in the development of hybrids. This necessitates strict quality control with respect to monitoring seed genetic purity at various production stages for the success of hybrid technology among stakeholders. Presently, EST-SSRs have emerged as an important category of molecular markers due to their ease of availability, their hyper variability nature, their aptness for high throughput analysis, their high rate of polymorphism and crosstransferability in comparison to other available markers (Poczai et al., 2013). EST-derived SSR markers possess great potential for use in marker assisted selection (MAS), for developing high yielding varieties, molecular mapping and quantitative trait loci (QTL) analysis (Varshney et al., 2005). In coconut, they are few reports on identification of EST-SSR markers in coconut (Xiao et al., 2013;Xia et al., 2014). However, the present study is the first report of hybrid authentication studies in coconut utilizing EST-SSR markers. Furthermore, the markers identified through this study could be utilized in assessments of purity of hybrid seedlings and identification and subsequent elimination of selfed progenies from seedling nurseries, resulting in considerable economy with respect to time and resources.