Diversity and population structure analysis in coconut (Cocos nucifera L) using molcular markers
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Date
2012
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College of Horticulture, Vellanikkara
Abstract
Assessment of genetic diversity and thus understanding the population structure is the mainstay for any crop improvement programmes. With the advent of molecular markers and suitable software for statistical analysis, this became a routine work in plant breeding. The technique is especially helpful in breeding of perennial heterozygous crops such as coconut. Hence the present study on ‘Diversity and population structure analysis in coconut (Cocos nucifera L.) using molecular markers’ was undertaken with the objective to estimate the genetic diversity among the selected cultivars using three marker systems viz., Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR). The experiment material included six tall cultivars (Laccadive Ordinary, Tiptur Tall, Komadan, Kuttiadi, Malappuram, Kasaragode), three dwarf cultivars (Chowghat Green Dwarf, Chowghat Orange Dwarf and Malayan Yellow Dwarf) and one intermediate (Gangabondam). Five palms from each cultivar were used for the study. The DNA isolated from each cultivar was amplified using selected primers for RAPD, ISSR and SSR. The DNA isolation procedure for coconut was standardized as it was difficult for isolating DNA from coconut due to the high polysaccharide and polyphenol content of its leaves. Several DNA isolation procedures were tried and among them, the protocol reported by Porebski et al. (1997) with suitable modifications yielded good quality DNA in sufficient quantity. The ten selected random primers for RAPD (after screening 35) generated 65 markers with an average of 40 percent polymorphism. The polymorphism information content (PIC) of selected primers ranged from 0.67 (RN4) to 0.89 (RN5, RN8). For ISSR assay, eleven primers were selected (after screening 41) which generated ninety amplicons. The average polymorphism per cent was 44.44 and the PIC value of selected primers ranged from 0.78 (UBC 855, UBCS2) to 0.90 (UBC 834). Ten sets of primers selected for the SSR analysis (after screening forty three) produced a total of 13 amplicons with 30.76 per cent polymorphism. Altogether, there were 168 markers produced by RAPD, ISSR and SSR assay of the 10 genotypes of which 70 bands (41.66%) were polymorphic with an average of 2.29 polymorphic markers per primer. The presence or absence of data was entered into a binary data matrix and was used for calculating the similarity coefficient using Dice coefficient (Nei and Li, 1979) using software DARwin (Version5.0) and Jaccard’s coefficient (Jaccard, 1908) using software NTSYS-PC (Rohlf, 1993). Cluster analysis was done using the UPGMA method and dendrograms (using DARwin and NTSYS) were constructed by neighbor joining. The genetic diversity values were calculated using the software, DARwin as the dendrogram based on DARwin showed more accuracy according to morphological characters. The dendrogram generated (using DARwin) from sixty five RAPD markers, grouped the cultivars into three major clusters. Cluster I consisted of four tall cultivars namely Kuttiadi, Tiptur Tall, Malappuram and Kasaragode. Cluster II included three dwarf cultivars viz., Chowghat Green Dwarf, Chowghat Orange Dwarf, Malayan Yellow Dwarf and the intermediate type, Gangabondam. Cluster III included the two tall cultivars, Laccadive Ordinary and Komadan. The dendrogram (using DARwin) based on ISSR markers grouped the cultivars into three major clusters which was slightly different from that of RAPD. Cluster I comprised of four tall cultivars viz., Kuttiadi, Tiptur Tall, Malappuram but instead of cv. Kasaragode in RAPD dendrogram, Laccadive Ordinary was included in first cluster of ISSR. The tall cv Kasragode was separated out as the third cluster. Cluster II is similar to that in RAPD except for the presence of one tall cultivar, Komadan. Based on all the 168 markers, dendrograms were constructed using DARwin and NTSYS software which grouped the cultivars into three clusters (Cluster I and III for Talls and Cluster II for Dwarfs and intermediate) by the former and into two (ClusterI for talls, Cluster II- Dwarfs and intermediate) by the latter. In the present study it was found that combination of three markers produced more accurate classification of genotypes. Based on 168 markers from three marker systems the overall genetic diversity observed among the three morphological groups viz.,Talls, Dwarfs and intermediate type palms in the selected population of coconut seed farm at Vellanikkara is comparatively less (GD=0.13). The highest genetic diversity was between Talls and Dwarfs (GD=0.14). The tall population consisting of six tall cultivars among which four are ecotypes of WCT showed the lowest diversity (GD-0.06). Within Talls, Kuttiadi is found to be similar to Tiptur Tall as well as Malappuram with least diversity (GD=0.04). The diversity observed within the Dwarf population of present study showed a higher diversity index (GD=0.09). This is mainly contributed by the dwarf cultivar Malayan Yellow Dwarf collected from RARS Pilicode which is found to be genetically most distant from the rest of the nine cultivars (average GD=0.16). The intermediate type Gangabondam is genetically diverse from Talls and is grouped under the cluster for dwarfs. Within this cluster it was found genetically closer to Malayan Yellow Dwarf. The two cultivars (Tiptur Tall and Malayan Yellow Dwarf) were identified for the production of a T x D hybrid based on the study as they were the most divergent among the cultivars selected.
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