Comparative genome analysis in coconut (Cocos nucifera Linn.) and marker development for distinguishing tall and dwarf coconut types

Abstract

Coconut is an important oil nut crop in the humid tropics of the world. Because of its inevitable uses as food, drink, fuel, and so on, this palm is known as ‘the tree of life or Kalpavriksha’. There are two ecotypes in coconut, tall and dwarf. Development of dwarf varieties with high yield, longer life span, field tolerance to biotic and abiotic stresses and good kernel and oil recovery, is the major breeding objective in this crop. Breeding attempts for dwarf palm stature are crippled with the non-availability of a precise methodology to identify the dwarf lines at the early plant stage itself. Development of molecular markers linked with this trait will enable the marker assisted selection for dwarf palms. The present study entitled “Comparative genome analysis in coconut (Cocos nucifera Linn.) and marker development for distinguishing tall and dwarf coconut types” was undertaken at Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Thrissur, during 2019 to 2022, with the objective to identify the differential genes and genomic regions among the tall and dwarf coconut genotypes through comparative whole genome sequence analyses and to develop molecular markers for distinguishing tall and dwarf coconut types. The coconut genome assemblies and raw reads were retrieved from various databases, quality of the assemblies and raw reads analyzed, raw reads trimmed and assembled using SOAPdenovo2, ABySS and Velvetoptimiser. Repeat masking was carried out on coconut genome assemblies, using RepeatMasker employing Dfam and RepBase as libraries. Since the library yielded insufficient masking percentage, de novo repeat library was prepared for the genome assemblies using RepeatModeller. The repeat libraries thus obtained have been merged to get a comprehensive and exhaustive repeat library for coconut and was used to perform repeat masking. Further, the efficiency of the combined repeat library for repeat masking in other palms was checked and found effective. The library was made publicly available at https://kau.in/repeat-libraries. Gene prediction was carried out for the repeat masked genomes using AUGUSTUS, a eukaryotic gene prediction tool. Comparative genome analyses were carried out by NCBI BLAST+, unique sequences obtained for dwarf and tall genomes were identified and extracted. The extracted sequences were annotated using online BLAST and functional annotation was carried out using BLAST2GO. Reverse BLAST was performed to ensure that the sequences thus obtained are unique and PCR primers were designed for the sequences. Leaf samples were collected from 10 coconut accessions, five each of the tall and dwarf types, from the parent palms at RARS Plicicode. DNA extracted from these accessions were screened using 27 primer combinations. Ten primer sets have amplified the markers in West Coast Tall only while the primer set Cocos_22 amplified the marker in West Cost Tall and Jawan Giant. Primer set Cocos_21 has amplified the markers in three tall samples, West Coast Tall, Jawan Giant, New Guinea and one dwarf sample Chowghat Green Dwarf. It is already recorded that Chowghat Green Dwarf is not a true dwarf ecotype but belongs to the semi-tall types. Marker Cocos_21 was successful in marker generation in tall accessions, except where this highly quantitative trait is governed by other major QTL. Further, in order to establish a universal marker linked to the height of the coconut palm, more whole genome sequences of tall, dwarf and intermediate ecotypes are required and insights from the sequence data (whole genome and transcriptome) could help in more refined classification of the palms. After validation with other tall and dwarf cultivars, the marker identified in this study may be used in marker assisted selection of coconut.