MORPHO-MOLECULAR CHARACTERISATION AND DETERMINATION OF RESISTANCE TO LETHAL YELLOWING DISEASE IN ELITE AND LOCAL COCONUT (COCOS NUCIFERA L.) VARIETIES

ABSTRACT

Breeding for resistance to LYD in coconut using the conventional method has not been very effective as it takes a very long time to make meaningful progress and the financial requirement is huge. A more effective and rapid method of detection of resistance to LYD is vital for sustainable coconut production. There is limited research work to develop durable coconut varieties using morphological markers and SSR markers (microsatellites). The aim of the study was to obtain an early, fast and effective method of detection of resistance using morphological traits and Simple Sequence Repeats  (SSR) markers (microsatellites). The specific objectives of the study were to; (I) Determine the cumulative LYD incidence in coconut genotypes, (II) Identify agronomic traits associated with resistance to lethal yellowing, (III) Identify SSR markers which are polymorphic for resistance and SSR markers which discriminate between resistance and susceptibility and (IV) Select coconut lines which are resistant to lethal yellowing at the seedlings stage. Materials used were 1871 first filial generation  (F1)  palms  on  field  trials  sited  in  disease  prevalent  locations  of  Nigeria  by Nigerian Institute for Oil Palm Research (NIFOR). The open pollinated varieties and dwarf varieties were planted one row of West African tall to two rows dwarf varieties in ten hectares. The hybrid varieties were planted in two hectares in a randomised complete block design. Varieties were replicated twice with eight palms per plot.   Spacing was 7.5  m triangular for the open pollinated and hybrid varieties. The varieties characterized were the West African Tall (WAT), Malayan Green Dwarf (MGD), Malayan Orange Dwarf (MOD), Malayan Yellow Dwarf (MYD), Sri-Lankan Brown Dwarf (SBD), Chowghat Green Dwarf (CGD); MGD x WAT, CGD x WAT, MYD x WAT, MYD x VTT (Vanuatu Tall), and Sri- Lankan Green Dwarf (SGD) x VTT hybrids.  Data were collected on 14 agronomic traits. The extraction of total genomic DNA from leaf samples, Polymerase Chain Reaction (PCR) Amplification and SSR marker analysis were done at the Bioscience Centre, International Institute of Tropical Agriculture (IITA), Ibadan. SSR marker data was analysed using PowerMarker version 3.25 software. Twenty six genomic DNA samples were analysed. Based on phenotypic assessment, the coconut varieties were clustered in four disease severity classes namely; highly resistant, moderately resistant, susceptible and highly susceptible. The SBD cultivar was highly resistant with 0% cumulative loss. The MGD and CGD were moderately resistant with 8.9 and 9.1% cumulative losses, respectively. The MGD x WAT and CGD x WAT hybrids were susceptible with 31.3% cumulative loss. The WAT, MYD and MOD genotypes and the SGD x VTT, MYD x WAT, MYD x VTT hybrids were highly susceptible with 98.5, 83.7 and 66.7, 62.5, 75.0, 84.4% losses, respectively. The susceptible genotypes differed significantly from the resistant genotypes at P ≤ 0.05 in height of trunk,

girth of trunk at 20 cm and at 1.5 m above soil level, length of leaf, length of petiole and crown size. Six out of the 20 SSR markers were polymorphic. Thirty-five amplified alleles were generated with a mean number of 6.0 alleles per locus. The primer CAC68 amplified the highest number of alleles (8) while CAC8 amplified the least number of alleles (4). The frequency of the major alleles per locus ranged from 0.3846 to 0.7308 with a mean of 0.6218. The mean genetic diversity for all loci was 0.5459 with a range of 0.4497 – 0.7101. The SSR markers had mean polymorphism information content (PIC) of 0.5109 with a range of 0.4103 to 0.6625. Primer CAC8 and CAC68 distinguished between resistant and susceptible coconut genotypes to LYD by showing resistance SSR markers in the resistant control and in the resistant  genotypes  at  130  and  180,  and  at  140  base pairs  levels,  respectively.  Rogers’ dissimilarity coefficient matrix revealed two main clusters. Principal Co-ordinates Analysis (PCoA) clustered the genotypes in two groups using the agronomic data based matrix and the SSR marker based matrix. The SSR genetic distance matrix ranged from 0.1667 to 1.000. The PCoA validated the earlier groupings in this study in clustering the genotypes in two main groups of resistance and susceptibility to LYD based on agronomic data and in two distinct  clusters  using  SSR  marker  data.  The  SSR  marker  based  PCoA  clustered  the genotypes more convincingly in two groups comprising ten resistant samples and eleven susceptible palms to LYD in each group. The SSR marker data based PCoA resistant group includes the SBD1, SBD2, SBD3, MGD1, MGD2, MGD3, MGD4, MGD5, CGD1, CGD2, and CGD3. The susceptible group comprises the CGD4, Susceptible bulk, MYD x VTT hybrid, SGD x VTT hybrid, WAT, CGD x WAT hybrid, MYD x WAT hybrid, MOD, MYD, MGD x WAT hybrid, and EWAT2. Resistant lines were SBD x WAT hybrid and WAT x SBD hybrid. It is evident that the genotypes studied constitute a genepool for resistance and susceptibility  to  lethal  yellowing  disease.  The  markers  which  discriminated  between resistance and susceptibility would be used routinely to select LYD resistant lines at the seedling stage and to select LYD resistant parents for resistance breeding taking cognizance of the genetically distant genotypes for maximum heterosis. This would enhance fast production of durable LYD-resistant coconut hybrids to boost coconut production and productivity in Nigeria besides saving time, space and cost of breeding programmes.

INTRODUCTION

The coconut palm (Cocos nucifera L., Arecaceae; 2n = 32) is an evergreen, monoecious, drupaceous, perennial, tropical tree made up of two main varieties – tall and dwarf.  The  tall  variety  is  out-crossing,  cultivated  both  for  household  and  commercial purposes with an economic lifespan of 60 – 70 years reaching a height of 20 – 30 meters while the dwarfs are shot (8 – 10 meters) when twenty years old. Dwarfs have characteristic petiole and fruit colours which differentiate them into green, orange, yellow and brown varieties (IPGRI, 1999) and are grown for ornamental and commercial uses.

Globally, coconut is grown in about 12.28 million hectares with an output of about

64.3 billion nuts and 2.1% of world vegetable oil (Arancon, 2013). About 96% of coconut is grown by more than 10 million resource-poor smallholder farmers tpending less than 4 hectares each (Eden-Green, 1999; Dery et al., 2005) in 86 countries of the world. Total world production of coconut is about 10 million tonnes of copra and 6 million tonnes of coconut oil. Among the major world producing countries, Indonesia (18,300,000 tonnes), Philippines (15,353,200 tonnes) and India (11,930,000 tonnes) are ranked first, second and third respectively.

In Africa it is planted to an estimated area of 526,700 ha with Tanzania (557,099 tonnes), Ghana (366,183 tonnes), Nigeria (265,000 tonnes), Mozambique (260,000 tonnes) and Cote d’Ivoire (195,000 tonnes) ranked  first to fifth respectively  (World Leaders in Coconut Production, 2016). The Asia and Pacific Coconut Community (APCC) countries occupied the first rank with an average area of 10,311,100 ha planted to coconut, followed by Africa (526, 700 ha), America (468,450 ha), Asia (average area of 82,600 ha), and the Pacific countries with 59,350 hectares (World leaders in coconut production, 2016). In Nigeria, an estimated 36,000 ha is presently under coconut cultivation (NIFOR, 2008, Uwubamwen et al., 2011). It is grown mostly in the riverine, coastal areas and homesteads in southern states of the country, but also is found beside streams, banks of rivers and stagnant pools of water in northern parts of Nigeria.  About 1.2 million hectares had been identified as suitable for coconut cultivation in Nigeria.

The types of coconut varieties had been investigated with DNA-based techniques, which had already supported the concept of wild-domestic-introgressed coconut types and currently identified two major gene pools representing sources of predominantly ‘wild-type’

and ‘domestic-type’ populations from which all modern cultivars had developed (Harries,

1995, Perera et al., 2009, Zizumbo et al., 2005). DNA analysis of world accessions and samples from under-sampled regions of Southern Indian Ocean using microsatellites showed that  cultivated  coconuts  had  two  independent  origins:  The  cross-pollinated  variety  in southern fringes of Indian sub-continent and the self-pollinated varieties in the Islands of Southeast Asia (Gunn et al., 2011). The palm is cultivated under diverse types of climate and different soil types – laterite, coastal sandy, alluvial, loamy, reclaimed marshy lowlands, although usually grown along the sea coast and in plain grounds; it tolerates wide range of pH (5.0 – 8.0). It can be grown in places up to 1,000 m above sea level and it tends to grow best in places with a mean annual temperature of 25°C -38°C and an annual rainfall of 2000 mm (Nair et al., 2003). More than 90% of the nation’s coconut belt is a continuation of the plantations or groves along the West African coast running from Cote D’Ivoire and southeast towards Ghana, Togo and Benin to Lagos state in a one kilometre wide strip.

More  than  80  million  people  of  the  world  depend  directly  on  coconut  and  its processing for their livelihood. Apart from the useful materials obtained from coir, functional coconut food products like copra, virgin coconut oil, protein isolate, skim milk, flour, feed concentrate, and coconut water; industrial coconut products such as cochin oil and biodiesel; activated carbon and shell charcoal, are some of its invaluable products. Natural coconut water contains many vitamins and minerals, including niacin, panthothenic acid, riboflavin, thiamin, potassium, magnesium, manganese, zinc, calcium, and vitamin C.  It is being marketed as an alternative to sports drinks in many advanced countries (USDA, 2016).

Lethal Yellowing Disease (LYD) is the most devastating disease of coconut palm in Nigeria affecting mainly palms of productive age. Since it was observed in Awka, Anambra State by Johnson (1917), it had spread to most parts of the country and destroyed more than

60% (Odewale et al., 2010) of the coconut population with the West African Tall (WAT) mostly affected. The symptoms are: abortion of nuts which progresses from the youngest to oldest bunches, blackening of spikelets; yellowing, browning and shedding of leaves in quick succession, and death of palm 3 – 6 months after the disease symptoms is first noticed in the palm. The effective control measure is by cultivation of resistant genotypes. Oxytetracycline injection on diseased palms is reported to have remission of symptoms within four weeks of treatment but the dosage has to be repeated every four months to prevent re-emergence of

symptoms and death of the affected palm (Ekpo and Ojomo, 1990, Eziashi and Omamor,

2010).  LYD  resistant  coconuts  are  a  prerequisite  for  sustainable  coconut  production  in Nigeria and other countries where the disease is endemic. Many exotic cultivars and some hybrids that were reported to be resistant to LYD in some countries had been found susceptible to it in Nigeria. The best strategy would be to conserve and exploit the diversity available in local populations either by developing resistant hybrids or to improve traditional cultivars (Devakumar et al., 2011, Upadhyay et al., 2004).

Screening of coconut for LYD resistance using field trials requires at least twenty years of evaluation in disease endemic and high pressure areas for the identification of genotypes that possess durable resistance alleles. The disease is regarded as the most significant factor impacting coconut production worldwide (Oropeza et al., 2005). The use of disease  resistant   coconut   varieties   is   a  prerequisite  for  sustainable   production   and productivity. Coconut genotypes on field trials in some sites located in LYD prevalent tracts in NIFOR had showed variable responses to the disease using phenotypic assessment. The use of morphological tools alone to select for pest resistance is less precise as it is influenced by both genetic and environmental factors. Breeding for resistance to lethal yellowing in coconut using the conventional method is less effective, very costly and time consuming. Coconut palms have long generation period, large crop size, low annual seed yield / palm; low plant density / hectare. Besides, the pathogen cannot be cultured in any axenic medium in the laboratory and has long incubation period. There is no permanent curative measure yet for infected palms and a more effective and rapid method of detection of resistance is vital for sustainable coconut production and productivity hence the need for a morpho-molecular characterisation and determination of resistance to LYD of elite and local coconut varieties. The aim of the study was to obtain an early, fast and effective method of detection of resistance to LYD. The objectives of the research were:

1.   To determine the cumulative incidence of LYD in coconut genotypes

2.   To identify phenotypic traits associated with resistance to lethal yellowing

3.   To identify SSR markers polymorphic for resistance to lethal yellowing and SSR

markers which discriminate between resistance and susceptibility 4.   To select for LYD resistance at the seedling stage.

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