GENETIC STUDIES ON DATE PALM (PHOENIX DACTYLIFERA L.) GERMPLASM GENE POOLS FROM JIGAWA, NIGERIA

ABSTRACT

Date palm is cultivated in arid and semiarid regions worldwide. In Nigeria, date palm is one of the most important tree crops of great socioeconomic importance in the Sahel, Sudan and Guinea Savannah ecologies. In order to assess the genetic diversity of the crop, survey and exploration were undertaken to collect the fruits from the populations of the female tree germplasm across the gene pools of the Nigerian Institute for Oil Palm Research (NIFOR), date palm research substation located in Dutse, Jigawa state, Nigeria. A total of 21 accessions were randomly collected across the gene pools. The fruits collected were analysed for their proximate and phytochemical compositions. The accessions were also evaluated for morphological characteristics using completely randomised block design (CRBD), Quantitative and qualitative characters were taken for the fruits following standard procedures. Random amplified polymorphic DNA (RAPD) technique was used to determine genetic diversity and sex identification. The cytological analysis was carried out using standard procedures. Results of the proximate composition showed that the accession R13P5 had the highest moisture content of 7.65 %. The highest carbohydrate content was observed in R4P12 with 82.32 %. The result of the mineral analysis showed that R24P9 had the highest calcium, magnesium, sodium and potassium with 119.39,80.55, 13.44  and 423 mg/100 g respectively.   The sugar content analysis showed that R5P8 had the highest glucose (320 mg/g) content while R15P6 recorded highest in fructose and sucrose (102 and 92 mg/g) content respectively. At late seedling stage, the highest plant height and girth size were observed in accessions R13P5 (39.00 cm) and R5P8 (10.00 cm). The results of the fruit characteristics showed that R7P1 had the highest fruit weight, fruit diameter and fruit thickness of 11.85 g, 2.10 mm, and 4.00 mm, respectively. The molecular diversity results revealed a total of 125 amplified fragments with 6 primers, of which 106 (84.3 %) were polymorphic and 19 (15.2  %) were  monomorphic. Out of the total of 21 accessions screened for sex determination, 12 accessions (R13P1, R4P12, R5P8, R3P22, R1P18, R7P1, R13P5, R13P9, R14P21, R4P29, R16P31 and zariya showed male pattern and 9 accessions (R24P9, R5P20, R2P4, R9P2, R6P20, R9P21, R5P24, R5P6, R1P10) were observed to be female. The morphological variations in the male accessions revealed a larger girth size and higher number of lower leaf spines while the female accessions showed a smaller girth size with little to absence of lower leaf spines. Cytological examination of some of the accessions confirmed 2n=36 number of chromosomes in accessions (R5P8 and R16P31) and 2n = 28 chromosomes in accession R1P18 with metacentric and submetacentric chromosomes respectively. The high genetic variability observed among the date palm germplasm for proximate, morphological and molecular characterisation has provided baseline information among the date palm germplasm in the gene pools. The developed sex determinant qualitative marker could be used for gender identification at the seedling stage of date palms in order to save time. Plant breeders and growers may adopt this marker as a potential tool for gender identification in date palm seedlings before they are transplanted in the field.

CHAPTER ONE

1.0       INTRODUCTION

1.1       Background to the Study

Date fruits are the products of date palm tree (Phoenix dactylifera L.), belonging to the family Arecaceae. It is one of the oldest cultivated plants in the world and is the most important subsistence crop in Northern Africa and the Middle East, although it is also cultivated in other parts of the world (Al-Shahib and Marshall, 2003). In Nigeria, date palm is one of the most important tree crops in Sahel, Sudan and Guinea Savannah ecologies where  it  has  remained  restricted  within  compounds,  homesteads  and  orchards  in  the Northern part of the country i.e. above latitude 10oN (Okolo et al., 2005). It is generally believed that the date palm was introduced into Nigeria in the early 17th century by traders and Muslim pilgrims on pilgrimage to the holy cities of Mecca and Madinah through the Trans-Saharan trade route from North Africa and Middle East (Omoti and Okolo, 2000).

Although  the  date  palm  is  not  indigenous  to  Nigeria,  but  with  over  400  years  of  its existence, the crop has been cultivated for sufficiently long time to have acquired agro – climatic adaptation and so evolved as landraces. Most national collections of date palm germplasm rely primarily on these land races. In Nigeria, several collection missions have been undertaken to capture these land races by the Nigerian Institute For Oil Palm Research (NIFOR) for  date palm  germplasm  conservation  and  crop  improvement  (Ataga  et  al., 2012). The States where date palm is grown in Northern Nigeria include Kaduna, Katsina, Kano, Sokoto, Kebbi, Jigawa, Yobe, Borno, Gombe, Bauchi and Adamawa. These are generally referred to as the Nigerian main date palm growing belt of the country. Other States include Plateau, Taraba, Nasarawa, Kaduna and Niger. These states are classified as marginal areas for date palm cultivation in the country (Abdulqadir et al., 2011). Date production in Nigeria has two fruiting seasons (dry season, that is from February to June; and wet season, from July to August), but only the dry season fruit is economically useful (Abdulqadir et al., 2011). Little or no research is carried out on wet season date fruit because it is harvested and consumed during the season in Nigeria.

The  date  palm  is  dioecious,  which  means  there  are  separate  male  and  female  plants, therefore pollination is required for fruit bearing (Maria et al., 2014). The flowers are yellowish, small and attached directly to the spikelets; male flowers are sweet-scented and have six stamens while the female flowers consist of three carpels with ovules, of which normally only one will develop into a fruit.

For fruit setting, fertilisation of the female flowers by male pollen is required. In date palm, pollination is not left to the wind or insects but is done traditionally by man where they insert a piece of spikelet of male flower at the moment when the female flowers are getting open. Besides traditional method, pollination can be done using machines, which has made the process quicker, easier, and efficient (Huntrods, 2011).

Date trees typically reach about 21–23 metres (69–75 ft) in height,  growing singly or forming a clump with several stems from a single root system. This tree typically has feathery leaves at the top which may be anywhere from 0.9 to 1.5 feet (3 to 5 m) in length. The trunk is usually very long and narrow  with rough-textured bark over most of its surface. There are no branches on a date palm tree. The leaves normally cascade downward from the crown of the plant to form a sort of canopy. They might spread anywhere from 19.7 to 32.8 feet (6 to 10 m) in diameter. This can often provide some shade for a person to sit in or for other small plants to grow in. This tree is believed to have originated in Africa or Asia (Wisegeek, 2011). Date fruits (dates) are oval-cylindrical, 3–7 cm long, and about an inch (2.5 cm) in diameter, ranging from bright red to bright yellow in colour, depending on variety. They are very sweet, containing about 75 percent of sugar when dry (Divya and Bichu, 2015).

Germplasm is a term used to describe living genetic resources such as seeds or tissue, maintained for the purpose of breeding, preservation and research. These resources may take the form of seed collections stored in seed banks, trees growing in nurseries, animal breeding lines maintained in animal breeding programmes or gene banks, etc. It provides potential diversity base in genetic resources of cultivated plants (Mahmut, 2012). Germplasm collections can range from collections of wild species to elite, domesticated breeding lines that have undergone extensive human selection.

Genetic diversity is the sum total of genetic characteristics within any species or genus (Rao and Hodgkins, 2002). Genetic diversity is required by breeders for the development of new, superior crop varieties with desirable qualities that can ensure a stable, abundant supply of food, feed and fibre (Jain et al., 2004). Therefore, genetic studies cannot be conducted if species do not show any variation. It is clear that gene erosion is a very dangerous and alarming feature of present-day exploitation of genetic resources (Bennett, 1965). This prospect alarms both geneticists and breeders, since lack of diversity severely impairs the future improvement of crops and/or limits the possibilities for addressing new production constraints (Vetelainen et al., 2008). The progress of any genetic preservation is dependent on understanding the amount and distribution of the genetic variation present in the gene pool (Jubrael et al., 2005).

Date fruits have great importance in human nutrition owing to their rich content of essential nutrients which include carbohydrate sugar, ranging from 65% to 80% on dry weight basis, and mostly of inverted form i.e glucose and fructose (Aldjain et al., 2011). In addition, date fruit has been reported to have other important components like proteins, fat, vitamins, dietary fiber, fatty acids, polyphenols, antioxidant and amino acids (Chandrasekaran et al., 2013). Date palm fruits have been reported to contain natural constituents like phytochemicals, sterols,  carotenes  and flavonoids, and have been screened for various medicinal activities to reduce the side-effects of artificial drugs that bring harm to human body  systems.  The  pulp  is  rich  in  iron,  calcium,cobalt,  copper,  fluorine,  manganese, sodium, copper and zinc.

Morphological characters of the tree are taken into consideration for cultivar identification. For the male trees, identification is a cumbersome process because they are mostly are seed-borne and are hardly identical to any female cultivar. Date palm varieties are very similar;  however,  studies  have  shown  that  there  are  clear  differences  based  on  the vegetative  characteristics  and  spathe  (Djerouni  et  al.,  2015).  Since  the  palm  leaf components look very different, the measurements which were taken from leaf palm like thorn length, pinna number and  leaf palm length have shown the similarities and the differences between the palms (Saker et al., 2006, Haider et al., 2015). In addition, several cultivars of date palm have been reported to be nearly morphologically similar and are of similar features (Ali et al., 2008).

The use of DNA markers provides a powerful tool to certify the identity of date palm cultivars at the seedling stage through the variety of fingerprinting. Random Amplified Polymorphic DNA (RAPD) markers can be used for fast screening of nuclear genome variations (Williams et al., 1990). RAPD markers have been used for germplasm characterisation in date palm (Tirfi et al., 2000; Askari et al., 2003; Soliman et al., 2003).

Moreover, Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) and Restriction Fragment Length Polymorphism (RFLPs), have been used to characterise date palm genotypes (Sedra et al., 1998; Ben Abdallah et al., 2000; Tirfi et al., 2000). Random amplified polymorphic DNAs (RAPD) are DNA fragments amplified by the polymerase chain reaction using short (usually 10 bp) synthetic primers of random sequence. RAPD markers have been used for identification and DNA fingerprinting of date palm varieties, (El-Tarras, 2007).

In Nigeria, sex determination in date palm at seedling stage has been very difficult to accomplish by date palm growers. Thus, many efforts have been reported to ascertain the sexes of date palm plants.  Al-Mahmoud et al. (2012) reported that the regions involved in sex determination in date palm employ a XX/XY (2n = 36) system with the male being heterogametic. Furthermore, they observed that the critical regions showed significant polymorphism between the male and female alleles. This polymorphism can be used in the development of assays to distinguish sexes at an early stage. Noppharat and Peerasak (2018) also used sex-specific DNA markers and concluded that such markers produced a reliable technique for sex determination in date palm, which could be used to predict the sex of date palm at the seedling stage. They also reported that this technique was easy to apply and accurate when tested repeatedly. It has shown to be successful when used to screen a large number of seedlings with results matching the theory of cross-breeding in dioecious plants where the ratio of male to female is 1:1.

1.2       Statement of the Research Problem

Despite the nutritional and economic potentials of date palm trees, the genetic resources have not been well utilised in enhancing the nutritional qualities of the crop. There is no significant information regarding the phytochemical study (e.g phytochemical screening) of the date fruits available in the germplasm gene pool in Jigawa, Nigeria. Morphological parameters are not sufficient to distinguish the closely related cultivars before fruiting. However, a large number of phenotypic characters need to be scored in date palms which are very laborious and time-consuming. Despite the fact that the germplasm gene pools at Jigawa have the largest diversity of date palm in Nigeria, the genetic diversity of the crop is yet to be ascertained, most especially using molecular markers.

The  dioecy  in  plants  represents  the  major  challenge  in  development  of  breeding programmes especially in crops like date palm. In Nigeria, although there is an increasing research efforts on a number of different plant species, very limited information is available on the molecular basis of gender determination of date palm at seedling stage so that growers can cultivate in their orchards a sufficiently large number of productive female trees with only a minimal number of male trees.

In Nigeria, inspite of many uses of date palm as an economically important crop, its cytogenetic study is rarely published because of the lack of understanding of the chromosomal behaviour in order to facilitate breeding for its improvement. Thus, Tahira et al. (2019) earlier reported that the limited number of roots, small and numerous numbers of chromosomes and the slow growth nature of palm trees have rendered the cytogenetic studies in date palm difficult.

1.3       Aim and Objectives

The aim of this study was to evaluate genetic characteristics of the date palm (Phoenix dactytlifera L.) germplasm gene pools in Jigawa State.

The objectives of this study were to:

(i)        quantify the proximate and phytochemical compositions of the different date palm fruits in the gene pool of Jigawa state.

(ii)       determine  the  morphological  parameters  and  fruit  characteristics  of  date palm samples.

(iii)      characterise the germplasm of date palm gene pool from Jigawa State into different genotypes using random amplified polymorphic DNA (RAPD) marker.

(iv)      determine the sex of date palm germplasm at seedling stage using sex specific RAPD marker

(v)       determine the cytological relationships among the accessions.

1.4       Justification for the Study

Date fruit is marketed all over the world as a high-value confectionery and remains an extremely important subsistence crop in most of the desert regions (Nadeem et al., 2011). Understanding the nutritional composition of various genotypes of the date palm within the gene  pool  would  enhance  and  facilitate  the  consumption  of  date  fruits;  this  will undoubtedly play an important role in human health since they may be a rich source of minerals and anti-oxidants which can serve as a great source of energy. Due to the high sugar content in the pulp, they can be used as a substitute for sugar in the food industry (Ricardo et al., 2019). The scientific clarification of the phytochemical screening in date fruits will provide information on dates as an alternative source of natural anti-oxidants that could improve health status.

Morphological parameters will be informative for description, cultivar characterisation, phenotypic diversity analysis and phylogenetic relationship exploration among date-palm ecotypes. Moreover, phenotypic diversity evaluation constitutes an available basic step for the elaboration of a programme to improve germplasm management and utilisation of the crop (Eissa et al., 2009). The use of data based on molecular markers such as Random Amplified Polymorphic DNA (RAPD) in the characterisation of date palm genotypes and for sex determination would accelerate genetic improvement and phylogenetic relationships in closely related groups of fruit trees through marker-assisted selection. Characterisation into main known genotypes is therefore, necessary to know some existing ones. This would be  most  important  for  speeding  up  breeding  and  thereby saving  time,  cost  and  other resources.  Similarly,  the  use  of  data  based  on  molecular  markers  such  as  Random Amplified Polymorphic DNA (RAPD) in the characterisation of date palm genotypes and for sex determination would accelerate genetic improvement and phylogenetic relationships in closely related groups of fruit trees through marker-assisted selection.

Cytology is still considered an important technique for the characterisation of plant species. The chromosomal data of date palm will serve as an important tool in understanding the similarities and differences on the basis of chromosome number, shape and size during chromosomal evolution and genetic diversity of the crop.

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