GENETIC DIVERSITY OF Digitaria horizontalis Willd. IN BUILT ENVIRONMENTS IN BENIN CITY, NIGERIA

he influence of humans on biodiversity at the genetic, species, community, and ecosystem levels is enormous. This is even more impactful on plant populations, which, being immobile, are at the mercy of their immediate environment. Digitaria horizontalis is a very common grass species in Benin City. Its ruderal nature accounts mostly for its widespread. The current study investigated the existence of possible genetic diversity of the grass in built environment in Benin City. Plant samples were randomly collected from 21 built environment locations within 6 Local Government Council Areas that make up Benin City. Test plants were allowed to acclimatize in experimental bowls and then allowed to grow for 4 weeks after which morphological characteristics were determined. Genetic diversity was determined by Random Amplification of Polymorphic DNA. Significant differences in plant height (33.0 – 59.0 cm) occurred when plants were compared based on location of collection. Changes in patterns of the association with weeds within the vicinity of the test plant was observed. The most prevalent plant species associated with the test plant was Pennisetum purpureum with a 12.18% prevalence. RAPD analysis using primer OPA 04 revealed a locus (about 600bp) that was absent in one or more of the locations with minimum disturbance. This 600pb fragment was however observed in most samples from highly built up and trampled environments. Primer OPA 03 showed monomorphic bands in 2 test samples with a band size of 400bp. It is therefore reported that there is an existence of the genetic diversity in a landscape of grass species (Digitaria horizontalis) found in different built environment in Benin City, Edo State, Nigeria.


INTRODUCTION
Industrialization has always seemed to be the key to prosperity and a better life, but studies have shown that, while it enhances living conditions in certain respects, it has a detrimental effect on the ecosystem and, as a result, threatens plant biodiversity (Pariente, 2002). Not only does it involve technological innovations, it also involves economic and social transformation of the human society (Borrelli, 2017). With industrialization come prospects as well as challenges. The challenges include managing changes in the environment (Linares et al., 2011). Due to these challenges, industrialization must take into account plant biodiversity disturbances and its consequences (Mligo, 2011). Plants play a key role in conserving ecosystems. They are a source of food and medicinal compounds while also providing raw materials for many industries. Rapid deforestation and industrialization, however, threaten plant biodiversity. In turn, this threatens the ecosystem (Maynard et al., 2013). Biodiversity of plants ensures a resource for new food crops and medicines. Plant life balances ecosystems, protects watersheds, mitigates erosion, moderates climate, and provides shelter for many animal species (Shrestha et al., 2012). One of such plants with these characteristics is Digitaria horizontalis.
Digitaria horizontalis is a tropical plant that can be found in tropical America as well as West and Central Africa. It can grow rapidly and has developed itself as a dominant species in intensively cultivated fields (Johnson, 1997). In terms of socioeconomic importance, the plant species belongs to one of the most important classes of Poaceae. The use of D. horizontalis as a source of fodder (Harun et al., 2017) and in pharmacopoeia are also important socio-economic factors (Pare et al., 2016). It can be found in a variety of environments, but open areas or grasslands tend to have the most diversity. Disturbed areas, such as gardens and cultivated fields, are suitable habitats for these grasses. Digitaria horizontalis is widely used as a lawn plant in horticulture, where it provides aesthetic value while also aiding in the improvement of soil nutrient capability (Ruprecht et al., 2009). Decoction of the plant is used in the treatment of gonorrhea. It is used as folk remedy for cataracts and debility. It is also said to be emetic. Fiber from the plant can be used for making paper. Its forage has an excellent quality and palatability (Latty et al., 2004). The plant's ability to grow and root makes it an ideal choice for soil erosion prevention strategies. The warm-season annual Digitaria horizontalis germinates, grows, and dies all in the same year. Each Digitaria horizontalis plant can produce up to 150,000 seeds during its growth. Those seeds remain in the field, ready to germinate the following season and repeat the cycle. Seeds that do not germinate right away can still be viable and germinate in subsequent years. These characteristics ensure its availability throughout the year.
Genetic diversity is of fundamental importance in the continuity of a species as it provides the necessary adaptation to the prevailing biotic and abiotic environmental conditions, and enables change in the genetic composition to cope with changes in the environment. Grasses are able to survive regular cutting or grazing as their growing points are situated at the base of the plant (Mishra et al., 2004). Lateral shoots (known as tillers) grow from the base of the main shoot, and when the upward growth of these is prevented by grazing; new buds are formed at their base and grow out laterally into new vegetative branches. In this way, grasses are adapted to tolerate grazing, cutting and trampling (Bell et al., 2016).
Many grassy areas are mown, especially in towns and cities. Mowing cuts the plants, and the cut material is often removed. Mowing may also have a crushing effect if heavy machinery and rollers are used. As a result of AJHSE 2(1) Ikhajiagbe, et al., 2021 | 26 trampling and mowing it is often easy to see a change in the height of the vegetation and both can result in changes to soil conditions as a result of compaction (Blanco et al., 2015). Plants growing on trampled areas have to be tolerant to soil compaction and its effects on soil conditions (Anonymous, n.d.). However, the species composition of the plant community will also be influenced by a number of other interacting factors (Bhuyan et al., 2003). Physical damage to plants by removal of growing tips and crushing occurs, so having the right growth form is important. Deposition of litter and dog fouling may cause changes in the soil mineral content though any change is difficult to measure without sophisticated equipment (Augusto et al., 2002). Relatively few species are likely to be able to survive when environmental conditions are extreme for example in most trampled areas where the chance of physical damage is high and the soil is very compacted. Also, in non-trampled areas where the vegetation is tall and there is strong competition for light. It is at the boundary between the two extremes that the greatest number of species is usually found (Bickham, 2013). At the edge of a trampled areas species are least affected by trampling but avoid too much competition with the more vigorously growing species. The aim of the study was to investigate possible existence of genetic diversity in a landscape grass species (Digitaria horizontalis) found in different built environment in Benin City, Edo State, Nigeria.

CAPITOL (A)
Botanic Garden located at Site B of the Ugbowo campus of the university, or otherwise referred to in the study as the Capitol is an area around the University of Benin that is minimally developed. It served as control because in comparison to other locations it did not contain buildings. Its bearing is 270 0 NW. It lies on latitude 6 0 20' 1.32'' N and longitude 5 0 36' 0.53''E. This area is very rich in biodiversity.

UNIVERSITY OF BENIN HALL 1 HOSTEL (B)
This is a built area, a hostel in the University of Benin for females. It is a highly disturbed area because the grasses are always mowed, trampled and all other soil factors. It is 119 0 SE. Its latitude is 6.39 65.8' 23'' and longitude 5.6 18' 69''

INE FILLING STATION (C)
Also located in Ovia North East L.G.A. It is a built area; a filling station. The area is highly disturbed.

EVIDENCE CHURCH (D)
The area was water-disturbed at the time. It is a highly disturbed location. Its bearing is 83 0 E. It lies on latitude N 6 0 25'24 and longitude E 5 0 36'6.

EGOR SECRETARIAT (E)
It is located at Uselu Market. along Mela Road. It is one of the serving eighteen L.G.A. Councils of the State. Court weddings are carried out here and other activities. This area is highly disturbed. It is constantly mowed and trampled.

MELA MOTEL (F)
This area is constantly trampled (especially by cars) and a very busy environment. Its bearing is 268 0 W.It lies on latitude 6 0 22'20''N and longitude 5 0 36'34''E.

EDAIKEN (G)
Although this area is built, it is not very disturbed. In fact, it is very rich in weed biodiversity. Its bearing is 251 0 SW.

EDOKPOLOR GRAMMAR SCHOOL (H)
This a secondary school in Oredo L.G.A, around the New Benin axis of the State Capital. It is a highly disturbed area.
This area is always trampled by vehicles and humans. Mowing is always done on this location. Its bearing is 305 0 NW'. It lies on latitude 6 0 21' 18''N and longitude 5 0 37'46''E.

NEW CONVENANT GOSPEL CHURCH, NEW BENIN (I)
This place is highly developed and highly disturbed. Most part of the land has been graded and completely devoid of plant life. It is on a bearing of 40 0 NE. It lies on a latitude 6 0 21'20''N and longitude 5 0 37'44''E.

NEW BENIN CEMETERY (J)
Located around the New Benin axis, it is a built area with very rich biodiversity. It is 81 0 E. It lies on a latitude 6 0 21'19''N and longitude 5 0 37'34''E.

OANDO FILLING STATION (K)
This petrol filling station is located outside New Benin, just on the axis of Ikpoba Slope. It is highly disturbed with cars driving through and it is always trampled. Its bearing is 81 0 E. It lies on latitude 6 0 21'19''N and longitude 5 0 37''E.

IKPOBA BRIDGE, AKPAKPAVA (L)
This is the bridge that crosses the Ikpoba River along the Akpakpava/Ikpoba Slope axis. This area is very rich in biodiversity although quite disturbed. Its bearing is 230 0 SW. It lies on latitude 6.3 51 16 0 N and longitude 5.6 4 70 0 E.

AVBIAMA ROAD (M)
Avbiama is a disturbed area that is extremely trampled. There are a lot of shops in this area. Its bearing is 94 0 E. It lies on latitude N6 0 20'51 and longitude E 5 0 38'52.

OREGBENI (N)
Oregbeni samples were collected around the popular Oregbeni Market axis around Ramat Park, Ikpoba Hill. Its bearing is on 131 0 SE. This is an urbanized area that has lots of shops around it. It lies on latitude and longitude.

RAMAT PARK (O)
This is a highly urbanized area on the Ikpoba Hill, with a lot of activities. Trampling is extreme in this location by local township vehicles and also humans. It lies on latitude 6.3 50 06 0 N and longitude5.6 6 12 0 E.

BYPASS ROAD (P)
The bypass is off Eyean on the Benin-Auchi axis. There was no land mark around this area. Although the area is highly disturbed, there were a diverse species of grass in this area. Its bearing is 164 0 SE. It lies on latitude N 6 0 22' 48 and longitude E 5 0 42' 22'.

PIPELINE ROAD (Q)
A lot of human activities take place here. It has a lot of built areas ranging from shops to family homes. It is very rich in biodiversity regardless. Its bearing is 121 0 NW. It lies on latitude N 6 0 23' 18' and longitude E 5 0 38' 19'.

IGUOMO ROAD (R)
This is an area that has experienced trampling and other human activities. It is 268 0 W and it is located at Orhionmwon L.G.A. It lies on latitude N 6 0 20' 17 and longitude E 5 0 44'14.

REHEEBOOTHS FOOD AFFAIR (S)
This is a popular restaurant located off Iguomo area in Orhionmwon local government area. Trampling activities is prevalent in this area, especially with parked cars. Its bearing is 121 0 N. It lies on latitude N 6 0 29' 11' and longitude E 5 0 33'' 15'

MAY-EWERE AND CO. GAS STATIONS (T)
This area is a gas station which means that it has lot of cars visiting the area. Trampling is also prevalent in this area.

COLLECTION OF PLANTS
Plants were collected from the various areas from A to U above. Three plants were uprooted each from these areas with a ball of earth. Care was taken to ensure that the plants collected did not show signs of chlorosis and necrosis.
Also, the plant had not started flowering when they were uprooted and taken immediately to the Screen House.

TRANSPLANTING AND STABILIZATION
The plants were brought into already prepared experimental bowls measuring 68 m in diameter and 43 m in depth.
Soils were collected from the botanical garden and were filled into nursery bags. The plants were transplanted into nursery bags to stabilize. Plants characteristics were eventually measured.

PLANT PARAMETERS ASSESSED
The cell elongation rate clearly influences overall plant height since cell division is restricted to a small portion of the shoots and roots. The prominent height was determined and was measured with a metre rule. The internode was determined as the portion of plant stem between nodes and was measured with a metre rule. Chlorophyll Content Index was determined by the aid of a chlorophyll content meter; CCM-200 plus, which is a non-destructive chlorophyll content measuring meter, which exploits the distinct optical absorbance characteristics of the chlorophyll in other to determine its relative concentration. The average meter reading of 3 leaves per plant was taken as the CCI. The leaves were counted and the total number recorded. The maximum length of inflorescence was measure from the point where the peduncle attached to the stem to the tip of the inflorescence. In order to determine the leaf area, the leaves were placed on a graph note book and traced. The area covered by the leaf on the graph trace were computed for leaf area.

RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)
DNA extraction from the leaf of test plants A -U was carried out using a ZR plant/seed DNA extraction kit according to manufacturer's instruction. Briefly, 150 mg of the test plant leaf was added to a ZR BashingBead™ containing 750 μl of the lysis solution. The leaf was then homogenized using a mini bead-beater for 10 minutes. After homogenization, the tubes were centrifuged at 10,000xg for 1 minute. 400 μl of the supernatant was transferred into a Zymo-Spin™ IV Spin Filter in a Collection Tube and centrifuged at 7,000 rpm for 1 minute. Tube and centrifuged at 10,000 x g for 1 minute. After repeating the wash step, the column was transferred to a clean 1.5 ml microcentrifuge tube and 100 μl of sterile water was added directly to the column matrix after which the tubes was centrifuged at 10,000 x g for 30 seconds to elute the DNA. Finally, the eluted DNA was filtered using a Zymo- Spin™ IV-HRC Spin Filter into a 1.5 ml microcentrifuge tube and centrifuged at 10,000 x g for 30 seconds.

POLYMERASE CHAIN REACTION
RAPD-PCR was carried out on the extracted DNA sample using the RAPD primer OPA 04 (5'-AAT CGG GCT G -3') and OPA 03 (5'-AGTCAGCCAC-3') The PCR reaction was carried out in a 20 μl reaction mixture containing 1X PCR buffer (Solis Biodyne), 2.5mM Magnesium Chloride, 0.2 mM of each dNTP, 40 pMol of primer, 1U Taq DNA polymerase, 10-200 ng of DNA, and sterile deionized water was used to make up the reaction mixture (Williams et al., 1990;Barakat et al., 2010;Yaycili and Alikamanoğlu, 2012). Amplification was carried out in an Eppendorf Nexus thermalcycler using the following cycling parameters; an initial denaturation at 95 ºC for 5 minutes which was followed by 40 consecutive cycles of 95 ºC for 1 minute, 30 ºC for 1 minute and 72ºC for 2 minutes. This was followed by a final extension of 72 ºC for 10 minutes. The PCR products were separated on a 1 % Agarose gel and 1 Kb DNA ladder (Fermentas) was used as DNA molecular weight standard.

STATISTICAL ANALYSIS
Results were presented in Mean of 5 replicates. Analysis of variance in complete by randomized design was done using the SPSS® version 20 statistical software, and means were separated by using the Least Significant Difference.
Plant diversity indices were determined using PAST version 2.17c (Hammer et al., 2001).

RESULTS
Morphological characteristics of test plant collected from the various sampling locations have been presented (Table   1). Prominent plant height ranged from 33.0 -59.0 cm with significant differences occurring when plants were compared based on location of collection. There were no significant differences in internode of plant collected and stabilized (7.5 -10.1 cm, p>0.05). Similarly, no significant differences in maximum length of inflorescence (cm) were observed in the study. An attempt was made to discover which of the measured plant parameters showed more variability when compared with the others (Fig. 1). Results showed that plant leaf area showed more variability.
The abundance of weeds associated with the test plant in the experimental bowls was reported ( in association with the test plant. Prevalence of the weeds as well as index of citation was presented in Table 3. The most prevalent plant species associated with the test plant was Pennisetum purpureum (12.18%). This weed species however had a lower citation index (0.476) than Eleusine indica (0.762). The least prevalence was obtained in Cyperus esculentus (5.34%).
Correspondence biplot and principal coordinates establishing relationship among sampling locations and associated weed species have been presented in Fig. 2(a) and 2(b) respectively. Results showed associateion of Lindernia crustacea with location K. Eleusine indica was mostly associated with O, M, and G locations. The association of the locations has been presented in Fig. 2(b).

DISCUSSION
The genetic variation of an entire species is often called genetic diversity. Plant immediate environment play an important role in the growth and development of the plant (Upadhyaya et al., 2013). Height of a plant is essential in determining the plants life span and time to maturity. It is a major determinant of a species' ability to compete for light (Moles et al., 2009). The height parameter is an essential trait in plant breeding and cultivar development. In this study, significant variation was observed in the height of test plants collected from various locations. Though, the internode and length of inflorescence across location did not vary significantly (Table 1). The differences in plant height may be attributed to the effect of alterations in various environments or locations examined. A similar observation was reported in the height of maize plants (Sabiel et al., 2014).
Understanding the genetic diversity of the test plant (crab grass) in relation to the various environments studied usually includes its association with other plants or organisms. This study identified weeds associated with the test plant from the sampled locations and the weed abundance. This was to further determine the level of variation of the plants between locations (Musa and Ikhajiagbe, 2020). Pennisetum purpureum was the most abundant weed across the built-up locations. Some locations with less human activity and more activity recorded none or minimal emergence of P. purpureumin pots planted out with test plants. This observation may be due to the absence or reduced presence of elephant grass seeds in the rhizophere of the test plants from the locations. It is however evident that the variation in the locations examined did not correlate with the type and abundance of weed observed.
RAPD markers were assessed in the test plants to reveal possible genetic diversity across locations. It was observed that primer OPA 04 revealed a locus (about 600bp) that was absent in location J with minimum disturbance.
Location J is a cemetery and it is not regularly trampled. This 600 pb fragment was however observed in most samples from highly built up and trampled environment e.g locations I and K (See figure 4). It may be inferred that the man- The samples with the highest polymorphism were from locations characterized by commercial activities and it may be deduced that the test genotypes obtained from these locations had denser genetic base when compared with plants from the control location, cemetery and probably those samples without amplification during PCR with OPA04.
Further genetic studies in the test plants will definitely unravel more information on possible mutation in the plants examined.

CONCLUSION
The result obtained from this study indicated that there was genetic diversity among Digitaria horizontalis that were obtained from the different built areas investigated in Benin City. It simply means that human activities affect the genetic diversity of this plant.