Biological Research Biotechnology Genetic assessment of Mangifera indica Linn. (Mango) from selected locations in Oyo State, Nigeria

This study characterized five (5) varieties of mango comprising 15 accessions collected from Ogbomosho, Saki, Ibadan and other locations in Oyo State. The field experiment was laid out in a Complete Randomized Design (CRD) with three replicates. Morphological characters were assessed on the stem, leaf and fruit. Also, Molecular studies (DNA amplification and sequencing) were carried out on 15 accessions of mango. The edited sequences were blasted in the National Center of Biotechnology Information (NCBI) data website. The Results showed variability in morphological characters of Mango. Ogbomosho Acc-2 performed best for leaf width (4.53cm) and lamina length (16.25cm) while Isehin Acc-1 had the highest number of leaves per seedling (7.76cm), leaf length (17.06cm), leaf area (38.84cm), petiole length (2.27cm), plant height (24.07cm) respectively. The Number of leaves had pos itive correlation with Leaf length (r=0.53), Leaf Area (r=0.59), Internodal Length (r= 0.55) and strong positive correlation with plant height (r=0.73) at p≤0.05. The success rate of amplified DNA products and sequencing was 77.78%. The query coverage of 99% and 100% confirmed positive amplification and sequencing of rbcL gene in the mango varieties. The sequences blasted in the NCBI data website were identified to be similar to accession KX871231.1. Sequences of rbcL marker showed genetic differences among samples; Grafe and OGBM Acc -1. Genetic distance between varieties from the same location was most often lower with Grafe mango being the most distant variety with genetic distance of 0.114-0.117. There were morphological and molecular variations in mango varieties and accessions. Isehin Acc-1, Saki Acc-1 and OGBM Acc-6 accessions had better growth performance.


INTRODUCTION
Mango (Mangifera indica L.) is a f avorite diploid f ruit tree with 20 pairs of chromosomes (2n = 40) and 439 Mbp genome size (Roy and Visweswaraiya, 1995;Mukherjee SK, and Litz, 2009). A perennial f ruit crop, rich source of vitamins, β-carotene, minerals, and antioxidants, of ten called "king of f ruits" f or its unmatchable taste and f lavor (a native of Southern Asian countries (Begum et al., 2014;Kaur et al., 2014). India is the largest producer in the world (18.0 million tons per year) (FAO, 2015), More than a thousand varieties of Mango have been identif ied all over the world (Rymbai et al., 2014). Mango was introduced to West Af rica in the 16th century by the Portuguese and since then it has become highly diversif ied and accepted f ruit in Nigeria and other Af rican countries (Okigbo, 2001;Fowomola, 2010). About 63 countries account f or more than 1000 million tons of mango f ruit production annually with India as the leading producer (FAOSTAT, 2015).
Morphological characterization is an important traditionally tools used to study variation in dif f erent crops (Gonzalez et al., 2002) including mango (Subedi et al., 2009). Morphological characteristics are still extremely usef ul f or identif ication and or dif f erentiation of cultivars, since mango published descriptors, lists are readily available (Hoogendijk and Williams, 2001;IPGRI, 2006). Also, being an important f ruit crop with huge diversity, the plant portends an important genetic resource that may be explored by breeders f or improvement purposes especially the f ruit characters (IITA, 2015). Genetic variation plays a key role in successf ul breeding programs of plants (Olawuyi et al., 2015).
DNA extraction is one of the methods used in molecular analysis of plants and the use of Sodium Dodecyl Sulf ate (SDS) and Proteinase K procedure described by Goldenberger et al., (1995) has been f ound promising in DNA extraction with high rate of ef f iciency. The Sodium Dodecyl Sulf ate (SDS) is strong anionic detergent that can solubilize the proteins and lipids that f orm the membranes to removes the negative ions f rom the protein and destroys its conf irmation (Goldenberger et al., 2005). Recently, the necessity of DNA sequencing became eminent as described by Francis Crick's theory that the sequence of nucleotides within a DNA molecule directly inf luenced the amino acid sequences of proteins (Mussane et al., 2010;Azim, et al., 2014).
Several studies on characterization of mango f ocused on morphology and use of molecular markers. There is need to provide more inf ormation on molecular sequence of mango. Hence, this study investigated the variability and relationship among the mango varieties and accessions evaluated in this study.

Plant collections and study location
Five (5) mango f ruit varieties comprising of 15 accessions were collected between March to May 2018 f ollowing the method described by IPGRI, (2006) ( Table 1). The geographic location of each of the sampled trees was recorded using a hand -held Global Positioning System (GPS) as shown in Table  1.

Experimental design and planting procedure
The f ield experiment was in Completely Randomized Design (CRD) in three replicates. The Mango seeds were processed using the procedure described by Verheij (2004). The planting was done in an open f ield using 1.0 m spacing within the row and column at the researc h f arm of the Department of Botany, University of Ibadan, Nigeria.

Determination of morphological characters
The morphological characters of all accessions were carried out f rom the f irst week to the twelf th week using the method described by IPGRI (2006).

Molecular studies
The Molecular experiments (DNA extraction, Amplif ication, purif ication and Sequencing) using rbcL with HIf and Fof ana primers were carried out for all accessions at Bioscience Unit of the International Institute of Tropical Agriculture (IITA), Ibadan, Oyo State.
The tubes were later removed and allow to cool f or 2 minutes bef ore adding 200µl of ice-cold 5M Potassium acetate and incubated on ice f or 20 minutes to precipitate protein later centrif uged at 10000rpm f or 10 minutes and then the supernatant was transf erred into f reshly labeled tubes. Ice-cold Isopropanol of 2/3 volume was added, mixed gently and incubated at -80 o c f or 15mins, centrif uged at 100000rpm f or 10 minutes to precipitate the DNA.
The supernatant was decanted until the last drop was released and 400µl of 70% ethanol was added to wash the DNA pellet and centrif uged at 10000rpm for 10minutes. The supernatant was decanted until the last drop and air dry the pellet. Also, 60µl of ultra-pure water or low salt TE was added to re-suspend the DNA with 2ul of RNase and incubated at 37 o C f or 30-40 minutes. Agarose gel of 0.8% was prepared f or checking DNA quality and to about 60 o c then 5µl ethidium bromide was added and gently mixed, later poured into the gel tray bef ore it polymerizes. Air bubbles was avoided in the middle of the gel. DNA of 3µl was mixed with 3µl of loading dye was pipetted into 0.8% agarose gel and run at 80 volts f or about 60 minutes. The gel picture was saved.

DNA amplification and primers
The amplif ication reaction were prepared f or 25µL PCR Reaction volume containing 2.0 µl of 100ng/µl DNA, 2.5 µl of 10× PCR buf f er, 1.5 µl of 50mM Mgcl2, 1.0 µl of 5pMol f orward primer, 1.0 µl of 5pMol reverse primer, 1.0 µl of DMSO, 2.0 µl of 2.5Mm DNTPs, Taq 5µ/µl 0.15, 13.85 µl of H2O f or 25µL. Amplif ication were perf ormed in thermocycler programmed f or a touch-down (TDSSR) protocol at the initial step of denaturation f or 5 minutes at 94°C f ollowed b y 9 cycles and later 35 cycles each consisting of denaturation step of 15 seconds at 94°C, an annealing step of 20 seconds at 65°C and an extension step of 30 seconds at 72°C. Seven minutes will be given af ter the last cycle of the extension step at 72°C to ensure the completion of primer extension reaction f ollowed by cold temperature at 10°C lasting f or inf inity. The primers considered are H1f F: CCACAAACAGAGACTAAAGC and Fof ana R: GTAAAATCAAGTCCA CCGCG.

PCR purification process and sequencing
The PCR product was purif ied by adding 20 µl of absolute ethanol to the PCR product and incubated at room temperature f or 15minutes later spined down at 10000rpm f or 15minutes, the supernatant was decanted, spined again at 10000rpm f or 15minutes then 40ul of 70% ethanol was added, the supernatant was later decanted, air dry. The amplicon was checked on 1.5% agarose (Zeugin and Hartley, 1985).
Purif ied samples were sequenced by Genetic analyser 3130×1 sequencer f rom Applied Biosystem using manuf acturer's manual, the sequencing kit of Big Dye terminator V3.1 cycle sequencing kit was considered while Bio edit sof tware (Mega 6) was used f or sequence editing.

Statistical analyses
Morphological Data were subjected to Analysis of Variance (ANOVA) using SAS 9.1 sof tware 2003 version. The Dif f erences in means were separated using Duncan Multiple Range Test (DMRT) at p˂0.05. Variation trends among the quantitative traits were established using Pearson Correlation Coef f icient and Principal Component Analysis (PCA).
The result in Table 2 shows the growth perf ormance of f ive Mango varieties. The variety of mango f rom Oyo is signif icantly (p<0.05) higher f or spouting days (0.58). The number of leaves per seedling (7.76), leaf area (34.86 cm 2 ), leaf length (17.06cm), Plant height (24.07cm) and lamina length (15.34cm) are higher in Ibadan variety. The Ogbomosho 2 variety was higher f or leaf length (16.32cm) and lamina length (16.25cm), while varieties f rom Ogbomosho 2 and Ibadan are signif icantly higher in leaf width at 4.53cm and 4.44cm respectively. The leaf area in varieties f rom Ogbomosho 2, Ibadan and Saki were higher, while the petiole length in varieties f rom Ogbomosho 1, Ogbomosho 2, Ibadan, Oyo and Saki were signif icantly higher. The Ogbomosho 1, Oyo and Saki varieties were signif icantly higher f or lamina length.
The growth perf ormance of Mango based on locations revealed signif icant dif f erence in table 3. The mangoes f rom Ogbomosho had the highest mean of 0.08 f or Sprouting Days, leaf length (15.30cm) and lamina length (14.74cm), while Saki produced the highest mean number of leaves per seedling (9.09), leaf area (32.57 cm 2 ), internodal length (26.74cm) and plant height (26.16 cm). The leaf width (4.63cm) and petiole length (2.12cm) had the highest f or Ibadan accession. Ogbomosho, Saki and Ibadan varieties were signif icantly higher for sprouting days (0.80), number of leaves per seedling (9.09), leaf length (15.30cm), leaf width (4.63), leaf area (32.57cm 2 ). The lamina length (14.74cm) in mango variety f rom Ogbomosho is significantly higher than other varieties. Table 4, shows the ef f ect of mean square interaction of location, replicate, varieties and weeks on growth related characters of Mango. The locations, accessions, weeks, f irst order interaction (location x accessions, location x week) and second order interaction (location x accessions x week) had higher signif icant (p<0.001) ef f ect on Sprouting days. The location, replicate, accessions and weeks, f irst order interaction (location x replicate, location x accessions, location x week, accessions x weeks) and second order (location x accessions x replicate, location x accessions x week) signif icantly af f ected the number of leaves per seedling. The leaf length, leaf width, leaf area, plant height and lamina length had higher signif icant ef f ect f or accessions, week, first order interaction (location x replicate, location x accessions, accessions x replicate, accessions x week) and second order interaction (location x accessions x replicate). The petiole length produced high signif icant ef f ect on location, replicate, accessions and f irst order interaction (location x accessions, location x week) ( Table 4).
Correlation coef f icient among the growth-related characters of Mango varieties at 5% level of signif icance (P≥0.05). The result of table 6 shows that the No of leaves per seedlings had a positive correlation with leaf length (r=0.53), leaf area (r=0.59), internodal length (r= 0.55) and strong positive correlation with plant height (r=0.73). Leaf length produced a strong positive association with leaf width (r=0.73), lamina length (r=0.99) has a positive correlation with plant height (r=0.53). Leaf width produced strong positive correlation of leaf length (r= 0.74); Leaf area produce positive correlation with plant height of (r=0.52).
The result in Table 7 shows the genetic distance among mango accessions. OROM Acc-3 (0.002), SHRIM Acc-1 (0.002) and OGBM Acc-6 with (0.002) genetic distance are closely related than OGBM Acc -1 (0.046) and GRAFE Acc-1 (0.114), while German Acc-2, OROM Acc-3, German Acc-3, SWMUI IDIA-2 and OYOM Acc-1 (0.000) are genetically related. Also, German Acc-3, OROM Acc-3, OGBM Acc-5, SWMUI IDIA-2 and OYOM Acc-2 (0.002) are closely related than OGBM Acc-1 (0.048) and Graf e (0.117). The GRAFE Acc-1 had higher genetic distance of 0.114 to 0.117 as compared to other mango accessions. Studies of genetic diversity based on molecular markers in the selected mango varieties revealed that location also played an important role in diversity. Genetic distance between varieties f rom the same location was most of ten lower. Sánchez-Guillén et al., (2011) had indicated the inf luence of location in genetic diversity studies, this might be responsible f or the close relationship between members originated f rom close locations.The success rate of amplif ied DNA p roducts and sequencing was 77.78%, and DNA sequencing showed 100% query cover which is identical to the mango on the Michigan Center f or Biological Inf ormation (MCBI) as similarly reported by Iquebal et al. (2017). Edited sequences were blasted in the NCBI data website and were identif ied to be similar to Mango accession KX871231.1, indicating the closeness of all varieties tested as shown in Table 8. However, the result f rom sequence analysis shows that sequencing region of amplif ied gene revealed genome size of 439Mbp, and this agrees with the reports of Singh (2016).
The result of each rbcL sequence f rom NCBI database shows that all the sequences of rbcL loci were identif ied as rbcL sequences of Mangifera indica in which most of them had identity of 99% and 100% coverage conf irming positive amplif ication and sequencing of the rbcL gene in mango varieties (Table 8).
Sequences of rbcL marker shows several genetic dif f erences among accessions especially in GRAFE Acc-1 and OGBM Acc-1 (LAUTECH 1) as they didn't cluster close to the other varieties (Figures 1 and 3).
The result in f igure 1 is the Dendrogram showing the relationships among accessions based on quantitative characters in f ruit. All accessions in the same clusters are similar or closely related to each other. Ogbomosho 11 (OGBM Acc-11) is more closely related to German 1 and related to German 2 as shown in cluster 1. Cluster 2 had 5 accessions with Oro Mango 3 more closely related to Cherry Mango (SHRIM Acc-1). Also, Lautech 1 (OGBM Acc-1) and Lautech 5 (OGBM Acc-5) are closely related to each other in sub clusters of 4. The result in f igure 2 is the Dendrogram showing the relationships among accessions based on quantitative characters in seed and pulp. It consists of two main clusters. Cluster 1 had 4 accessions while cluster 2 had 11 accessions. German 1 and Oyo Mango 3 are more closely related to each other and related to Oyo Mango 3 in sub cluster of 1. In Cluster 2, cherry mango (SHRIM) 1 and Oro Mango 3 (OROMAcc-3) are more closely related. Also, Surulere 7 (OGBM Acc-7) and Sweet Mango UI 2 are more closely related in dif f erent sub cluster of 2.
The dendrogram showing the relatedness between the 15 accessions of Mango is shown in Figure 3. The dendogram showed that the plant produced a close cluster with their most identical sequence in the NCB I except f or LAUTECH 6 (OGBM Acc-6), LAUTECH 1 (OGBM Acc-1) and Graf e Acc-1 which f ormed an out grouped. This implies that they were the most distantly related but closer to sweet mango UI (SWMUI IDIA-2), LAUTECH 5 (OGBM Acc-5) and Oyo mango 1 (OYOM Acc-1) this agrees with the observation made by Hartana (2010). The main group f ormed 2 major cluster with Oro Mango, German 3 mango, sweet mango and Surulere mango clustering together and closely related to the ref erence mango sequence while Oro Mango Acc-2, Oyo mango Acc-1, Sweet Mango UI, LAUTECH 5 and German Mango Acc-1 clustering together. Plate 1 ph otograph shows the gel obtained with Primer which reveals variation in mango accessions

CONCLUSION
There were morphological and molecular variations in mango varieties and accessions. Isehin Acc-1, Saki Acc-1 and OGBM Acc-6 accessions had better growth perf ormance. The mango f rom Ogbomoso and Saki locations had higher growth characters. The leaf length, leaf area, internodal length, plant height and number of leaves per seeding were best characters to be selected f or f urther breeding of mango. Hif and Fof ana were promising genes f or molecular analysis of mango.