Analysis of the Genetic Structure of Five Eucalyptus Grandis Populations and Generation of F1 Progenies From E. Grandis × E. Urophylla

Abstract

Eucalypts account for about 39600 ha of land in Kenya while other exotic species, Cupressus and Pinus, at 28,900 and 6,800 ha of land, respectively in the year 1999 though in 2009, the total area under eucalypts cultivation stood at 100, 000 ha. Nonetheless, in 2012, wood supply was estimated to be 31 million cubic meters while the demand was about 42 million cubic meters but, future forecasting shows that wood supply is set to increase to about 36 million cubic meters and a demand of about 51 million cubic meters by the year 2032. This demand could be addressed using planation forestry with trees such as eucalypts, but there is no active breeding program in Kenya due to insufficient information on genetic diversity and pollination techniques. The objectives of this study were to: i) determine the E. grandis genetic structure from selected candidate plus trees and their progenies using microsatellite markers from five main seed collection centers; and ii) generating F1 progenies from E. grandis and E. urophylla genotypes in Muguga, Kenya as a strategy of increasing genetic diversity. The genetic fingerprinting work, entailed genomic DNA extraction using CTAB method, DNA quality check and quantification using Nano drop nucleic acid analyzer, model Shimadzu Bio Spec-nano 206-26300-48, SSR screening and reconstitution using fluorescently labeled EMBRA primers, PCR amplification using Veriti™ 96-well thermal cycler and capillary electrophoresis by Applied Biosystems 3500 Genetic Analyzer. Data on allele frequency, allelic richness, gene diversity, heterozygosity and Polymorphic Information Content (PIC) then analyzed by Power Marker v3.2.5 Genetic distance matrices were generated by GenAlEx v6.5 and used for computing Principal Coordinates Analysis (PCoA) to visualize variations and similarities. Neighbor-joining trees were constructed using DARwin v6.0.21 where the resulting trees were visualized. Variation partitioning within and among group components was computed using Analysis of Molecular Variance (AMOVA) in GenAlEx v6.5 which facilitated estimation of broad sense heritability (H2), standardized allelic patterns across different xix families and populations and components of variance significance levels using 999 permutations. From analysis, results generally indicated high genetic diversity of 0.844 suggesting high genetic variability for possible exploitation in future breeding programs. The work on pollination involved collecting ripe flowers at anthesis stage of development, pollen extraction and examination of its characteristics, in-vitro pollen germination on liquid media to determine viability, pollen short-term storage at 4⁰C, controlled pollination by conventional and one-stop techniques and lastly seed extraction at maturity. Data was also collected on various parameters including morphological traits of pollen and mother parents, pollen germination rate on nutrient agar medium, flower diameter and length of artificially and naturally pollinated flowers. From this analysis, a success rate of around 28.6% was realized indicating that more Eucalyptus breeding programs can be established using Kenyan germplasm. Conclusively, the study confirmed that there is need for maximum utilization of genotypes from the main seed collection zones for heterosis or targeting of specific traits, and also ratified that different breeding objectives such as fast growth and disease resistance can be achieved through controlled pollination.