Pinus patula is the single most important commercial plantation forest tree species in South Africa. It accounts for 52% approximately (700,000 ha) of total commercial plantation area in the country and is utilised for saw logs and pulp and paper production. P. patula is a tropical pine species indigenous to Mexico. Excellent ex situ conservation and range-wide provenance trials have been established for P. patula in South Africa and South America. These highly organised trials provide the opportunity to perform association genetic studies with the long term aim to identify trait linked markers for future molecular improvement of P. patula. In this study, the first gene-based assessment of allelic diversity in P. patula was performed. This pilot study focused on two cellulose biosynthetic genes as representatives of wood formation genes and assayed molecular evolution parameters such as nucleotide diversity, allelic diversity and linkage disequilibrium (LD) in a species-wide reference population of P. patula. Two novel cellulose synthase (CesA) genes were isolated and characterised in P. patula. One of these genes, PpCesA1, is putatively involved in the biosynthesis of secondary cell walls of tissues such as xylem (wood), while the other, PpCesA2 is proposed to be associated with primary cell wall formation in rapidly growing tissue types. The genomic DNA copies of PpCesA1 and PpCesA2 were 6025 bp and 6365 bp in length, respectively. The corresponding cDNA sequences encoded 1083 and 1058 amino acids, respectively, and differed considerably from each other (73% amino acid identity). Both amino acid sequences contained the key domains and motifs characteristic of functional CESA proteins isolated in other higher plants. Phylogenetic analysis revealed that PpCesA1 was most similar (99%) to its putative ortholog in Pinus taeda, PtCesA3, and PpCesA2 was highly similar to a putative ortholog in Pinus radiata, PrCesA2 (99% identity). This phylogenetic analysis supported previous findings that the divergence between the primary and secondary cell wall associated CESA proteins occurred before the divergence of angiosperms and gymnosperms approximately 300 million years ago. A fragment of a putative paralogous gene copy of PpCesA1, named PpCesA1-B was also isolated. The PpCesA1-B gene fragment was found to differ from PpCesA1 by 22 nucleotide polymorphisms and its non-allelic (paralogous) status was confirmed by segregation analysis in P. patula. In order to gain an understanding of molecular genetic variation that might affect wood formation in P. patula, we sequenced multiple allelic variants of PpCesA1, PpCesA1-B and PpCesA2, which we sampled from a species-wide reference population of P. patula. The average levels of nucleotide diversity were found to be low for all three genes (π ≈ 0.0015), which may be a property of functional members of the CesA gene family. As a result of the low nucleotide diversity, only small numbers of pair-wise informative sites were available for LD analysis and the decay in LD could only be studied in PpCesA2 where it was found to decay very rapidly (within 200 bp). Tests of neutrality suggested that the exon sequences of PpCesA1 and PpCesA2 were under significant positive (adaptive) selection. Comparison of levels of nucleotide diversity and selection in different parts of the two genes indicated that the highest levels of adaptive selection occurred in areas where amino acid substitutions could alter protein structure or function. This study provides valuable insights for designing future allele discovery efforts in P. patula with the ultimate goal of developing gene-based markers for the molecular improvement of wood formation in this tree species.
Dissertation (MSc (Genetics))--University of Pretoria, 2009.