Barry Goldfarb

Association Mapping of Quantitative Disease Resistance in a Natural Population of Loblolly Pine (Pinus taeda L.)

Genetic resistance to disease incited by necrotrophic pathogens is not well understood in plants. Whereas resistance is often quantitative, there is limited information on the genes that underpin quantitative variation in disease resistance. We used a population genomic approach to identify genes in loblolly pine (Pinus taeda) that are associated with resistance to pitch canker, a disease incited by the necrotrophic pathogen Fusarium circinatum. A set of 498 largely unrelated, clonally propagated genotypes were inoculated with F. circinatum microconidia and lesion length, a measure of disease resistance, data were collected 4, 8, and 12 weeks after inoculation. Best linear unbiased prediction was used to adjust for imbalance in number of observations and to identify highly susceptible and highly resistant genotypes (“tails”). The tails were reinoculated to validate the results of the full population screen. Significant associations were detected in 10 single nucleotide polymorphisms (SNPs) (out of 3938 tested). As hypothesized for genes involved in quantitative resistance, the 10 SNPs had small effects and proposed roles in basal resistance, direct defense, and signal transduction. We also discovered associated genes with unknown function, which would have remained undetected in a candidate gene approach constrained by annotation for disease resistance or stress response.

Morphological and chemical variations between juvenile wood, mature wood, and compression wood of loblolly pine (Pinus taeda L.)

Abstract To better understand the within-tree variations between juvenile wood, mature wood, and compression wood, wood from a 35-year-old mature bent loblolly pine was separated into seven groups by different positions in the tree. Morphological and chemical structure analyses, including fiber quality, X-ray diffraction, sugar and lignin content analysis, as well as nitrobenzene oxidation, ozonation, and advanced NMR spectroscopy, were performed. Fiber properties were significantly different for tree-top juvenile normal wood and tree-bottom juvenile normal wood, juvenile normal and mature normal wood, juvenile compression and mature compression wood. However, differences in the chemical structure and composition were less significant within the specific tissues indicated above.

Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants

A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata cv. ‘Crandon’) suspension cultures and regeneration of transformed plants is described. Transformants were recovered when suspension cultures were inoculated with Agrobacterium tumefaciens at a density of 107 colony-forming units ml-1, cocultivated for 48 h, and plated to cellulose acetate filters on Woody Plant Medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 250 mg l-1 cefotaxime. Levels of cefotaxime greater than 250 mg l-1 were unnecessary for control of residual bacteria and inhibited callus growth. Transgenic plants were regenerated by culturing the transformed callus on media containing 0.11 to 27 μM thidiazuron. In contrast to thidiazuron, N6-benzyladenine had a negative effect on shoot regeneration; the callus became necrotic when we attempted to induce shoots with concentrations of 1.1 to 8.9 μM, and growth was inhibited when concentrations of 0.11 or 0.22 μM were used to regenerate callus from suspension cultures. Following cocultivation of poplar suspension cultures, we recovered transgenic plants containing the maize transposon Ac, and callus containing an insect toxin gene from Bacillus thuringiensis.

Association genetics of the loblolly pine (Pinus taeda, Pinaceae) metabolome

The metabolome of a plant comprises all small molecule metabolites, which are produced during cellular processes. The genetic basis for metabolites in nonmodel plants is unknown, despite frequently observed correlations between metabolite concentrations and stress responses. A quantitative genetic analysis of metabolites in a nonmodel plant species is thus warranted. Here, we use standard association genetic methods to correlate 3563 single nucleotide polymorphisms (SNPs) to concentrations of 292 metabolites measured in a single loblolly pine (Pinus taeda) association population. A total of 28 single locus associations were detected, representing 24 and 20 unique SNPs and metabolites, respectively. Multilocus Bayesian mixed linear models identified 2998 additional associations for a total of 1617 unique SNPs associated to 255 metabolites. These SNPs explained sizeable fractions of metabolite heritabilities when considered jointly (56.6% on average) and had lower minor allele frequencies and magnitudes of population structure as compared with random SNPs. Modest sets of SNPs (n = 1-23) explained sizeable portions of genetic effects for many metabolites, thus highlighting the importance of multi-SNP models to association mapping, and exhibited patterns of polymorphism consistent with being linked to targets of natural selection. The implications for association mapping in forest trees are discussed.

An auxin-inducible gene from loblolly pine (Pinus taeda L.) is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein.

We have isolated a gene from loblolly pine, 5NG4, that is highly and specifically induced by auxin in juvenile loblolly pine shoots prior to adventitious root formation, but substantially down-regulated in physiologically mature shoots that are adventitious rooting incompetent. 5NG4 was highly auxin-induced in roots, stems and hypocotyls, organs that can form either lateral or adventitious roots following an auxin treatment, but was not induced to the same level in needles and cotyledons, organs that do not form roots. The deduced amino acid sequence shows homology to the MtN21 nodulin gene from Medicago truncatula. The expression pattern of 5NG4 and its homology to a protein from Medicago involved in a root-related process suggest a possible role for this gene in adventitious root formation. Homology searches also identified similar proteins in Arabidopsis thaliana and Oryza sativa. High conservation across these evolutionarily distant species suggests essential functions in plant growth and development. A 38-member family of genes homologous to 5NG4 was identified in the A. thaliana genome. The physiological significance of this redundancy is most likely associated with functional divergence and/or expression specificity of the different family members. The exact biochemical function of the gene is still unknown, but sequence and structure predictions and 5NG4::GFP fusion protein localizations indicate it is a transmembrane protein with a possible transport function.

Association genetics of carbon isotope discrimination, height and foliar nitrogen in a natural population of Pinus taeda L

Loblolly pine, Pinus taeda L., is one of the most widely planted, commercially and ecologically important tree species in North America. We took an association genetics approach, using an unimproved population of 380 clonally replicated unrelated trees, to test 3938 single nucleotide polymorphisms (SNPs) in as many genes for association with phenotypic variation in carbon isotope discrimination, foliar nitrogen concentration and total tree height after two growing seasons. Best linear unbiased prediction (BLUP) was used with a spatial adjustment to remove environmental variation from phenotypic data derived from a common garden experiment. After correction for multiple testing, a total of 14 SNPs were associated with the traits of carbon isotope discrimination (n=7), height (n=1) and foliar nitrogen concentration (n=6) using 380 clones. Tails of the population phenotypic distribution were compared for allele frequency differences, revealing 10 SNPs with allele frequency in at least one tail significantly different from the overall population. Eight associated SNPs were in sequences similar to known genes, such as an AP2 transcription factor related to carbon isotope discrimination and glutamate decarboxylase associated with foliar nitrogen concentration, and others were from unknown genes without homologs in Arabidopsis.

Comparison of morphological and chemical properties between juvenile wood and compression wood of loblolly pine

Abstract In conifers, juvenile wood (JW) is always associated with compression wood (CW). Due to their similar properties, there is a common belief that JW is the same as CW. To resolve whether JW is identical to CW, 24 rooted cuttings of one loblolly pine clone were planted in growth chambers under normal, artificial bending, and windy environments. The results show that the morphology of JW is significantly different from CW. Furthermore, chemical analyses revealed that JW and CW are significantly different in chemical composition. Our results indicate that JW is different from CW, and the wood formed under a controlled windy environment is a mild type of compression wood.

Genetic analysis of early field growth of loblolly pine clones and seedlings from the same full-sib families

Field trials established with clones and seedlings from the same families provide an opportunity for comparing full-sib family performance across propagule types. More than 1200 different clones together with over 14 000 zygotic seedlings from the same 61 full-sib families of loblolly pine (Pinus taeda L.) were tested on multiple sites across Florida and Georgia. The genetic variance associated with several early growth traits partitioned differently depending on propagule type. Most of the genetic variance associated with growth in the clonal population was additive, while the estimate of dominance in the seedling population was greater than estimates of dominance in the clonal population, based on single-site analyses. Apparently, a lack of randomization of the seedlings prior to field establishment caused full-sib families to appear more different, inflating estimates of dominance genetic variance. Parental and full-sib family ranks were stable regardless of propagule type as indicated by type B geneti...

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons

SummaryThe effects of high-concentration, 2-h liquid pulses of N6-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 μM BA, and 100 and 200 μM TD. Shoots developed best from buds induced with 300, 400, and 800 μM BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 μM BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.

Genetic gain from selection for rooting ability and early growth in vegetatively propagated clones of loblolly pine

A successful clonal forestry program for loblolly pine based on rooted cutting technology needs to consider selection for both rooting ability and subsequent field growth. Rooting ability and second-year height were assessed in more than 2,000 clones from 70 full-sib families of loblolly pine. The bivariate analysis of rooting ability from five rooting trials and field growth from six field trials allowed for estimation of the genetic covariance between rooting ability and second-year height for parental effects, full-sib family effects, and the total genetic value of clones within full-sib family. There was a positive genetic relationship between rooting ability and second-year height at all three genetic levels. The genetic correlation at the parental level between rooting ability and second-year height