Huhu grubs (Prionoplus reticularis) are wood-feeding beetle larvae endemic to New Zealand and belonging to the family Cerambycidae. Compared to the wood-feeding lower termites, very little is known about the diversity and activity of microorganisms associated with xylophagous cerambycid larvae. To address this, we used pyrosequencing to evaluate the diversity of metabolically active and inactive bacteria in the huhu larval gut. Our estimate, that the gut harbors at least 1,800 phylotypes, is based on 33,420 sequences amplified from genomic DNA and reverse-transcribed RNA. Analysis of genomic DNA-and RNA-derived data sets revealed that 71% of all phylotypes (representing 95% of all sequences) were metabolically active. Rare phylotypes contributed considerably to the richness of the community and were also largely metabolically active, indicating their participation in digestive processes in the gut. The dominant families in the active community (RNA data set) included Acidobacteriaceae (24.3%), Xanthomonadaceae (16.7%), Acetobacteraceae (15.8%), Burkholderiaceae (8.7%), and Enterobacteriaceae (4.1%). The most abundant phylotype comprised 14% of the active community and affiliated with Dyella ginsengisoli (Gammaproteobacteria), suggesting that a Dyella-related organism is a likely symbiont. This study provides new information on the diversity and activity of gutassociated microorganisms that are essential for the digestion of the nutritionally poor diet consumed by wood-feeding larvae. Many huhu gut phylotypes affiliated with insect symbionts or with bacteria present in acidic environments or associated with fungi.

Development of genome-wide resources for application in genomic selection or genome-wide association studies, in the absence of full reference genomes, present a challenge to the forestry industry, where longer breeding cycles could benefit from the accelerated selection possible through marker-based breeding value predictions. In particular, large conifer megagenomes require a strategy to reduce complexity, whilst ensuring genome-wide coverage is achieved. Using a transcriptome-based reference template, we have successfully developed a high density exome capture genotype-by-sequencing panel for radiata pine (Pinus radiata D.Don), capable of capturing in excess of 80,000 single nucleotide polymorphism (SNP) markers with a minor allele frequency above 0.03 in the population tested. This represents approximately 29,000 gene models from a core set of 48,914 probes. A set of 704 SNP markers capable of pedigree reconstruction and differentiating individual genotypes were tested within two full-sib mapping populations. While as few as 70 markers could reconstruct parentage in almost all cases, the impact of missing genotypes was noticeable in several offspring. Therefore, 60 sets of 110 randomly selected SNP markers were compared for both parentage reconstruction and clone differentiation. The performance in parentage reconstruction showed little variation over 60 iterations. However, there was notable variation in discriminatory power between closely related individuals, indicating a higher density SNP marker panel may be required to elucidate hidden relationships in complex pedigrees.

BackgroundNon-key traits (NKTs) in radiata pine (Pinus radiata D. Don) refer to traits other than growth, wood density and stiffness, but still of interest to breeders. Branch-cluster frequency, stem straightness, external resin bleeding and internal checking are examples of such traits and are targeted for improvement in radiata pine research programmes. Genomic selection can be conducted before the performance of selection candidates is available so that generation intervals can be reduced. Radiata pine is a species with a long generation interval, which if reduced could significantly increase genetic gain per unit of time. The aim of this study was to evaluate the accuracy and predictive ability of genomic selection and its efficiency over traditional forward selection in radiata pine for the following NKTs: branch-cluster frequency, stem straightness, internal checking, and external resin bleeding.ResultsNine hundred and eighty-eight individuals were genotyped using exome capture genotyping by sequencing (GBS) and 67,168 single nucleotide polymorphisms (SNPs) used to develop genomic estimated breeding values (GEBVs) with genomic best linear unbiased prediction (GBLUP). The documented pedigree was corrected using a subset of 704 SNPs. The percentage of trio parentage confirmed was about 49% and about 50% of parents were re-assigned. The accuracy of GEBVs was 0.55–0.75 when using the documented pedigree and 0.61–0.80 when using the SNP-corrected pedigree. A higher percentage of additive genetic variance was explained and a higher predictive ability was observed when using the SNP-corrected pedigree than using the documented pedigree. With the documented pedigree, genomic selection was similar to traditional forward selection when assuming a generation interval of 17 years, but worse than traditional forward selection when assuming a generation interval of 14 years. After the pedigree was corrected, genomic selection led to 37–115% and 13–77% additional genetic gain over traditional forward selection when generation intervals of 17 years and 14 years were assumed, respectively.ConclusionIt was concluded that genomic selection with a pedigree corrected by SNP information was an efficient way of improving non-key traits in radiata pine breeding.

There is a wide diversity of bioinformatic tools available for the assembly of next generation sequence and subsequence variant calling to identify genetic markers at scale. Integration of genomics tools such as genomic selection, association studies, pedigree analysis and analysis of genetic diversity, into operational breeding is a goal for New Zealand’s most widely planted exotic tree species, Pinus radiata. In the absence of full reference genomes for large megagenomes such as in conifers, RNA sequencing in a range of genotypes and tissue types, offers a rich source of genetic markers for downstream application. We compared nine different assembler and variant calling software combinations in a single transcriptomic library and found that Single Nucleotide Polymorphism (SNPs) discovery could vary by as much as an order of magnitude (8,061 SNPs up to 86,815 SNPs). The assembler with the best realignment of the packages trialled, Trinity, in combination with several variant callers was then applied to a much larger multi-genotype, multi-tissue transcriptome and identified 683,135 in silico SNPs across a predicted 449,951 exons when mapped to the Pinus taeda ver 1.01e reference.

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.