Haploid, diploid, and pooled exome capture recapitulate features of biology and paralogy in two non-model tree species

Despite their suitability for studying evolution, many conifer species have large and repetitive giga-genomes (16-31Gbp) that create hurdles to producing high coverage SNP datasets that captures diversity from across the entirety of the genome. Due in part to multiple ancient whole genome duplication events, gene family expansion and subsequent evolution within Pinaceae, false diversity from the misalignment of paralog copies create further challenges in accurately and reproducibly inferring evolutionary history from sequence data. Here, we leverage the cost-saving benefits of pool-seq and exome-capture to discover SNPs in two conifer species, Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirb.) Franco, Pinaceae) and jack pine (Pinus banksiana Lamb., Pinaceae). We show, using minimal baseline filtering, that allele frequencies estimated from pooled individuals show a strong positive correlation with those estimated by sequencing the same population as individuals (r > 0.948), on par with such comparisons made in model organisms. Further, we highlight the use of haploid megagametophyte tissue in identifying sites that are likely due to misaligned paralogs. Together with additional minor filtering, we show that it is possible to remove many of the loci with large frequency estimate discrepancies between individual and pooled sequencing approaches, improving the correlation further (r > 0.973). Our work addresses bioinformatic challenges in non-model organisms with large and complex genomes, highlights the use of megagametophyte tissue for the identification of paralog sites when sequencing large numbers of populations, and suggests the combination of pool-seq and exome capture to be robust for further evolutionary hypothesis testing in these systems.

Utilization of Tissue Ploidy Level Variation in de Novo Transcriptome Assembly of Pinus sylvestris

Compared to angiosperms, gymnosperms lag behind in the availability of assembled and annotated genomes. Most genomic analyses in gymnosperms, especially conifer tree species, rely on the use of de novo assembled transcriptomes. However, the level of allelic redundancy and transcript fragmentation in these assembled transcriptomes, and their effect on downstream applications have not been fully investigated. Here, we assessed three assembly strategies for short-reads data, including the utility of haploid megagametophyte tissue during de novo assembly as single-allele guides, for six individuals and five different tissues in Pinus sylvestris. We then contrasted haploid and diploid tissue genotype calls obtained from the assembled transcriptomes to evaluate the extent of paralog mapping. The use of the haploid tissue during assembly increased its completeness without reducing the number of assembled transcripts. Our results suggest that current strategies that rely on available genomic resources as guidance to minimize allelic redundancy are less effective than the application of strategies that cluster redundant assembled transcripts. The strategy yielding the lowest levels of allelic redundancy among the assembled transcriptomes assessed here was the generation of SuperTranscripts with Lace followed by CD-HIT clustering. However, we still observed some levels of heterozygosity (multiple gene fragments per transcript reflecting allelic redundancy) in this assembled transcriptome on the haploid tissue, indicating that further filtering is required before using these assemblies for downstream applications. We discuss the influence of allelic redundancy when these reference transcriptomes are used to select regions for probe design of exome capture baits and for estimation of population genetic diversity.

Utilization of tissue ploidy level variation in de novo transcriptome assembly of Pinus sylvestris

Compared to angiosperms, gymnosperms lag behind in the availability of assembled and annotated genomes. Most genomic analyses in gymnosperms, especially conifer tree species, rely on the use of de novo assembled transcriptomes. However, the level of allelic redundancy and transcript fragmentation in these assembled transcriptomes, and their effect on downstream applications have not been fully investigated. Here, we assessed three assembly strategies, including the utility of haploid (megagametophyte) tissue during de novo assembly as single-allele guides, for six individuals and five different tissues in Pinus sylvestris. We then contrasted haploid and diploid tissue genotype calls obtained from the assembled transcriptomes to evaluate the extent of paralog mapping. The use of the haploid tissue during assembly increased its completeness without reducing the number of assembled transcripts. Our results suggest that current strategies that rely on available genomic resources as guidance to minimize allelic redundancy are less effective than the application of strategies that cluster redundant assembled transcripts. The strategy yielding the lowest levels of allelic redundancy among the assembled transcriptomes assessed here was the generation of SuperTranscripts with Lace followed by CD-HIT clustering. However, we still observed some levels of heterozygosity (multiple gene fragments per transcript reflecting allelic redundancy) in this assembled transcriptome on the haploid tissue, indicating that further filtering is required before using these assemblies for downstream applications. We discuss the influence of allelic redundancy when these reference transcriptomes are used to select regions for probe design of exome capture baits and for estimation of population genetic diversity.

DNA content for Asian pines parallels New World relatives

This hypothesis is based on an observed correlation between DNA content and thermal regime for fish, zooplankton, salamanders, and some flowering plants. The Asian pine results provided no support for the latitudinal hypothesis; Asian tropical pine species did not have smaller genomes than their temperate or boreal relatives. DNA content of haploid megagametophyte tissue varied from 21.85 pg/C for hard pine Pinus densiflora Sieb. & Zucc. to 29.59 pg/C for soft pine Pinus bungeana Zucc. Pinus merkusii Jung. & De Vriese (29.63 pg/C) was the exceptional hard pine, with a genome size larger than many soft pines. The mean DNA content of Asian soft pines exceeded Asian hard pines (Δ 3.22 pg/C), a parallel to the previously reported trends for New World pines. No continental effect was detected. Based on 46 pines species sampled in centers of species diversity in Asian and the New World, soft pines had mean DNA content which exceeded hard pines by 4.97 pg/C.Key words: gymnosperms, conifers, laser flow cytometry, megagametophytes, C values, phylogeny.

Embryogenesis from protoplasts of haploid European larch

Haploid megagametophyte-derived lines of European larch (Larixdecidua Mill.) displaying different degrees of embryogenicity had their protoplasts isolated and cultured. Embryoids were produced from two lines. Colonies of dividing cells but no embryoids were produced from the third, least embryogenic, line. The isolation procedure consisted of preplasmolysis in 0.44 M mannitol for 1 h, followed by enzyme digestion of the cell walls using cellulase and Macerozyme. The released protoplasts were separated from the digestion mixture by screening through cheesecloth and Nitex meshes and by differential gradient centrifugation. The protoplasts were plated in modified half-strength LM medium solidified with agarose. At the end of 1 week the agarose was suspended in a regeneration medium that had lower osmolarity. Osmotic levels were progressively lowered over a period lasting 3 weeks. An alternative method for osmotic reduction was used in which protoplasts were transferred directly from regeneration to maintenance medium. In either method, regeneration of protoplasts occurred in agarose. Regenerated embryoids underwent limited development in suspension, so maturation was completed by plating them onto semisolid half-strength LM medium, and they were raised in the dark for another month. Development from single protoplasts is described, and time-lapse illustrations of colony development from multinucleate protoplasts (coenocytes) are also included.

Inheritance and linkage of allozymes in seed tissue of whitebark pine

Allozyme variants were examined for 12 enzyme systems in haploid megagametophyte tissue of whitebark pine (Pinus albicaulis Engelm.). Inheritance and linkage information is reported for 11 loci (Aat-3, Aco, Adh, Fle-2, Idh, Mdh-2, Mdh-3, Mdh-4, Mdr, Pgi-2, and Pgm-2). Deviations from a 1:1 segregation ratio were observed at Fle-2, Pgi-2, and between two of the three alleles at Mdh-4. Significant linkage was observed for two pairs of loci (Aco–Fle-2 and Adh–Pgi-2), but estimates of recombination frequencies were relatively high.Key words: Pinus albicaulis, isozymes.

Inheritance and linkage relationships of some isozymes of black spruce in New Brunswick

Thirteen polymorphic loci from eleven enzyme systems in black spruce (Piceamariana (Mill.) B.S.P.) were studied by gel electrophoresis using embryos and haploid megagametophyte tissue. Two loci (Mdh-1 and Sdh) showed deviations from the expected 1:1 segregation ratio of megagametophytes from heterozygous maternal plants that were significant at the 1 % level and a third (Aat) at the 5% level. Of the 78 possible pairwise comparisons for the linkage analysis, 58 could be tested for jointly independent segregation. Linkage could be detected between three pairs of loci: Aat–Pgi, Gdh–Idh, and Fum–Pgm.

Inheritance and linkage of isozymes in white spruce (Picea glauca)

Variation patterns for 26 loci of white spruce (Picea glauca (Moench) Voss) from 16 enzyme systems were studied using haploid megagametophyte tissue. Inheritance models for 17 polymorphic loci were formulated and tested for deviations from Mendelian segregation. Linkage analyses were conducted for 12 of the polymorphic loci in pairwise comparisons. Significant joint segregation was found for three pairs of loci: Pgi-2 with Aat-1, Gdh with Idh, and one of three families tested with Pgi-2 and 6pg-2,3.