TcMYB1 , TcMYB4 , and TcMYB8 participate in the regulation of lignan biosynthesis in Taiwania cryptomerioides Hayata Abstract Taiwania (Taiwania cryptomerioides Hayata) is a conifer species that is rich in bioactive secondary metabolites. Lignans, which are the major extracts, are especially important, but the biosynthetic gene regulation and downstream biosynthesis pathways remain unclear. In a previous study, we identified three pinoresinol-lariciresinol reductase (PLR) genes in Taiwanina, and in the present study, the regulation of these PLR genes was investigated. Through next-generation transcriptome sequencing, gene co-expression network analysis was performed to predict the relationship between putative transcription factor genes and upstream biosynthetic genes. Three MYB genes were identified as putative lignan biosynthetic regulators and named TcMYB1, TcMYB4, and TcMYB8. The gene expression of TcMYBs and TcPLRs was further confirmed with real-time PCR. Finally, an Agrobacteria-mediated transient overexpression experiment was conducted, and the activation of proTcPLR3::YFP by TcMYBs suggested that TcMYB1, TcMYB4, and TcMYB8 are biosynthetic regulators of lignan. These lignan regulators can be used in further research and applied to achieve a higher lignan content.

Transcriptome-wide analysis to dissect the transcription factors orchestrating the phase change from vegetative to reproductive development in Larix kaempferi Abstract The timing of phase change from vegetative to reproductive development during aging of forest trees is important for wood and seed production. In a previous study, we investigated the effects of aging on wood formation by measuring the transcriptomic changes in the uppermost main stems of 1-, 2-, 5-, 10-, 25-, and 50-year-old Larix kaempferi. Based on the published transcriptome data, here we investigated the transcriptomic differences between the juvenile vegetative (1- and 2-year-old) and adult reproductive (25- and 50-year-old) phases to determine the molecular mechanisms underlying the phase change. In total, 12,789 transcripts were identified as differentially expressed genes, including 573 transcription factors. Further analysis showed that 27 transcription factors belonging to 8 families were common to all four comparisons between old and young life stage categories: (I) 25 vs 1 year old; (II) 50 vs 1 year old; (III) 25 vs 2 years old; and (IV) 50 vs 2 years old. The analysis of their expression patterns in six age categories showed that members of the AP2 and Dof families were expressed highly in 1- and 2-year-old trees, weakly in 25- and 50-year-old trees, while members of the C3H, G2-like, GRAS, MYB-related, and MADS families had the opposite patterns. Notably, one member of the MADS family was only detected in 25- and 50-year-old trees. These results suggest that the phase change might (1) occur in the early stage of the L. kaempferi lifetime and (2) be controlled by a complex regulatory network of different transcription factors, some of which are known to play roles in the phase change in model plants. These findings not only provide molecular markers to distinguish different stages of tree growth and development and potential targets for genetic manipulation to improve the reproductive traits of trees, but also improve our understanding of the phase change with aging in trees.

Genetic diversity and structure of Populus nigra populations in two highly fragmented river ecosystems from Turkey Abstract European black poplar (Populus nigra L.) is an important tree species in terms of social, economic, and ecological interest in Turkey. Although large poplar plantations meet the needs of the economy, natural genetic resources of the species have been highly degraded due to anthropogenic effects such as overexploitation and habitat fragmentations. To assess genetic diversity and structure of fragmented populations, 124 naturally distributed European black poplar trees from two major rivers (Kızılırmak and Göksu) in Turkey were sampled and screened by using 20 nuclear microsatellite DNA loci. To detect the possibility of natural hybridization, 10 reference Populus deltoids L. trees were studied with six microsatellite loci. Out of 124, five trees were determined as F2 hybrids with diagnostic and informative alleles. Studied populations appear to have experienced a recent bottleneck event which is likely to cause to reduction in allelic diversity and to increase heterozygosity (mean Ho = 0.80). Four populations representing upstream, middle and downstream parts of the Kızılırmak River were found to be differentiated from Göksu river population, evidenced by genetic differentiation (FST = 0.06), geographical distances in principal coordinate analysis and clustering pattern in structure analysis. Traditional management of European black poplar coupled with bottleneck and hybridization events has played an important role in reduced genetic diversity and degradation of the genetic resources of the species in two river systems. The study has provided invaluable information to prevent further genetic degradation, to maintain existing genetic diversity of the species in its natural habitats, and to implement efficient conservation strategies and breeding programs for future generations.

An integrated genetic linkage map of Bixa orellana L. Abstract Bixa orellana L. is a plant from the Bixaceae family commercialized for its high bixin contents. Recently, demand for this pigment has led to the development of strategies for its genetic improvement. Through molecular markers obtained by sequence-related amplified polymorphism (SRAP), one β-cyclase marker and the two-way “pseudo-testcross” strategy an integrated genetic linkage map of Bixa orellana L. has been developed. Genetic maps were constructed for each parent and an integrated map using an F1 population with 157 individuals was produced. A total of 167 markers were used in linkage analysis. The maternal map covers a length of 306 cM, consisting of 52 markers with an average distance between them of 13.21 cM, distributed over seven linkage groups. The paternal map covers a length of 509 cM, consisting of 70 markers with an average distance between them of 12.28 cM, distributed over 12 linkage groups. The integrated genetic map covers a length of 879.054 cM, comprising 103 markers (102 SRAP and the β-cyclase), of which, 68% segregated in a 1:1 ratio and 32% in a 3:1 ratio, with an average distance of 14.6 cM between them, and distributed over 20 linkage groups. The size of the B. orellana genome was estimated between 1514 and 1728 cM. This study provides a basis for future construction of a high-density map and for genomic studies of B. orellana, in relation to bixin production and fruit dehiscence. It will also be useful in marker-assisted selection (MAS) within breeding programs of B. orellana and other species.

RAD designer : a workflow to select the optimal sequencing methodology in genotyping experiments on woody plant species Abstract Polymorphism detection in woody plant species is important in phylogenetics and an essential stage in plant breeding programs. Genotyping has benefitted from the development of next-generation sequencing (NGS) techniques. Methodologies based upon single and double-digested DNA sequencing (RADseq and ddRADseq) allow the detection of polymorphisms on thousands of loci in non-model organisms for which a reference genome is not available, such as many woody plant species. However, experiments using these methodologies are not safe from technical errors, and require careful designs in order to reduce library preparation and sequencing costs at the same time unbiased results are obtained. In order to avoid undesired effects of technical errors on genotyping, it is advisable to incorporate error quantification through pilot sequencing of technical replicates, along with filtering strategies and parameter tuning, so the technical conditions of the whole experiment can be properly defined a priori. However, these kinds of approaches to account for technical errors have been frequently disregarded in the literature. Here, we present RADdesigner, a workflow that combines pilot sequencing of replicate samples with a bioinformatic pipeline to select the most suitable methodology by quantifying genotyping errors, to optimize polymorphism detection parameters, and to perform post-filtering of the results produced by the commonly used RADseq bioinformatic pipeline IpyRAD. The workflow was validated using a pilot study that analyzed a subset of replicate samples of Quercus suber, Q. ilex, and their hybrids.

Construction of a high-density genetic map of tree peony ( Paeonia suffruticosa Andr. Moutan ) using restriction site associated DNA sequencing (RADseq) approach Abstract High-density genetic maps play a vital role in dissecting genetic components of biologically or agronomically important traits and molecular marker-assisted selection breeding, especially for a species with limited genomic information. Tree Peony (Paeonia suffruticosa Andr. Moutan DC.) is a traditional flowering plant in China, with important ornamental value, which have been spread around the world. However, less genetic and genomic information is available for the tree peony molecular studies. Here, we performed restriction site-associated DNA sequencing (RADseq) of an F1 population, derived from a traditional variety “Qing Long Wo Mo Chi (QL)” and a variety “Mo Zi Lian (MZL)”. High-density genetic maps were developed using RADseq markers for female (1471 markers) and male (793 markers) parents. They covered 965.69 and 870.21 cM, with the average marker intervals of 0.66 and 1.10 cM along the seven and five linkage groups (LGs), respectively. Furthermore, the identified markers were assigned to 1671 bins, representing the unique genetic positions in LGs, and inter-marker distances smaller than 5 cM covered 97.93% of genetic maps. This study suggests that rapid de novo construction of genetic maps in peony could be conducted through RADseq approach, which provides an important tool for further linkage mapping and genomic structure analysis, and enable development of molecular markers for molecular breeding in tree peony.

Genetic diversity of ten black walnut ( Juglans nigra L.) cultivars and construction of a mapping population Abstract Black walnut (Juglans nigra L.) nut production stands on the brink of potentially great market expansion that can only be realized with significant genetic and horticultural improvement, such that commercial production becomes feasible. The goal of this study was to incorporate microsatellite/simple sequence repeat (SSR) markers to genotype 11 cultivars and selections, and establish a mapping population from a cross of two cultivars, “Football” × “Sparrow.” Morphology of these two cultivars coupled with phylogenetic analysis supports the notion that a cross between them may yield desirable hybrids. Of the 51 SSR markers amplified, 23 were polymorphic and useful for genotyping. A selection mislabeled as “Mintyle” was definitively identified as “Mintle”; therefore, the number of cultivars was reduced to 10. Furthermore, seven of the 23 markers were used to identify 91 intraspecific hybrids (“Football” × “Sparrow”) resulting from open pollination, thereby establishing the first mapping population of black walnut using SSR markers.

Structure and genetic diversity in wild and cultivated populations of Zapote mamey ( Pouteria sapota , Sapotaceae) from southeastern Mexico: its putative domestication center Abstract Tropical fruit trees are an important component of the human diet; however, little is known about their genetic diversity levels. Zapote mamey (Pouteria sapota) is a tree native to southeastern Mexico and Central America, and Mexico is the leading producer in the world. Studies of the genetic diversity of Zapote mamey have been based on cultivated materials using morphological and biochemical characterization or dominant molecular markers. To gain a deeper understanding about the conservation status of Zapote mamey in its center of origin and domestication, we collected 188 individuals from eight wild and five cultivated populations in southeastern Mexico and characterized them using eight microsatellite loci. STRUCTURE, 3D-PCoA, and neighbor-joining analyses showed three groups in the wild gene pool and one group in the cultivated gene pool. FST values were significant between wild and cultivated gene pools, among the four groups observed and among the 13 populations collected (0.13, 0.25, and 0.36, respectively). Overall, we found low levels of genetic diversity (A = 2.77, HO = 0.29, HE = 0.39), permutation tests did not show significant differences between wild and cultivated gene pools. The Garza–Williamson index showed low values in both gene pools (wild = 0.16, cultivated = 0.11) and the Bottleneck program indicated a decrease in genetic diversity in both gene pools (wild, P = 0.027; cultivated, P = 0.054); both analyses suggest a potential genetic bottleneck within this species. This study can help to generate adequate sampling techniques and to develop effective management strategies for Zapote mamey of southeastern Mexico.

Characterizing the genetic diversity of Atlas cedar and phylogeny of Mediterranean Cedrus species with a new multiplex of 16 SSR markers Abstract Cedar is an emblematic Mediterranean forest tree. Genetic research activities on Mediterranean Cedrus species are progressively developed in relation to conservation, restoration, and exploitation plans of these forest tree species. However, currently available molecular biology tools supporting genetic research in these fields are still scarce and have a limited genetic resolution potential. We developed a new set of 12 nuclear microsatellite markers (nSSRs) on Cedrus atlantica that we combined with four previously developed ones in three multiplexes. We checked their monogenic inheritance in controlled crosses. We used a collection of 131 samples from six populations of C. atlantica to estimate null allele frequencies and probability of identity and to characterize the structure of genetic diversity in the fragmented distribution range of this species. We also tested the transferability of the markers to another set of 36 samples from the other Mediterranean Cedrus species and performed a phylogenetic analysis. The three multiplexes reached a high level of resolution potential that we used to evaluate sampling quality. Null allele frequency estimates showed no specific pattern across populations or across species and did not affect the results of biogeographic and phylogenic analyses. Our results reveal a very clear geographical genetic structure within C. atlantica, and the phylogenic tree matched previous analyses based on other markers. Our results confirm the potential interest of these nSSR multiplexes for genotyping in Cedrus.

Consequences of habitat fragmentation on genetic diversity and structure of Salix alba L. populations in two major river systems of Turkey Abstract Salix alba L. (white willow) is an indicator species of a healthy riparian ecosystem with great renewable energy potential in Turkey though habitats of the species in many river ecosystems are highly degraded or fragmented. Impacts of this degradation of river ecosystems on the magnitude and pattern of genetic diversity are not known. This study was aimed at assessing the genetic structure of S. alba populations in two highly degraded and fragmented river systems (the Göksu and Kızılırmak rivers) in Turkey with the use of 20 nuclear simple sequence repeat (SSR) loci. Fifteen of them were used for the first time in this study. Out of the 20 SSR loci, 10 loci significantly deviated from the Hardy-Weinberg equilibrium frequencies and five of them contributed greatly to the differentiation of populations. Generally, high levels of genetic diversity were found in populations of both Göksu and Kızılırmak river systems and moderate genetic differentiation (FST = 0.07) between the river systems. On the contrary to expectations, genetic diversity was higher in middle populations of the rivers (Ho = 0.67 of GRMID (Göksu river middle population), Ho = 0.68 in KRMID1 (Kızılırmak river middle population 1), and 0.65 in KRMID2 (Kızılırmak river middle population 2)) than in downstream populations (Ho = 0.65 in GRDOWN (Göksu river downstream population), Ho = 0.62 in KRDOWN1, 2 (Kızılırmak downstream populations 1, 2)). These could be due to experienced past bottlenecks, extensive vegetative material movements, and habitat fragmentation by constructed dams in the natural ecosystems of the two river systems. The genetic structure results revealed that the white willow populations in the two different river systems may have evolved from two different founder populations. A low level of genetic admixture between the river systems but high admixture within the river systems were observed due to extensive human-mediated vegetative material movements. The current study has provided valuable genetic data and information that could contribute insights to efficient conservation, management, utilization, and breeding of genetic resources of the species.