A single-nucleotide substitution of CjTKPR1 determines pollen production in the gymnosperm plant Cryptomeria japonica.

Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species. The wind-pollinated Japanese cedar (Cryptomeria japonica) is the largest contributor to severe pollinosis in Japan, where increasing proportions of people have been affected in recent decades. The MALE STERILITY 4 (MS4) locus of Japanese cedar controls pollen production, and its homozygous mutants (ms4/ms4) show abnormal pollen development after the tetrad stage and produce no mature pollen. In this study, we narrowed down the MS4 locus by fine mapping in Japanese cedar and found TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution ("T" to "C" at 244-nt position) of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plants not only for Japanese cedar but also for broader plant species.

Different photorespiratory mechanisms in conifer leaves, where peroxisomes have intrinsically low catalase activity.

Photorespiration is an essential metabolic mechanism associated with photosynthesis; however, little is known about the photorespiratory pathway of conifer gymnosperms. Metabolite analyses of the leaves of 27 tree species showed that the mean glycerate content in conifer leaves was lower than that in angiosperm leaves. We performed experiments where [13 C]-serine was fed to detached shoots of a conifer (Cryptomeria japonica), via the transpiration stream, and compared the labeling patterns of photorespiratory metabolites with those of an angiosperm tree (Populus nigra), because glycerate is produced from serine via hydroxypyruvate in peroxisomes. In P. nigra, hydroxypyruvate, glycerate and glycine were labeled with 13 C, whereas in C. japonica, glycolate and a non-canonical photorespiratory metabolite, formate, were also labeled, suggesting that an H2 O2 -mediated non-enzymatic decarboxylation (NED) reaction occurs in C. japonica. We analyzed changes in the metabolite contents of leaves kept in the dark and leaves exposed to illuminated photorespiration-promoting conditions: a positive relationship between formate and serine levels in C. japonica implied that the active C1 -metabolism pathway synthesizes serine from formate. Leaf gas exchange analyses revealed that CO2 produced through NED was recaptured by chloroplasts. Database analysis of the peroxisomal targeting signal motifs of an H2 O2 -scavenging enzyme, catalase, derived from various species, including nine coniferous species, as well as analyses of peroxisomal fractions isolated from C. japonica and P. nigra leaves indicated that conifer peroxisomes had less catalase activity. These results suggest that NED and the subsequent C1 metabolism are involved in the photorespiratory pathway of conifer leaves, where peroxisomes have intrinsically low catalase activity.

A DNA barcode reference library for the native woody seed plants of Japan.

DNA barcode databases are increasingly available for a range of organisms, facilitating the wide application of DNA barcode-based studies. Here we announce the development of a comprehensive DNA barcode reference library of Japanese native woody seed plants representing 43 orders, 99 families, 303 genera and 834 species, and comprising 77.3% of the genera and 72.2% of the species of native woody seed plants in Japan. A total of 6216 plant specimens were collected from 223 sites across the subtropical, temperate, boreal and alpine biomes in Japan with most species represented by multiple accessions. This reference library utilized three chloroplast DNA regions (rbcL, trnH-psbA and matK) and consists of 14,403 barcode sequences. Individual regions varied in their identification rates, with species-level and genus-level rates for rbcL, trnH-psbA and matK based on blast being 57.4%/96.2%, 78.5%/99.1% and 67.8%/98.1%, respectively. Identification rates were higher using region combinations, with total species-level rates for two region combinations (rbcL & trnH-psbA, rbcL & matK and trnH-psbA & matK) ranging between 90.6% and 95.8%, and for all three regions being equal to 98.6%. Genus-level identification rates were even higher, ranging between 99.7% and 100% for two region combinations and being 100% for the three regions. These results indicate that this DNA barcode reference library is an effective resource for investigations of native woody seed plants in Japan using DNA barcodes and provides a useful template for the development of libraries for other components of the Japanese flora.

Stress-responsive retrotransposable elements in conifers.

Conifers are important in many forest ecosystems. They have a long generation time and are immobile; therefore, they require considerable plasticity to adapt to environmental stresses. Moreover, conifers have a large genome, a high proportion of which is occupied by repetitive elements. Retrotransposons are the most highly represented repetitive elements in conifers whose whole-genome sequences have been examined. These retrotransposons are usually silenced, to maintain genome integrity; however, some are activated by environmental stress. The insertion of retrotransposons into genic regions is associated with phenotypic and genetic diversity. The large number and high diversity of retrotransposons in conifer genomes suggest that they play a role in adaptation to the environment. In this review, progress in research on the roles of retrotransposons in the stress responses of conifers is reviewed, and potential future work is discussed.

Genotype-by-environment interaction and genetic dissection of heartwood color in Cryptomeria japonica based on multiple common gardens and quantitative trait loci mapping.

The heartwood color of a major plantation tree Cryptomeria japonica shows high variability among clones and cultivars, and brighter heartwood has higher value in the usage of non-laminated wood such as in traditional construction, which makes heartwood color an important trait in breeding of this species. However, the genetic basis of the interactions between genetics and the environment on heartwood color has been understudied while these are necessary for effective breeding programs in multiple environmental condition. The objectives of the present study were to evaluate the effects of genetics and environments on heartwood color and how they interact in contrasting environments, and to identify genomic regions controlling heartwood color in C. japonica across multiple environments. Heartwood color in terms of L*a*b* color space and spectral reflectance was measured in common gardens established in three contrasting sites. Quantitative trait loci (QTL) that affect heartwood color were identified using previously constructed highly saturated linkage maps. Results found that heartwood color was largely genetically controlled, and genotype-by-environment interaction explained one-third of the total genetic variance of heartwood color. The effect of the environment was small compared to the effect of genetics, whereas environmental effects largely varied among heartwood color traits. QTL analysis identified a large number of QTLs with small to moderate effects (phenotypic variation explained of 6.6% on average). Some of these QTLs were stably expressed in multiple environments or had pleiotropic effects on heartwood color and moisture content. These results indicated that genetic variation in phenotypic plasticity plays an important role in regulating heartwood color and that the identified QTLs would maximize the breeding efficiency of heartwood color in C. japonica in heterogeneous environments.

Construction of a reference transcriptome for the analysis of male sterility in sugi (Cryptomeria japonica D. Don) focusing on MALE STERILITY 1 (MS1).

Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. As the genome of this species is 11 Gbps, it is too large to assemble within a short timeframe. Transcriptomics is one approach that can address this deficiency. Here we designed a workflow consisting of three stages to de novo assemble transcriptome using Oases and Trinity. The three transcriptomic stage used were independent assembly, automatic and semi-manual integration, and refinement by filtering out potential contamination. We identified a set of 49,795 cDNA and an equal number of translated proteins. According to the benchmark set by BUSCO, 87.01% of cDNAs identified were complete genes, and 78.47% were complete and single-copy genes. Compared to other full-length cDNA resources collected by Sanger and PacBio sequencers, the extent of the coverage in our dataset was the highest, indicating that these data can be safely used for further studies. When two tissue-specific libraries were compared, there were significant expression differences between male strobili and leaf and bark sets. Moreover, subtle expression difference between male-fertile and sterile libraries were detected. Orthologous genes from other model plants and conifer species were identified. We demonstrated that our transcriptome assembly output (CJ3006NRE) can serve as a reference transcriptome for future functional genomics and evolutionary biology studies.

Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don).

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Genotype and transcriptome effects on somatic embryogenesis in Cryptomeria japonica.

Somatic embryogenesis (SE), which is in vitro regeneration of plant bodies from somatic cells, represents a useful means of clonal propagation and genetic engineering of forest trees. While protocols to obtain calluses and induce regeneration in somatic embryos have been reported for many tree species, the knowledge of molecular mechanisms of SE development is still insufficient to achieve an efficient supply of somatic embryos required for the industrial application. Cryptomeria japonica, a conifer species widely used for plantation forestry in Japan, is one of the tree species waiting for a secure SE protocol; the probability of normal embryo development appears to depend on genotype. To discriminate the embryogenic potential of embryonal masses (EMs) and efficiently obtain normal somatic embryos of C. japonica, we investigated the effects of genotype and transcriptome on the variation in embryogenic potential. Using an induction experiment with 12 EMs each from six genotypes, we showed that embryogenic potential differs between/within genotypes. Comparisons of gene expression profiles among EMs with different embryogenic potentials revealed that 742 differently expressed genes were mainly associated with pattern forming and metabolism. Thus, we suggest that not only genotype but also gene expression profiles can determine success in SE development. Consistent with previous findings for other conifer species, genes encoding leafy cotyledon, wuschel, germin-like proteins, and glutathione-S-transferases are likely to be involved in SE development in C. japonica and indeed highly expressed in EMs with high-embryogenic potential; therefore, these proteins represent candidate markers for distinguishing embryogenic potential.

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don.

OBJECTIVE: Due to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used to produce a pollen-free cultivar. In order to reduce the time and cost needed for production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica to rapidly identify pollen-free mutants. RESULTS: We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.

Transcriptome analysis of heat stressed seedlings with or without pre-heat treatment in Cryptomeria japonica.

With global warming as a major environment concern over the coming years, heat tolerance is an important trait for forest tree survival during the predicted future warmer weather conditions. Cryptomeria japonica is a coniferous species widely distributed throughout Japan, and thus, can adapt to a wide range of air temperatures. To elucidate genes involved in heat response in Cryptomeria japonica, transcriptome analysis was conducted for seedlings under heat shock conditions. To test whether heat acclimation affects levels of gene expression, half of the seedlings were pretreated with moderately high temperatures prior to heat shock. De novo assembly of the transcriptome generated 107,924 unigenes and the analysis of differentially expressed genes was conducted using these unigenes. A total of 5217 differentially expressed genes were identified. Most genes upregulated by heat shock, regardless of pre-heat treatment, were conserved to heat response genes of angiosperm species, such as heat shock factors (Hsf) and heat shock proteins (Hsp). Pre-heating of seedlings affected expression levels of several Hsfs and their induction was lower in pre-heated seedlings than in seedlings without pre-heat treatment. This suggests a conserved role of Hsfs in heat response and heat acclimation in seed plants. On the other hand, many unknown genes were upregulated in only seedlings without pre-heat treatment after heat exposure. Notably, expression of gypsy/Ty3 type retrotransposons was dramatically induced. These findings provide valuable information to develop a better understanding of the molecular mechanisms of heat response and acclimation in C. japonica.

Oxygen response of leaf CO2 compensation points used to determine Rubisco specificity factors of gymnosperm species.

Rubisco specificity factor (Sc/o), a measure of the relative capacities of an enzyme to catalyze carboxylation and oxygenation of ribulose-1,5-bisphosphate, determines the extent of photosynthetic CO2 assimilation and photorespiratory CO2 release. The current model of C3 photosynthesis, the Farquhar-von Caemmerer-Berry (FvCB) model, requires a species-specific Sc/o. However, Sc/o values have never been reported in conifers, likely because in vitro kinetic analysis of conifer Rubisco presents difficulties. To estimate the Sc/o of conifers and compare it with angiosperm Sc/o, we measured changes in leaf CO2 compensation points (Γ) in response to O2 partial pressure for a variety of leaves, with different rates of day respiration (Rday) and maximum Rubisco carboxylation (Vcmax) in gymnosperms (Ginkgo biloba), conifers (Metasequoia glyptostroboides and Cryptomeria japonica), and angiosperms (Nicotiana tabacum and Phaseolus vulgaris). As predicted by the FvCB model, the slope of a linear function of Γ vs O2 partial pressure, d, increased alongside increasing Rday/Vcmax. The Sc/o was obtainable from this relationship between d and Rday/Vcmax, because the d values at Rday/Vcmax = 0 corresponded to α/Sc/o, where α was the photorespiratory CO2 release rate per Rubisco oxygenation rate (generally assumed to be 0.5). The calculated Sc/o values of N. tabacum and P. vulgaris exhibited good agreement with those reported by in vitro studies. The Sc/o values of both conifers were similar to those of the two angiosperm species. In contrast, the Sc/o value of G. biloba was significantly lower than those of the other four studied species. These results suggest that our new method for Sc/o estimation is applicable to C3 plants, including those for which in vitro kinetic analysis is difficult. Furthermore, results also suggest that conifer Sc/o does not differ significantly from that of C3 angiosperms, assuming α remains unchanged.

Climate sensitivity of Cryptomeria japonica in two contrasting environments: Perspectives from QTL mapping.

Long-lived forest tree species experience a wide range of environmental conditions throughout their lifespan. Evaluation of the underlying growth and development mechanisms of these species is essential to predict tree growth under climate change. This study investigated climate sensitivity to temperature, precipitation, dry periods, and the associated genomic regions in Cryptomeria japonica, Japan's most commercially important tree. We used tree rings and common garden experiments with three clonal replicates planted in two contrasting environments in Kyushu (Kumamoto site) and Honshu (Chiba site), Japan. Tree growth showed a significant negative correlation with the dry period (>4 days) in March of the year of tree-ring formation at the Chiba site. In contrast, temperature and precipitation had little influence on tree growth. Quantitative trait locus (QTL) analysis was performed to investigate climate sensitivity to dry periods at the Chiba site, revealing 13 significant QTLs. One QTL showed a substantially large contribution to the overall climate sensitivity, accounting for 12.4% of the total phenotypic variation. The phenotypic variance explained (PVE) by other QTLs ranged from 0.9% to 2.9%, and the total PVE by all QTLs was 35.6%. These findings indicate that the tree population at the Chiba site could be vulnerable to drought in early spring and that the QTL showing the greatest impact on climate sensitivity may be closely related to genes associated with tolerance or adaptation to drought stress. The QTLs identified in this study could be useful for molecular breeding, forest management, and predicting the growth of C. japonica under a changing climate.

Scanning RNA-Seq and RAD-Seq approach to develop SNP markers closely linked to MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Fine mapping of the male-sterile genes (MS1, MS2, MS3, and MS4) and development of SNP markers for marker-assisted selection in Japanese cedar (Cryptomeria japonica D. Don).

Pollinosis caused by Japanese cedar (Cryptomeria japonica) is a widespread social problem in Japan. To date, 23 male-sterile C. japonica trees have been selected in Japan to address pollinosis, from which four male-sterility loci (MS1, MS2, MS3, and MS4) have been identified from test crossing results. For efficient breeding of male-sterile C. japonica trees, more male-sterile individuals and individuals heterozygous for male-sterile genes are required. Therefore, we aimed to develop DNA markers for marker-assisted selection of four types of male-sterile genes from populations without a family structure. First, for four families exhibiting segregation of each male-sterile locus (MS1, MS2, MS3, and MS4), genome-wide single-nucleotide polymorphism and insertion/deletion (indel) genotyping was performed using the Axiom myDesign Targeted Genotyping Array method. Four high-density linkage maps for mapping the MS1, MS2, MS3, and MS4 families were constructed, which included 4923, 1722, 1896, and 2247 markers, respectively. In these maps, 15, 4, 2, and 2 markers were located 0.0, 3.3, 1.1, and 0.0 cM from the MS1, MS2, MS3, and MS4 loci, respectively. Second, for the markers located 0.0 cM from a male-sterile locus (i.e., MS1 and MS4), to clarify the most tightly linked markers, we calculated the prediction rate of male-sterile gene genotypes from marker genotypes for 78 trees. The markers with the highest prediction rates were AX-174127446 (0.95) for MS1 and AX-174121522 (1.00) for MS4. The AX-174121522 marker was considered to be suitable for selecting trees homozygous or heterozygous for the MS4 gene from plus-trees without a pollination test, which requires a large amount of time and effort. The nearest markers to the male-sterile loci found in this study may facilitate the isolation of male-sterile genes in C. japonica via combination with the draft genomic sequence that is currently being collated.

Comprehensive analysis of small RNAs expressed in developing male strobili of Cryptomeria japonica.

Deep sequencing of small RNAs (sRNAs) in developing male strobili of second-generation offspring originating from a nuclear genic male sterile tree of Cryptomeria japonica were performed to characterize sRNA populations in the male strobili at early pollen developmental stages. Comparing to sequences of microRNA (miRNA) families of plant species and sRNAs expressed in the reproductive organs of representative vascular plants, 37 conserved miRNA families were detected, of which eight were ubiquitously expressed in the reproductive organs of land plant species. In contrast, miR1083 was common in male reproductive organs of gymnosperm species but absent in angiosperm species. In addition to conserved miRNAs, 199 novel miRNAs candidates were predicted. The expression patterns of the obtained sRNAs were further investigated to detect the differentially expressed (DE) sRNAs between genic male sterile and fertile individuals. A total of 969 DE sRNAs were obtained and only three known miRNA families were included among them. These results suggest that both conserved and species-specific sRNAs contribute to the development of male strobili in C. japonica.

Genetic differentiation and evolutionary adaptation in Cryptomeria japonica.

Local adaptation of plant species is a central issue for survival during global climate change, especially for long-lived forest trees, with their lengthy regeneration time and spatially limited gene flow. Identification of loci and/or genomic regions associated with local adaptation is necessary for knowledge of both evolution and molecular breeding for climate change. Cryptomeria japonica is an important species for forestry in Japan; it has a broad natural distribution and can survive in a range of different environments. The genetic structure of 14 natural populations of this species was investigated using 3930 SNP markers. Populations on the Pacific Ocean side of Japan are clearly different from those on the Japan Sea side, as discussed in previous studies. Structure analysis and population network trees show that peripheral populations, including the most northerly and southerly ones, have unique features. We found that the genetic differentiation coefficient is low, FST = 0.05, although it must account for the presence of important genes associated with adaptation to specific environments. In total, 208 outlier loci were detected, of which 43 were associated with environmental variables. Four clumped regions of outlier loci were detected in the genome by linkage analysis. Linkage disequilibrium (LD) was quite high in these clumps of outlier loci, which were found in linkage groups (LGs) 2, 7, 10, and 11, especially between populations of two varieties, and when interchromosomal LD was also detected. The LG7 region is characteristic of the Yakushima population, which is a large, isolated, peripheral population occupying a specific environment resulting from isolation combined with volcanic activity in the region. The detected LD may provide strong evidence for selection between varieties.

Demonstration of genome-wide association studies for identifying markers for wood property and male strobili traits in Cryptomeria japonica.

Genome-wide association studies (GWAS) are an alternative to bi-parental QTL mapping in long-lived perennials. In the present study, we examined the potential of GWAS in conifers using 367 unrelated plus trees of Cryptomeria japonica D. Don, which is the most widely planted and commercially important tree species in Japan, and tried to detect significant associations between wood property traits and quantity of male strobili on the one hand, and 1,032 single nucleotide polymorphisms (SNPs) assigned to 1,032 genes on the other. Association analysis was performed with the mixed linear model taking into account kinship relationships and subpopulation structure. In total, 6 SNPs were found to have significant associations with the variations in phenotype. These SNPs were not associated with the positions of known genes and QTLs that have been reported to date, thus they may identify novel QTLs. These 6 SNPs were all found in sequences showing similarities with known genes, although further analysis is required to dissect the ways in which they affect wood property traits and abundance of male strobili. These presumptive QTL loci provide opportunities for improvement of C. japonica, based on a marker approach. The results suggest that GWAS has potential for use in future breeding programs in C. japonica.

A second generation framework for the analysis of microsatellites in expressed sequence tags and the development of EST-SSR markers for a conifer, Cryptomeria japonica.

BACKGROUND: Microsatellites or simple sequence repeats (SSRs) in expressed sequence tags (ESTs) are useful resources for genome analysis because of their abundance, functionality and polymorphism. The advent of commercial second generation sequencing machines has lead to new strategies for developing EST-SSR markers, necessitating the development of bioinformatic framework that can keep pace with the increasing quality and quantity of sequence data produced. We describe an open scheme for analyzing ESTs and developing EST-SSR markers from reads collected by Sanger sequencing and pyrosequencing of sugi (Cryptomeria japonica). RESULTS: We collected 141,097 sequence reads by Sanger sequencing and 1,333,444 by pyrosequencing. After trimming contaminant and low quality sequences, 118,319 Sanger and 1,201,150 pyrosequencing reads were passed to the MIRA assembler, generating 81,284 contigs that were analysed for SSRs. 4,059 SSRs were found in 3,694 (4.54%) contigs, giving an SSR frequency lower than that in seven other plant species with gene indices (5.4-21.9%). The average GC content of the SSR-containing contigs was 41.55%, compared to 40.23% for all contigs. Tri-SSRs were the most common SSRs; the most common motif was AT, which was found in 655 (46.3%) di-SSRs, followed by the AAG motif, found in 342 (25.9%) tri-SSRs. Most (72.8%) tri-SSRs were in coding regions, but 55.6% of the di-SSRs were in non-coding regions; the AT motif was most abundant in 3' untranslated regions. Gene ontology (GO) annotations showed that six GO terms were significantly overrepresented within SSR-containing contigs. Forty-four EST-SSR markers were developed from 192 primer pairs using two pipelines: read2Marker and the newly-developed CMiB, which combines several open tools. Markers resulting from both pipelines showed no differences in PCR success rate and polymorphisms, but PCR success and polymorphism were significantly affected by the expected PCR product size and number of SSR repeats, respectively. EST-SSR markers exhibited less polymorphism than genomic SSRs. CONCLUSIONS: We have created a new open pipeline for developing EST-SSR markers and applied it in a comprehensive analysis of EST-SSRs and EST-SSR markers in C. japonica. The results will be useful in genomic analyses of conifers and other non-model species.

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don.

BACKGROUND: High-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems. RESULTS: We constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM. CONCLUSIONS: Using the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.

Isolation and characterization of 52 polymorphic EST-SSR markers for Callitris columellaris (Cupressaceae).

PREMISE OF THE STUDY: We developed simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) for Callitris columellaris sensu lato (s.l.) to elucidate population genetic structure and detect outlier loci by genome scan. METHODS AND RESULTS: mRNA from an individual seedling was subjected to cDNA synthesis and then de novo pyrosequencing. Two hundred and nineteen primer pairs bordering sequence regions were designed from the obtained sequence data. In total, 52 showed polymorphism within 16 individuals representative of the species' entire range, with the number of alleles per locus and expected heterozygosity ranging from two to 10 and 0.06 to 0.84, respectively. CONCLUSIONS: The EST-SSR markers developed in this study will be useful for evaluating the range-wide genetic structure of C. columellaris s.l. and detecting outlier loci under selection, as well as providing useful markers to investigate the conservation genetics and reproductive ecology of the species.