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The moso bamboo genome contains the two structurally intact and thus potentially functional mariner-like elements Ppmar1 and Ppmar2. Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast (Saccharomyces cerevisiae). We have designed a two-component system consisting of a transposase expression cassette and a non-autonomous transposon on two separate plasmids. We demonstrate that the Ppmar1 transposase Pptpase1 catalyses excision of the non-autonomous Ppmar1NA element from the plasmid and reintegration at TA dinucleotide sequences in the yeast chromosomes. In addition, we generated 14 hyperactive Ppmar1 transposase variants by systematic single amino acid substitutions. The most active transposase variant, S171A, induces 10-fold more frequent Ppmar1NA excisions in yeast than the wild type transposase. The Ppmar1 transposon is a promising tool for insertion mutagenesis in moso bamboo and may be used in other plants as an alternative to the established transposon tagging systems.

Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo (Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types (NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity. Electronic supplementary material The online version of this article (10.1186/s13100-019-0179-y) contains supplementary material, which is available to authorized users.

The sequences of primers used in the study to amplify the enhanced cyan fluorescent protein (ECFP) with a nuclear localization signal (NLS) (ECEP-NLS) and to mutate the NES sequences of Ppmar1 and Ppmar2 transposases. (DOCX 17 kb)

The sequences of primers used in the study for amplification of Ppmar1NA, Ppmar2 and Ppmar2NA transposons. (DOCX 16 kb)

The nucleotide sequences of Ppmar1 and Ppmar2 transposases and their amino acid sequences. (DOCX 18 kb)

The nucleotide sequences of Ppmar1NA and Ppmar2NA transposons (non-autonomous). (DOCX 15 kb)

The fluorescence intensity of both blue and red colour of ECFP and EYFP channels, respectively. (DOCX 18 kb)

DNA contents of Chinese 134 bamboo species were first time announced, and FISH of 45S rDNA shows that paleotropical woody bamboo species may be originated from a multi-source system. Woody bamboo mainly includes two types of species: the temperate woody bamboo and the tropical woody bamboo. Both vary widely in genome sizes, chromosome number, and ploidy. In this study, the DNA contents of 135 bamboo species (22 genera) mainly distributed in China were analyzed by the flow cytometry. The DNA contents of the 134 bamboo species were first time announced. In contrast to the paleotropical woody bamboos, the temperate woody species is larger in genome size. Both the average arm ratio and the average symmetric coefficient of karyotype of the temperate woody species are less than those of the paleotropical woody bamboos, suggesting that the karyotypes of temperate woody bamboo species are more symmetrical than that of paleotropical bamboos. Also, FISH (fluorescence in situ hybridization) was applied to evaluate the distribution of 45S rDNA in the chromosomes of the representative bamboo species. Only one pair of sites was detected for 45S rDNA in temperate woody bamboo species, while three pairs of sites in paleotropical woody bamboo species, implying that the paleotropical woody bamboo species may be originated from a multi-source system. This study exposed a corner of the complex genomic relationships in the woody bamboos and provided an initial framework for future phylogenic studies.

Key message Endo-1,4-β-glucanasegene was isolated fromPhyllostachys heterocyclavar.pubescensinternodes. It is engaged in the culm or stem development by regulation of the biosynthesis of cellulose.

Quercus gilva (Blume) is an evergreen oak species that is native to Japan, China, and Taiwan. Because of a long history of human impact, this species is threatened with extinction in several regions of Japan. The objective of this study was to identify the genetic structure of Q. gilva based on chloroplast DNA (cpDNA) sequencing analysis. We collected 123 samples from 25 populations in Japan, 8 samples from 1 population in China, and 46 samples from 5 populations in Taiwan. Approximately 1815 bp of cpDNA was sequenced for each of the 177 samples. Thirteen haplotypes were detected, with no cross-region distribution of haplotypes among the three geographically separated countries. There were large genetic differences among populations (GST = 0.824, [Formula: see text] = 0.937). Six haplotypes (haplotypes 1, 2, 3, 4a, 4b, and 5) were detected in Japan; haplotype 4a was the most common, detected from 20 populations, and the other rare haplotypes, except for haplotype 4b, occurred at the edge of the species’ distribution. In addition, four haplotypes (haplotypes 1, 2, 3, and 5) were quite different from the predominant haplotype (haplotype 4a), with more than four cpDNA mutations except for a mononucleotide repeat, suggesting that populations with these rare haplotypes should be conserved separately.

Most bamboo species are distinguished by the rapid growth, e.g., more than 100 cm/d in Phyllostachys pubescens. The latest research showed that the genome of P. pubescens is large and contains large and diverse families of transposable elements, which were assumed to effect on its morphogenesis and development. Mariner-like elements (MLEs) are class II transposable elements found in almost all eukaryotic genomes. We have characterized 82 amplification fragments and 79 full-length mariner-like transposases representing MLEs derived from 79 representative bamboo species from 38 genera within six subtribes of the Bambusoideae. Phylogenetic analysis of these MLE transposase sequences shows that MLEs are widespread, diverse, and abundant in the Bambusoideae. There is horizontal transfer between distantly related species or an ancestral MLE polymorphism followed by divergent evolution and stochastic loss. Two full-length MLEs were isolated with typical ITR consensus sequences of plant MLEs, intact DNA-binding motifs, and DD39D catalytic domain, and many residues are previously shown to be critical for transposase activity from P. pubescens, implying that both transposons are likely natively active. Transformation into Arabidopsis thaliana showed that the MLE transposons left the primary site, jump, and produce footprints in the A. thaliana genome. The active bamboo Tc1/mariner will provide a foundation for future comparative analyses of animal and plant elements, a new wide host range transposable element available for plant gene tagging.

We report the isolation of two full-length mariner-like elements (MLEs) from a bamboo species (Phyllostachys edulis) by chromosome walking using a modified magnetic enrichment procedure. Ppmar1 is 3472 base pairs (bp) in length with 28-bp perfect inverted terminal repeats (ITRs) and a 1986-bp open reading frame (ORF) encoding transposase, whereas Ppmar2 is 2679 bp in length with the 27-bp perfect ITRs and the 1877-bp ORF encoding transposase. Ppmar1 and Ppmar2 share the typical ITR consensus sequences of plant MLEs (5′-CTC CCT CCR T-3′, where R is A or G) and the typical TA/AT target site duplications. Phylogenetic analysis demonstrated that Ppmar1 belongs to clade A2 and Ppmar2 to clade C of plant MLE subfamilies. The two transposases encoded contain intact DNA-binding motifs and a DD39D catalytic domain and many residues previously shown to be critical for transposase activity are conserved in Ppmar1 and Ppmar2. All the above results imply that both transposons are likely to be naturally active....

Transposable elements (transposons) are fragments of DNA sequences which can move within host genome. Miniature inverted-repeat transposable elements (MITEs) are widespread and high-copy transposable elements in eukaryotic genomes. Tourist-like MITEs are especially abundant in plant kingdom. Earlier genome-wide analysis has shown that MITEs are widely distributed in the moso bamboo genome and preferentially inserted into gene regions. In the present study, in order to examine the potential influence of MITEs on the moso bamboo gene expressions, a highly conserved Tourist-like MITE family, which distributed near genes, was selected as research focus and named PhTst-3 (Phyllostachys edulis Tourist-like element 3). The MITEs’ insertion sites were tested in moso bamboo half-sib seedlings by real-time fluorescence quantitative PCR. Amplification polymorphisms were found in a copy of PhTst-3 (PhTst-3-55) which was located in the intron of PH01002699G0010. This inserted PhTst-3-55 had a significant impact on the gene expression revealed by the real-time fluorescence quantitative PCR. The gene expression levels were four times higher in the absence of PhTst-3-55 than those in the presence of it. This finding suggests that the PhTst-3 located in the intron is involved in the regulation of the gene. In order to examine the impact of PhTst-3-55 on the near genes, the PhTst-3-55 was inserted into a promoter analysis vector, pxk7S2D, between the two promoter sequences. The Agrobacterium-mediated transient expression showed that PhTst-3-55 insertion decreases the expression level of upstream GUS gene and downstream GFP gene. So, PhTst-3-55 can have a silencing role by bidirectionally inhibiting gene expression.

Bamboo is one of the most important non-timber forest species in the world, but their molecular breeding lags far behind in contrast to other economic plants. Regarding the difficulties of hybridization and gene modification, the transposon-based insertional mutagenesis might be an alternative, feasible way for molecular breeding of bamboo. A systematic search for potential active transposons identified two full-length mariner-like elements (MLEs) (Ppmar1 and Ppmar2) from moso bamboo in the previous study. Both MLEs contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase. Two transposases contain intact DNA-binding motifs and a DD39D catalytic domain which indicates that Ppmar1 and Ppmar2 are likely active. Here, we deployed a heterologous transposition system of Arabidopsis thaliana to study the transposition activity of Ppmar1 and Ppmar2. The results show that both MLEs could transpose in A. thaliana. Excisions of Ppmar1 and Ppmar2 are usually unperfect as they leave 1–4 bp in excision sites. The reinsertions of both Ppmar1 and Ppmar2 occur at TA dinucleotides and prefer to insert into the TA-rich regions. The insertion sites are dispersed and non-linked. Two active bamboo transposons identified here not only could be applied to construction of the bamboo mutant libraries but also would provide another choice for other plant transposon-based gene tagging.

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR&rsquo, s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5&ndash, 2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

In Gramineae plants, carbon sequestration is closely related to the internodes development which is regulated by the gibberellins (GAs). Chitin-inducible gibberellin-responsive genes (CICR) are a member of the GRAS superfamily involved in the gibberellin signal transduction pathway and play roles in the development and defense of the plant. In this study, a CICR homolog gene (named BvCIGR) was cloned from the shoot tip of Bambusa ventricosa McClure with shorten internodes by rapid amplification of cDNA ends, which was 2143 bp in length encoding 545 amino acids with typical plant GRAS domain. Expression levels of BvCIGR varied in different positions of internodes in B. ventricosa (highest in the upper, followed by the middle and lowest in the bottom internodes). The results indicated that BvCIGR is possibly involved in the regulation of the internodal elongation of B. ventricosa, and has possible influence on the carbon sequestration potential in bamboo plantations.

With 1 figure and 1 table Abstract Public sequence databases provide a convenient and inexpensive resource for the rapid development of microsatellite markers. We analysed 3406 publically available expressed sequence tags (ESTs) from caespitose bamboo species (Bambusa edulis and B. oldhamii) and found 245 non-redundant simple sequence repeats (SSRs) in 205 EST contigs that were used to develop 15 EST-SSR markers. The transferability of the markers was 59.6% among 14 additional caespitose bamboo species (4 within the same genus and 10 in other genera) and Phyllostachys pubescens. The successfully transferred markers showed 51.4% polymorphism. Markers BOM01 and BOM02 transferred successfully to most of the caespitose bamboo species showed rich polymorphism, and are therefore potentially valuable as species-specific alleles for the identification of caespitose bamboo interspecies hybrids.

Mariner-like elements (MLEs) are the most diverse and widespread transposable elements, with members of the MLE superfamily found in fungi, plants, ciliates and animals. In a previous study, we characterized 82 MLE transposase gene fragments (average length 383 bp) in 44 bamboo species, indicating that MLEs are widespread, abundant and diverse in the Bambusoideae subfamily. In this study, we isolated 79 full-length MLE transposase genes from 63 bamboo species representing 38 genera in six subtribes mainly found in China. The transposases were highly conserved, mostly uniform in length and contained intact DNA-binding motifs and DD39D catalytic domains with few notable frameshift, indel and nonsense mutations. This suggested the MLEs are probably still mobile, not yet affected by vertical inactivation. A phylogenetic tree of the Bambusoideae subfamily established using ribosomal DNA internal transcribed spacer sequences was incongruent with a second tree based on the MLE transposase genes. This evidence, together with the presence of near-identical MLEs in distantly related species and diverse MLEs in closely related species, indicates that MLEs have evolved in a distinct manner, probably independently of speciation events in the subfamily. The evolution and diversity of MLE transposase genes in the Bambusoideae subfamily is discussed.

PIF-like transposable elements were classified as members of the PIF/Harbinger superfamily that has been characterized in many eukaryotes. Complete and partial sequences of PIF-like transposable elements have been isolated from hundreds of plant species, but only two have been identified in bamboo species. We isolated 139 sequences from 44 bamboo species, and phylogenetic analysis showed that PIF-like transposable elements are widespread, diverse and abundant in the Bambusoideae subfamily. A molecular phylogeny of the Bambusoideae subfamily was also established using the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA, and was incongruent with the tree derived from PIF-like transposase genes. These results indicate that PIF-like transposable elements may have been involved in horizontal transfer events between phylogenetically distant bamboo species, or that an ancestral polymorphism has been followed by divergent evolution and stochastic loss of PIF-like transposable elements in different bamboo species. Neutralist tests show that PIF-like transposase gene coding sequences were negatively selected during evolution and introns of PIF-like transposase genes evolve neutrally with minus negative selection.

Mariner-like elements (MLEs) are class II transposable elements found in almost all eukaryotic genomes including those of plants, from which hundreds of complete and partial sequences have been elucidated. We have characterized 82 amplification fragments representing MLEs derived from diverse members of the Bambusoideae subfamily. Phylogenetic analysis of MLE transposase sequences shows that MLEs are widespread, diverse and abundant in the Bambusoideae. In addition, a molecular phylogeny of the Bambusoideae subfamily was established by using the internal transcribed spacer sequences of nuclear ribosomal DNA (ITS) information. The phylogenetic tree constructed using MLE fragment sequences was incongruent with a second tree based on ITS information. These results suggest horizontal transfer between distantly related species or the existence of an ancestral MLE polymorphism followed by divergent evolution and stochastic loss.

The PIF/IS5 is a recently discovered superfamily of DNA transposons which include Pong-like elements and PIF-like elements and has been successively detected in the genomes of many flowering plants, fungi and diverse animals. Here we present the first comprehensive characterization and analysis of Pong-like elements in Bambusoideae subfamily. Eighty-two Pong-like elements were cloned and sequenced from 44 representative species of Bambusoideae. Phylogenetic analysis of 82 distinct Pong-like elements sequences showed that Pong-like elements were widespread, diverse and abundant in Bambusoideae. A molecular phylogeny of Bambusoideae was established by using the internal transcribed spacer sequence of nuclear ribosomal DNA (ITS) information. The comparison between ITS and Pong-like elements based trees reveals obviously incongruent. The results suggest that 1) there are multiple Pong-like element families in Bambusoideae; 2) a single Pong-like element family could be present in multiple bamboo species; 3) Pong-like elements from the same family from different bamboo species could be more similar than elements from different families in the same bamboo species or closely related species.

Ty1-copia retroelements have been found in all major plants and are largely responsible for the huge differences in the genome size. In this study we isolated and sequenced Ty1-copia reverse transcriptase (rt) gene fragments from 44 representative species of bamboo and nine cultivars or forms of Phyllostachys pubescens. Phylogenetic analysis of 72 distinct Ty1-copia rt sequences showed that Ty1-copia retroelements were widespread, diverse and abundant in these species of Bambusoideae subfamily. In addition, a molecular phylogeny of the species of the Bambusoideae subfamily was established by using the internal transcribed spacer sequences of nuclear ribosomal DNA (ITS) sequences. The comparison between ITS- and Ty1-copia rt- based trees is obviously incongruent. The results suggested either the existence of horizontal transfer events between phylogenetically distant species, or an ancestral Ty1-copia retroelement polymorphism followed by different evolution and stochastic losses.

Bamboo shoots grow quickly through the rapid elongation of internodes, but the precise molecular mechanisms underlying this process remain unknown. We used a combination of suppressive subtractive hybridization (SSH), dot blotting, sequencing and bioinformatics to identify Phyllostachys pubescens genes that are differentially expressed in rapidly elongating vs. static internodes (SIs). We isolated 1020 expressed sequence tags (ESTs) by SSH, 173 of which were shown to be differentially expressed by dot blotting. We then sequenced the 20 ESTs showing the greatest difference in expression, 13 of which were preferentially expressed in elongating internodes and seven in SIs. Functional characterization of the ESTs showed that rapid internode elongation requires meristem initiation and proliferation, high-level protein synthesis, cellular respiration, and cell wall synthesis, as well as the regulation of the activated methyl cycle, gibberellin and brassinosteroid biosynthesis, and their signal transduction pathways.

Public sequence databases provide a rapid, simple and cost-effective source of microsatellite markers. We analyzed 1,532 bamboo (Phyllostachys pubescens) sequences available in public domain DNA databases, and found 3,241 simple sequence repeat (SSR) loci comprising repeats of two or more nucleotides in 920 genomic survey sequences (GSSs) and 68 cDNA sequences. This corresponded to one SSR per 336 bp of GSS DNA and one SSR per 363 bp of cDNA. The SSRs consisted of 76.6 and 74.5% dinucleotide repeats, 20.0 and 22.3% trinucleotide repeats, and 3.4 and 3.2% higher-number repeats in the GSS DNA and cDNA sequences, respectively. The repeat motif AG/CT (or GA/TC) was the most abundant. Nineteen microsatellite markers were developed from Class I and Class II SSRs, showing that the limited polymorphism in Ph. pubescens cultivars and provenances could be attributed to clonal propagation of the bamboo plant. The transferability of the microsatellites reached 75.3%, and the polymorphism of loci successfully transferred was 66.7% for six additional Phyllostachys species. Microsatellite PBM014 transferred successfully to all six species, showed rich polymorphism, and could serve as species-specific alleles for the identification of Phyllostachys interspecies hybrids.

The ability to initiate stem elongation at the early growth stages of a plant is the major survival mechanism of floating rice in the presence of flooding. We previously identified two quantitative trait loci (QTLs) for this “early elongation ability” on chromosomes 3 and 12 using a Patnai23 (a non-floating cultivar) × Goai (a floating cultivar) F2 population. The objectives of the present study were: (1) to identify QTLs for early elongation ability in the cross Habiganj Aman VIII (a floating cultivar) × Patnai23, and (2) to confirm the results obtained in our previous diallel analysis indicating that Habiganj Aman VIII might harbour additional gene(s) for early elongation ability compared with Goai. The genotype of a total of 192 F2 plants was investigated using 85 (9 RFLP and 76 SSR) markers and the plants were assessed for early elongation ability. We scaled early elongation ability based on the position of the lowermost internode that exhibited elongation (“lowest elongated internode”, LEI), since the LEI position parallels the time of the onset of stem elongation. As a result, a large QTL detected on chromosome 12 seemed to be common to the two crosses. A small QTL detected on chromosome 1 was unique to the Habiganj Aman VIII × Patnai23 cross, which was consistent with the results obtained in our previous diallel analysis. No QTLs were found on chromosome 3 in this cross. These results suggested that (1) the QTL on chromosome 12 might be the most important one for the expression of early elongation ability in terms of the to percentage of phenotypic variation and consistency in different backgrounds, and (2) diallel analysis might be an effective method for examining differences in genetic control among diverse parents.

In rice, genetic variation in “phyllochron” (the time interval between the appearance of successive leaves) affects many aspects of shoot system development. The objective of our study was to identify quantitative trait loci (QTLs) that control phyllochron in a Lemont (japonica) × IR36 (indica) F2 population. Composite interval mapping detected four phyllochron QTLs located on chromosomes 1, 2, 9 and 11. Individually, these QTLs accounted for 8-14% of the phenotypic variation, indicating that phyllochron is controlled by multiple QTLs rather than by a major gene system. At these QTLs, the presence of a Lemont allele increased phyllochron. The only exception was for the QTL on chromosome 9, where the Lemont allele decreased phyllochron. Based on the synteny between rice and maize genomes, the ortholog of the maize terminal ear 1 gene was considered a candidate gene for the QTL on chromosome 1.

Genetic variation in seed/cone production among clones was studied in a hinoki (Chamaecyparis obtusa Endl.) seed orchard containing 25 plus-trees by analyzing the number of cones, the yield of cones and seeds of individual ramets for 5 successive years (1982 to 1986). There was significant variation among clones each year and parental contribution in the seed orchard. Specifically, in the years 1982, 1983, 1984, 1985, and 1986, 20% of the clones produced 37.2, 60.6, 36.0, 44.3, and 44.8% of the total cones, respectively. The size of the crop greatly influenced the parental balance in the resulting seed/cone crops. The product moment correlation coefficients and Spearman’s coefficients of rank correlation were small and insignificant between consecutive years, but large and highly significant between alternate years, suggesting the presence of carry-over effects in seed/cone production. The broad-sense heritability on a clone mean basis was 0.74 ± 0.15 for the number of cones, 0.72 ± 0.14 for the yield of cones, and 0.68 ± 0.13 for the yield of seeds. The corresponding heritabilities from analyses combined over all years were 0.24, 0.558, and 0.724, respectively. These results indicate that seed/cone production in hinoki is under strong genetic control. Several managerial measures are discussed that maintain the genetic diversity in seedlots used for reforestation, by reducing the variation in seed/cone production among clones and producing seed crops with equal contributions from all parents.

The effects of nursery practices on genetic composition and structure were studied inChamaecyparis obtusa by analyzing seeds and one-, two-, and three-year-old seedlings. Enzyme polymorphisms of the6Pgd-2, G6pd, Got, Gk, Shd-2, Pod, Dia-1, andPgm loci were used. OnlyDia-1 indicated significantly different allelic frequencies between seeds and three-year-old seedlings. Most of the genetic diversity of the parental clones in this seed orchard could be transferred to the offsprings. Genetic diversity on the basis of an average expected heterozygosity did not differ between seeds and seedlings. A slight excess of homozygotes in seeds and a significant excess of heterozygotes in seedlings of different ages to the expected panmictic proportions were found. Most of the differences in genotypic distributions among the seed and seedling stages, probably resulted from viability selection favoring outcrosses, and selective removal of inbred offsprings including self-fertilized ones prior to their transplantation in the nursery.

Current databases of Phyllostachys pubescens full-length cDNAs (FL-cDNAs) provide a rich source of sequences for the development of potential FL-cDNA simple sequence repeat (SSR) markers. We screened 10,608 P. pubescens cDNAs, discovering 1614 SSRs in 1382 SSR-containing FL-cDNAs. The SSRs were more abundant within transposable elements (TEs) than expressed sequence tags (ESTs) and genome survey sequences (GSSs), and specific dinucleotide repeats tended to associate with particular TE families: (TA)n with En/Spm and (CT)n with Mutator. A selected panel of 100 FL-cDNAs containing type I SSRs yielded 68 functional SSR markers with an average polymorphism information content (PIC) value of 0.12, among which 22 loci contained polymorphisms. These markers became less transferrable (83.1% → 69.9% → 49.3%) but more polymorphic (79.4% → 92.3% → 92.8%) with increasing phylogenetic distance (intra-genus → intra-subtribe → intra-family). Transferability and polymorphism also depended on the location of the marker, with those located in the coding region being more transferrable (69.1%) and less polymorphic (89.4%) than those in the 5′-UTR (63.4% transferable, 90.7% polymorphic) and the 3′-UTR (61.8% transferable, 91.4% polymorphic). As proof of principle, we were able to use our FL-cDNA SSR markers to identify the parental stocks in interspecific hybrids of bamboo within and beyond P. pubescens, and estimate the outcrossing rate for P. pubescens. Our research should facilitate molecular breeding in bamboo species where original genetic markers are scarce.