CRISPR/Cas9 mutants delineate roles of Populus FT and TFL1/CEN/BFT family members in growth, dormancy release and flowering.

Vegetative and reproductive phase change and phenology are economically and ecologically important traits. Trees typically require several years of growth before flowering and, once mature, seasonal control of the transition to flowering and flower development is necessary to maintain vegetative meristems and for reproductive success. Members of two related gene subfamilies, FLOWERING LOCUST (FT) and TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN)/BROTHER OF FT AND TFL1 (BFT), have antagonistic roles in flowering in diverse species and roles in vegetative phenology in trees, but many details of their functions in trees have yet to be resolved. Here, we used CRISPR/Cas9 to generate single and double mutants involving the five Populus FT and TFL1/CEN/BFT genes. The ft1 mutants exhibited wild-type-like phenotypes in long days and short days, but after chilling, to release dormancy, they showed delayed bud flush and GA3 could compensate for the ft1 mutation. After rooting and generating some phytomers in tissue culture, both cen1 and cen1ft1 mutants produced terminal as well as axillary flowers, indicating that the cen1 flowering phenotype is independent of FT1. The CEN1 showed distinct circannual expression patterns in vegetative and reproductive tissues and comparison with the expression patterns of FT1 and FT2 suggests that the relative levels of CEN1 compared with FT1 and FT2 regulate multiple phases of vegetative and reproductive seasonal development.

SSR individual identification system construction and population genetics analysis for Chamaecyparis formosensis.

Chamaecyparis formosensis is an endemic species of Taiwan, threatened from intensive use and illegal felling. An individual identification system for C. formosensis is required to provide scientific evidence for court use and deter illegal felling. In this study, 36 polymorphic simple sequence repeat markers were developed. By applying up to 28 non-linked of the developed markers, it is calculated that the cumulative random probability of identity (CPI) is as low as 1.652 × 10-12, and the identifiable population size is up to 60 million, which is greater than the known C. formosensis population size in Taiwan. Biogeographical analysis data show that C. formosensis from four geographic areas belong to the same genetic population, which can be further divided into three clusters: SY (Eastern Taiwan), HV and GW (Northwestern Taiwan), and MM (Southwestern Taiwan). The developed system was applied to assess the provenance of samples with 88.44% accuracy rate and therefore can serve as a prescreening tool to reduce the range required for comparison. The system developed in this study is a potential crime-fighting tool against illegal felling.

Isolation and characterization of SSR and EST-SSR loci in Chamaecyparis formosensis (Cupressaceae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) and expressed sequence tag (EST)-SSR markers were developed as tools for marker-assisted selection of Chamaecyparis formosensis and for the molecular differentiation of cypress species. METHODS AND RESULTS: Based on the SSR-enriched genomic libraries and transcriptome data of C. formosensis, 300 primer pairs were selected for initial confirmation, of which 19 polymorphic SSR and eight polymorphic EST-SSR loci were chosen after testing in 92 individuals. The number of alleles observed for these 27 loci ranged from one to 17. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.903, respectively. Most markers also amplified in C. obtusa var. formosana. CONCLUSIONS: The developed SSR and EST-SSR sequences are the first reported markers specific to C. formosensis. These markers will be useful for individual identification of C. formosensis and to distinguish cypress species such as C. obtusa var. formosana.

Higher DNA insert fragment sizes improve mitogenomic assemblies from metagenomic pyrosequencing datasets: an example using Limenitidinae butterflies (Lepidoptera, Nymphalidae).

A large number of diverse mitogenomic sequences can be obtained more easily and affordably via mitochondrial metagenomics, which generates high-throughput sequences directly from sheared DNA extractions and assembles mitogenomic sequences using a few bioinformatic processing steps. However, following de novo assembly analysis, the optimal DNA fragment insert size is unclear. In this study, four extracted Limenitidinae butterfly DNA samples were sonically fragmented, and two fragment size ranges (200-400 and 400-600 bp) of each sample were tagged with different barcodes, producing pyrosequencing datasets. The results show that the datasets generated from longer DNA insert fragments result in better coverage and more complete mitogenomic sequences, and the phylogenetic analysis shows high support at nodes, revealing that Athyma butterflies do not represent a monophyletic group. Therefore, we recommend using longer insert DNA fragment sizes to generate high-throughput datasets for obtaining complete mitogenomic sequences which can improve phylogenetic studies.

The MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase.

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.

Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus.

Members of the CENTRORADIALIS (CEN)/TERMINAL FLOWER 1 (TFL1) subfamily control shoot meristem identity, and loss-of-function mutations in both monopodial and sympodial herbaceous plants result in dramatic changes in plant architecture. We studied the degree of conservation between herbaceous and woody perennial plants in shoot system regulation by overexpression and RNA interference (RNAi)-mediated suppression of poplar orthologs of CEN, and the related gene MOTHER OF FT AND TFL 1 (MFT). Field study of transgenic poplars (Populus spp.) for over 6 years showed that downregulation of PopCEN1 and its close paralog, PopCEN2, accelerated the onset of mature tree characteristics, including age of first flowering, number of inflorescences and proportion of short shoots. Surprisingly, terminal vegetative meristems remained indeterminate in PopCEN1-RNAi trees, suggesting the possibility that florigen signals are transported to axillary mersitems rather than the shoot apex. However, the axillary inflorescences (catkins) of PopCEN1-RNAi trees contained fewer flowers than did wild-type catkins, suggesting a possible role in maintaining the indeterminacy of the inflorescence apex. Expression of PopCEN1 was significantly correlated with delayed spring bud flush in multiple years, and in controlled environment experiments, 35S::PopCEN1 and RNAi transgenics required different chilling times to release dormancy. Considered together, these results indicate that PopCEN1/PopCEN2 help to integrate shoot developmental transitions that recur during each seasonal cycle with the age-related changes that occur over years of growth.