Understanding the Potential Gene Regulatory Network of Starch Biosynthesis in Tartary Buckwheat by RNA-Seq.

Starch is a major component of crop grains, and its content affects food quality and taste. Tartary buckwheat is a traditional pseudo-cereal used in food as well as medicine. Starch content, granule morphology, and physicochemical properties have been extensively studied in Tartary buckwheat. However, the complex regulatory network related to its starch biosynthesis needs to be elucidated. Here, we performed RNA-seq analyses using seven Tartary buckwheat varieties differing in starch content and combined the RNA-seq data with starch content by weighted correlation network analysis (WGCNA). As a result, 10,873 differentially expressed genes (DEGs) were identified and were functionally clustered to six hierarchical clusters. Fifteen starch biosynthesis genes had higher expression level in seeds. Four trait-specific modules and 3131 hub genes were identified by WGCNA, with the lightcyan and brown modules positively correlated with starch-related traits. Furthermore, two potential gene regulatory networks were proposed, including the co-expression of FtNAC70, FtPUL, and FtGBSS1-3 in the lightcyan module and FtbHLH5, C3H, FtBE2, FtISA3, FtSS3-5, and FtSS1 in the brown. All the above genes were preferentially expressed in seeds, further suggesting their role in seed starch biosynthesis. These results provide crucial guidance for further research on starch biosynthesis and its regulatory network in Tartary buckwheat.

Identification of Genetic Locus Underlying Easy Dehulling in Rice-Tartary for Easy Postharvest Processing of Tartary Buckwheat.

As a highly nutritious crop, Tartary buckwheat (Fagopyrum tartaricum) strongly adapts and grows in adverse environments and is widely grown in Asia. However, its flour contains a large proportion of the hull that adheres to the testa layer of the groats and is difficult to be removed in industrial processing. Fortunately, rice-Tartary, with the loose and non-adhering hull, provides potentiality of improving Tartary buckwheat that can dehull easily. Here, we performed high-throughput sequencing for two parents (Tartary buckwheat and rice-Tartary) and two pools (samples from the F2 population) and obtained 101 Gb raw sequencing data for further analysis. Sequencing reads were mapped to the reference genome of Tartary buckwheat, and a total of 633,256 unique SNPs and 270,181 unique indels were found in these four samples. Then, based on the Bulked Segregant Analysis (BSA), we identified a candidate genetic region, containing 45 impact SNPs/indels and 36 genes, that might underly non-adhering hull of rice-Tartary and should have value for breeding easy dehulling Tartary buckwheat.

Comparative transcriptome among Euscaphis konishii Hayata tissues and analysis of genes involved in flavonoid biosynthesis and accumulation.

BACHGROUND: Euscaphis konishii Hayata, a member of the Staphyleaceae Family, is a plant that has been widely used in Traditional Chinese Medicine and it has been the source for several types of flavonoids. To identify candidate genes involved in flavonoid biosynthesis and accumulation, we analyzed transcriptome data from three E. konishii tissues (leaf, branch and capsule) using Illumina Hiseq 2000 platform. RESULTS: A total of 91.7, 100.3 and 100.1million clean reads were acquired for the leaf, branch and capsule, respectively; and 85,342 unigenes with a mean length of 893.60 bp and N50 length of 1307 nt were assembled using Trinity program. BLASTx analysis allowed to annotate 40,218 unigenes using public protein databases, including NR, KOG/COG/eggNOG, Swiss-Prot, KEGG and GO. A total of 14,291 (16.75%) unigenes were assigned to 128 KEGG pathways, and 900 unigenes were annotated into 22 KEGG secondary metabolites, including flavonoid biosynthesis. The structure enzymes involved in flavonoid biosynthesis, such as phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase, shikimate O-hydroxycinnamoyltransferase, coumaroylquinate 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, flavonolsynthese, dihydroflavonol 4-reductase, anthocyanidinreductase, leucoanthocyanidin dioxygenase, leucoanthocyanidin reductase, were identified in the transcriptome data, 40 UDP-glycosyltransferase (UGT), 122 Cytochrome P450 (CYP) and 25 O-methyltransferase (OMT) unigenes were also found. A total of 295 unigenes involved in flavonoid transport and 220 transcription factors (97 MYB, 84 bHLH and 39 WD40) were identified. Furthermore, their expression patterns among different tissues were analyzed by DESeq, the differentially expressed genes may play important roles in tissues-specific synthesis, accumulation and modification of flavonoids. CONCLUSION: We present here the de novo transcriptome analysis of E. konishii and the identification of candidate genes involved in biosynthesis and accumulation of flavonoid. In general, these results are an important resource for further research on gene expression, genomic and functional genomics in E. konishii and other related species.

Functions of stone cells and oleoresin terpenes in the conifer defense syndrome.

Conifers depend on complex defense systems against herbivores. Stone cells (SC) and oleoresin are physical and chemical defenses of Sitka spruce that have been separately studied in previous work. Weevil oviposit at the tip of the previous year's apical shoot (PYAS). We investigated interactions between weevil larvae and trees in controlled oviposition experiments with resistant (R) and susceptible (S) Sitka spruce. R trees have an abundance of SC in the PYAS cortex. SC are mostly absent in S trees. R trees and S trees also differ in the composition of oleoresin terpenes. Transcriptomes of R and S trees revealed differences in long-term weevil-induced responses. Performance of larvae was significantly reduced on R trees compared with S trees under experimental conditions that mimicked natural oviposition behavior at apical shoot tips and may be attributed to the effects of SC. In oviposition experiments designed for larvae to feed below the area of highest SC abundance, larvae showed an unusual feeding behavior and oleoresin appeared to function as the major defense. The results support a role for both SC and oleoresin terpenes and possible synergies between these traits in the defense syndrome of weevil-resistant Sitka spruce.

The evolution of aluminum accumulation in ferns and lycophytes.

PREMISE OF THE STUDY: This paper investigates the occurrence and evolution of aluminum (Al) accumulation within ferns and lycophytes, which is characterized by Al concentrations above 1000 mg·kg-1 in aboveground plant tissues. We hypothesize that this feature is more common in ferns than in angiosperms, and potentially correlated with growth form and other chemical elements. METHODS: Aluminum concentrations were obtained from novel analyses and literature for a total of 354 specimens and 307 species. Moreover, a semiquantitative aluminon test was applied for a subset of 105 species and validated against exact Al measurements. KEY RESULTS: Molecular phylogenetic analyses showed that the major Al-accumulating groups were primarily found in the Gleicheniales and Cyatheales, and largely absent in the Polypodiales. At the species and generic level, Al accumulation was typically either absent or present, and mixed results within a single species and genus were limited to less than 30% of the species and genera tested. Epiphytic ferns had significantly lower Al levels than terrestrial ferns, although this finding was not significant after phylogenetic correction. In addition, a significant, positive correlation was found between Al and iron, while Al was negatively correlated with phosphorus and potassium concentrations. CONCLUSIONS: Aluminum accumulation is most common outside of the Polypodiales and occurs in 38% of the species studied, indicating that this trait is indeed common within subtropical and tropical ferns, a finding that could be in line with their role as pioneer species on landslides and soils with high levels of soluble Al.

Lake sedimentary DNA accurately records 20th Century introductions of exotic conifers in Scotland.

Sedimentary DNA (sedDNA) has recently emerged as a new proxy for reconstructing past vegetation, but its taphonomy, source area and representation biases need better assessment. We investigated how sedDNA in recent sediments of two small Scottish lakes reflects a major vegetation change, using well-documented 20th Century plantations of exotic conifers as an experimental system. We used next-generation sequencing to barcode sedDNA retrieved from subrecent lake sediments. For comparison, pollen was analysed from the same samples. The sedDNA record contains 73 taxa (mainly genus or species), all but one of which are present in the study area. Pollen and sedDNA shared 35% of taxa, which partly reflects a difference in source area. More aquatic taxa were recorded in sedDNA, whereas taxa assumed to be of regional rather than local origin were recorded only as pollen. The chronology of the sediments and planting records are well aligned, and sedDNA of exotic conifers appears in high quantities with the establishment of plantations around the lakes. SedDNA recorded other changes in local vegetation that accompanied afforestation. There were no signs of DNA leaching in the sediments or DNA originating from pollen.

Molecular authentication and quantitative analysis of Sarcandra glabra and adulterated chloranthus products using SNP markers.

BACKGROUND: Sarcandra glabra (Thunb.) Nakai is one of the most popular and valuable plant species in the oriental medicinal herb market. Chloranthus (Chloranthaceae) species are the most widely used adulterants, but they are known to have hepatotoxicity effects and different medicinal values. OBJECTIVE: The aim of this study is to develop a robust and accurate DNA marker for the qualitative and quantitative analyses of their products. MATERIALS AND METHODS: Four single nucleotide polymorphism (SNP) sites specific to Sarcandra glabra, Chloranthus spicatus, Chloranthus serratus and Chloranthus henryi were exploited from the trnL-F region in chloroplast DNA, which have a higher copy number in the products than the nuclear DNA. Based on the SNP sites, specific primers were designed to identify the products of Sarcandra glabra, Chloranthus spicatus, Chloranthus serratus and Chloranthus henryi in mixed solutions via multiplexed PCR. The primers were also used to quantitatively analyse the ratio of chloroplast DNA in the mixed products using real-time PCR. RESULTS: The established multiplexed-PCR and real-time PCR methods were determined to be effective for the authentication and relative quantitative assessments of the products of Sarcandra glabra, its adulterants, and their mixtures. CONCLUSION: We therefore present an effective method for monitoring the quality of these products.

Genetic variation in Rhodomyrtus tomentosa (Kemunting) populations from Malaysia as revealed by inter-simple sequence repeat markers.

Kemunting (Rhodomyrtus tomentosa) from the Myrtaceae family, is native to Malaysia. It is widely used in traditional medicine to treat various illnesses and possesses significant antibacterial properties. In addition, it has great potential as ornamental in landscape design. Genetic variability studies are important for the rational management and conservation of genetic material. In the present study, inter-simple sequence repeat markers were used to assess the genetic diversity of 18 R. tomentosa populations collected from ten states of Peninsular Malaysia. The 11 primers selected generated 173 bands that ranged in size from 1.6 kb to 130 bp, which corresponded to an average of 15.73 bands per primer. Of these bands, 97.69% (169 in total) were polymorphic. High genetic diversity was documented at the species level (H(T) = 0.2705; I = 0.3973; PPB = 97.69%) but there was a low diversity at population level (H(S) = 0.0073; I = 0 .1085; PPB = 20.14%). The high level of genetic differentiation revealed by G(ST) (73%) and analysis of molecular variance (63%), together with the limited gene flow among population (N(m) = 0.1851), suggests that the populations examined are isolated. Results from an unweighted pair group method with arithmetic mean dendrogram and principal coordinate analysis clearly grouped the populations into two geographic groups. This clear grouping can also be demonstrated by the significant Mantel test (r = 0.581, P = 0.001). We recommend that all the R. tomentosa populations be preserved in conservation program.

Genetic characterization and authentication of Dimocarpus longan Lour. using an improved RAPD technique.

Dimocarpus longan Lour. is an edible and traditional herb in China, commonly referred to as longon. An improved randomly amplified polymorphic DNA (RAPD) protocol was here developed in order to determine the geographical origins of D. longan samples collected from 5 provinces in the southern and southwestern areas of China, including Sichuan, Hainan, Fujian, Guangdong, and Guangxi. Generally, the improved RAPD method generated good fingerprinting of the 5 samples using the selected 17 primers. In particular, primers SBS-A5, SBS-A13, SBS-I9, SBS-I20, SBS-M1, and SBS-Q12 produced distinguishable bands that clearly separated all 5 cultivars, suggesting that there are variations in RAPD genetic sites among the samples. The similarity index ranged from 0.69 to 0.76. The Sichuan and Hainan clades clustered together with a 0.73 similarity index. The Guangxi and Fujian clades clustered together with a 0.76 similarity index, and they formed the sister clade to the Sichuan/Hainan clade with a 0.71 similarity index. The Guangdong clade was in a basal polytomy with a 0.70 similarity index. Based on the abundant DNA polymorphisms, these longan accessions are distinguishable using our improved RAPD technique. Therefore, RAPD analysis is an effective technique in distinguishing the geographical origins of D. longan. Moreover, the improved method could also be employed for a variety of applications including genetic diversity and fingerprinting analyses.

Contemporary pollen-mediated gene immigration reflects the historical isolation of a rare, animal-pollinated shrub in a fragmented landscape.

Fragmentation is generally considered to have negative impacts on widespread outbreeders but impacts on gene flow and diversity in patchy, naturally rare, self-compatible plant species remain unclear. We investigated diversity, gene flow and contemporary pollen-mediated gene immigration in the rare, narrowly distributed endemic shrub Calothamnus quadrifidus ssp. teretifolius. This taxon occurs in an internationally recognized biodiversity hotspot subjected to recent human-induced fragmentation and the condition of the remnants ranges from intact to highly degraded. Using microsatellites, we found that inbreeding, historically low gene flow and significant population differentiation have characterized the genetic system of C. quadrifidus ssp. teretifolius. Inbreeding arises from self-pollination, a small amount of biparental inbreeding and significant correlation of outcross paternity but fecundity was high suggesting populations might have purged their lethals. Paternity analyses show that pollinators can move pollen over degraded and intact habitat but populations in both intact and degraded remnants had few pollen parents per seed parent and low pollen immigration. Genetic diversity did not differ significantly between intact and degraded remnants but there were signs of genetic bottlenecks and reduced diversity in some degraded remnants. Overall, our study suggests human-induced fragmentation has not significantly changed the mating system, or pollen immigration to, remnant populations and therefore genetic connectivity need not be the highest conservation priority. Rather, for rare species adapted to higher levels of inbreeding, conservation efforts may be best directed to managing intact habitats and ecosystem processes.

Pinaceae-like reproductive morphology in Schizolepidopsis canicularis sp. nov. from the Early Cretaceous (Aptian-Albian) of Mongolia.

PREMISE OF THE STUDY: Seed cone scales assigned to the genus Schizolepidopsis are widespread in Late Triassic to Cretaceous Eurasian deposits. They have been linked to the conifer family Pinaceae based on associated vegetative remains, but their exact affinities are uncertain. Recently discovered material from the Early Cretaceous of Mongolia reveals important new information concerning Schizolepidopsis cone scales and seeds, and provides support for a relationship between the genus and extant Pinaceae. METHODS: Specimens were collected from Early Cretaceous (probable Aptian-Albian) lignite deposits in central Mongolia. Lignite samples were disaggregated, cleaned in hydrofluoric acid, and washed in water. Specimens were selected for further study using light and electron microscopy. KEY RESULTS: Schizolepidopsis canicularis seed cones consist of loosely arranged, bilobed ovulate scales subtended by a small bract. A single inverted seed with an elongate micropyle is borne on each lobe of the ovulate scale. Each seed has a wing formed by the separation of the adaxial surface of the ovulate scale. CONCLUSIONS: Schizolepidopsis canicularis produced winged seeds that formed in a manner that is unique to Pinaceae among extant conifers. We do not definitively place this species in Pinaceae pending more complete information concerning its pollen cones and vegetative remains. Nevertheless, this material suggests that Schizolepidopsis may be important for understanding the early evolution of Pinaceae, and may potentially help reconcile the appearance of the family in the fossil record with results based on phylogenetic analyses of molecular data.

Aberrant Classopollis pollen reveals evidence for unreduced (2n) pollen in the conifer family Cheirolepidiaceae during the Triassic-Jurassic transition.

Polyploidy (or whole-genome doubling) is a key mechanism for plant speciation leading to new evolutionary lineages. Several lines of evidence show that most species among flowering plants had polyploidy ancestry, but it is virtually unknown for conifers. Here, we study variability in pollen tetrad morphology and the size of the conifer pollen type Classopollis extracted from sediments of the Triassic-Jurassic transition, 200 Ma. Classopollis producing Cheirolepidiaceae were one of the most dominant and diverse groups of conifers during the Mesozoic. We show that aberrant pollen Classopollis tetrads, triads and dyads, and the large variation in pollen size indicates the presence of unreduced (2n) pollen, which is one of the main mechanisms in modern polyploid formation. Polyploid speciation may explain the high variability of growth forms and adaptation of these conifers to different environments and their resistance to extreme growth conditions. We suggest that polyploidy may have also reduced the extinction risk of these conifers during the End-Triassic biotic crisis.

Application of the ITS2 Region for Barcoding Medicinal Plants of Selaginellaceae in Pteridophyta.

BACKGROUND: Selaginellaceae is a family of nonseed plants with special evolutionary significance. Plants of the family Selaginellaceae are similarly shaped and easily confused, complicating identification via traditional methods. This study explored, for the first time, the use of the DNA barcode ITS2 to identify medicinal plants of the Selaginellaceae family. METHODOLOGY/PRINCIPAL FINDINGS: In our study, 103 samples were collected from the main distribution areas in China; these samples represented 34 species and contained almost all of the medicinal plants of Selaginellaceae. The ITS2 region of the genome was amplified from these samples and sequenced using universal primers and reaction conditions. The success rates of the PCR amplification and sequencing were 100%. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 regions, while the presence of a barcoding gap was obvious. Using the BLAST1 and nearest distance methods, our results proved that the ITS2 regions could successfully identify the species of all Selaginellaceae samples examined. In addition, the secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Selaginellaceae. Furthermore, cluster analysis using the ITS2 barcode supported the relationship between the species of Selaginellaceae established by traditional morphological methods. CONCLUSION: The ITS2 barcode can effectively identify medicinal plants of Selaginellaceae. The results provide a scientific basis for the precise identification of plants of the family Selaginellaceae and the reasonable development of these resources. This study may broaden the application of DNA barcoding in the medicinal plant field and benefit phylogenetic investigations.

Transcriptome/degradome-wide identification of R. glutinosa miRNAs and their targets: the role of miRNA activity in the replanting disease.

Rehmannia glutinosa, a traditional Chinese medicine herb, is unable to grow normally in a soil where the same species has recently been cultivated. The biological basis of this so called "replanting disease" is unknown, but it may involve the action of microRNAs (miRNAs), which are known to be important regulators of plant growth and development. High throughput Solexa/Illumina sequencing was used to generate a transcript library of the R. glutinosa transcriptome and degradome in order to identify possible miRNAs and their targets implicated in the replanting disease. A total of 87,665 unigenes and 589 miRNA families (17 of which have not been identified in plants to date) was identified from the libraries made from a first year (FP) and a second year (SP) crop. A comparison between the FP and SP miRNAs showed that the abundance of eight of the novel and 295 of the known miRNA families differed between the FP and SP plants. Sequencing of the degradome sampled from FP and SP plants led to the identification of 165 transcript targets of 85 of the differentially abundant miRNA families. The interaction of some of these miRNAs with their target(s) is likely to form an important part of the molecular basis of the replanting disease of R. glutinosa.

Recurrent abnormalities in conifer cones and the evolutionary origins of flower-like structures.

Conifer cones are reproductive structures that are typically of restricted growth and either exclusively pollen-bearing (male) or exclusively ovule-bearing (female). Here, we review two common spontaneous developmental abnormalities of conifer cones: proliferated cones, in which the apex grows vegetatively, and bisexual cones, which possess both male and female structures. Emerging developmental genetic data, combined with evidence from comparative morphology, ontogeny and palaeobotany, provide new insights into the evolution of both cones and flowers, and prompt novel strategies for understanding seed-plant evolution.

Patterns of pollen and seed dispersal in a small, fragmented population of the wind-pollinated tree Araucaria angustifolia in southern Brazil.

Paternity analysis based on eight microsatellite loci was used to investigate pollen and seed dispersal patterns of the dioecious wind-pollinated tree, Araucaria angustifolia. The study sites were a 5.4 ha isolated forest fragment and a small tree group situated 1.7 km away, located in Paranalpha State, Brazil. In the forest fragment, 121 males, 99 females, 66 seedlings and 92 juveniles were mapped and genotyped, together with 210 seeds. In the tree group, nine male and two female adults were mapped and genotyped, together with 20 seeds. Paternity analysis within the forest fragment indicated that at least 4% of the seeds, 3% of the seedlings and 7% of the juveniles were fertilized by pollen from trees in the adjacent group, and 6% of the seeds were fertilized by pollen from trees outside these stands. The average pollination distance within the forest fragment was 83 m; when the tree group was included the pollination distance was 2006 m. The average number of effective pollen donors was estimated as 12.6. Mother-trees within the fragment could be assigned to all seedlings and juveniles, suggesting an absence of seed immigration. The distance of seedlings and juveniles from their assigned mother-trees ranged from 0.35 to 291 m (with an average of 83 m). Significant spatial genetic structure among adult trees, seedlings, and juveniles was detected up to 50 m, indicating seed dispersal over a short distance. The effective pollination neighborhood ranged from 0.4 to 3.3 ha. The results suggest that seed dispersal is restricted but that there is long-distance pollen dispersal between the forest fragment and the tree group; thus, the two stands of trees are not isolated.

Estimating pollen flow using SSR markers and paternity exclusion: accounting for mistyping.

Highly informative genetic markers, such as simple sequence repeats (SSRs), can be used to directly measure pollen flow by parentage analysis. However, mistyping (i.e. false inference of genotypes caused by the occurrence of null alleles, mutations, and detection errors) can lead to substantial biases in the estimates obtained. Using computer simulations, we evaluated a direct method for estimating pollen immigration using SSR markers and a paternity exclusion approach. This method accounts for mistyping and does not rely on assumptions about the distribution of male reproductive success. If ignored, even minor rates of mistyping (1.5%) resulted in overestimating pollen immigration by up to 150%. When we required at least two mismatching loci before excluding candidate fathers from paternity, the resulting pollen immigration estimates had small biases for rates of mistyping up to 4.5%. Requiring at least three mismatches for exclusion was needed to minimize the upward biases of pollen immigration caused by rates of mistyping up to 10.5%. The minimum number of highly variable SSR loci needed to minimize cryptic gene flow and obtain reliable estimates of pollen immigration varied from five to seven for a sampling scheme applicable to most conifers (i.e. when paternal haplotypes can be unambiguously determined). Between five and nine highly variable SSR loci were needed for a more general sampling scheme that is applicable to all diploid seed plants. With moderately variable SSR markers, consistently accurate estimates of pollen immigration could be obtained only for rates of mistyping up to 4.5%. We developed the POLLEN FLOW (PFL) computer program which can be used to obtain unbiased and precise estimates of pollen immigration under a wide range of conditions, including population sizes as large as 600 parents and mistyping rates as high as 10.5%.