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Shrub willows are widely planted for landscaping, soil remediation, and biomass production, due to their rapid growth rates. Identification of regulatory genes in wood formation would provide clues for genetic engineering of willows for improved growth traits on marginal lands. Here, we conducted an expression quantitative trait locus (eQTL) analysis, using a full sibling F1 population of Salix suchowensis, to explore the genetic mechanisms underlying wood formation. Based on variants identified from simplified genome sequencing and gene expression data from RNA sequencing, 16,487 eQTL blocks controlling 5505 genes were identified, including 2148 cis-eQTLs and 16,480 trans-eQTLs. eQTL hotspots were identified, based on eQTL frequency in genomic windows, revealing one hotspot controlling genes involved in wood formation regulation. Regulatory networks were further constructed, resulting in the identification of key regulatory genes, including three transcription factors (JAZ1, HAT22, MYB36) and CLV1, BAM1, CYCB2;4, CDKB2;1, associated with the proliferation and differentiation activity of cambium cells. The enrichment of genes in plant hormone pathways indicates their critical roles in the regulation of wood formation. Our analyses provide a significant groundwork for a comprehensive understanding of the regulatory network of wood formation in S. suchowensis. [ABSTRACT FROM AUTHOR]

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), as the rate-limiting enzyme in the mevalonate pathway, is essential for the biosynthesis of shikonin in Lithospermum erythrorhizon. However, in the absence of sufficient data, the principles of a genome-wide in-depth evolutionary exploration of HMGR family members in plants, as well as key members related to shikonin biosynthesis, remain unidentified. In this study, 124 HMGRs were identified and characterized from 36 representative plants, including L. erythrorhizon. Vascular plants were found to have more HMGR family genes than nonvascular plants. The phylogenetic tree revealed that during lineage and species diversification, the HMGRs evolved independently and intronless LerHMGRs emerged from multi-intron HMGR in land plants. Among them, Pinus tabuliformis and L. erythrorhizon had the most HMGR gene duplications, with 11 LerHMGRs most likely expanded through WGD/segmental and tandem duplications. In seedling roots and M9 cultured cells/hairy roots, where shikonin biosynthesis occurs, LerHMGR1 and LerHMGR2 were expressed significantly more than other genes. The enzymatic activities of LerHMGR1 and LerHMGR2 further supported their roles in catalyzing the conversion of HMG-CoA to mevalonate. Our findings provide insight into the molecular evolutionary properties and function of the HMGR family in plants and a basis for the genetic improvement of efficiently produced secondary metabolites in L. erythrorhizon. [ABSTRACT FROM AUTHOR]

Willows, in the genus Salix, are widespread on the earth with significant ecological and economic values for humans. Although about 500 Salix species have been estimated, the genomic foundation of their adaptations to environments with diverse stresses has been underexplored. Here, we applied a pan-transcriptome approach to investigate the phylogenetic relationships and genetic variations among 16 willow species. A pan-transcriptome of 29,668 gene families was assembled, 69% of which exhibited presence/absence variation across the analyzed species. In comparison to core genes present in all species, shell gene families absent in at least one species were enriched with genes in pathways of signaling transduction and response to stimuli, suggesting their functions in the interaction with diverse environmental factors. A phylogenetic tree of 16 willow species was constructed with high confidence based on 870 single-copy orthologous genes, providing detailed evolutionary relationships of willow sections. The willow species were further assigned into four species clusters using the gene numbers in each family. The diversity of gene family size and gene expression levels among the willow species are closely associated with their geographical distributions. The gene family members involved in DNA repair and cellular response to DNA damage stimuli were expanded in willow species from high-altitude regions in southwestern China, which may contribute to their tolerance to ultraviolet radiation stress. Our study generates a comprehensive pan-transcriptome resource for a large set of Salix species and provides insights into the adaptations of willows to diverse environments, which will be valuable for comparative analysis with other related woody and herbaceous plants. [ABSTRACT FROM AUTHOR]

Expansins are a group of cell wall enzyme proteins that help to loosen cell walls by breaking hydrogen bonds between cellulose microfibrils and hemicellulose. Expansins are essential plant proteins that are involved in several key processes, including seed germination, the growth of pollen tubes and root hairs, fruit ripening and abscission processes. Currently, there is a lack of knowledge concerning the role of expansins in woody plants. In this study, we analyzed expansin genes using Populus genome as the study target. Thirty-six members of the expansin gene family were identified in Populus that were divided into four subfamilies (EXPA, EXPB, EXLA and EXLB). We analyzed the molecular structure, chromosome localization, evolutionary relationships and tissue specificity of these genes and investigated expression changes in responses to phytohormone and abiotic stresses of the expansin genes of Populus tremula L. (PtEXs). Molecular structure analysis revealed that each PtEX protein had several conserved motifs and all of the PtEXs genes had multiple exons. Chromosome structure analysis showed that the expansin gene family is distributed on 14 chromosomes. The PtEXs gene family expansion patterns showed segmental duplication. Transcriptome data of Populus revealed that 36 PtEXs genes were differently expressed in different tissues. Cis-element analysis showed that the PtEXs were closely associated with plant development and responses to phytohormone and abiotic stress. Quantitative real-time PCR showed that abscisic acid (ABA) and low-temperature treatment affected the expression of some PtEXs genes, suggesting that these genes are involved in responses to phytohormone and abiotic stress. This study provides a further understanding of the expansin gene family in Populus and forms a basis for future functional research studies. [ABSTRACT FROM AUTHOR]

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) containing both AGP-like glycated domains and fasciclin (FAS) domains, which are involved in plant growth and development and synthesis of the cell wall. However, these proteins have not been identified or analyzed in willow, Salix, the sister genus of Populus. In this study, we performed a whole genome study of the FLA gene family of Salix suchowensis and compared it with the FLA gene family of Populus deltoides. The results showed the presence of 40 and 46 FLA genes in P. deltoides and S. suchowensis, distributed on 17 and 16 chromosomes, respectively. Four pairs of tandem repeat genes were found in willow, while poplar had no tandem repeat genes. Twelve and thirteen pairs of duplicated gene fragments were identified in poplar and willow, respectively. The multispecies phylogenetic tree showed that the FLA gene family could be divided into four groups (I–IV), with Group 1 showing significant expansion in woody plants. A gene expression analysis showed that PdeFLA19/27 in Group I of poplar was highly expressed, specifically during the secondary growth period of the stem and the rapid elongation of seed hairs. In the Group I genes of S. suchowensis, SsuFLA25/26/28 was also highly expressed during the secondary growth period, whereas increased expression of SsuFLA35 was associated with seed hair tissue. These results provide important clues about the differences in the FLA gene family during the evolution of herbs and woody plants, and suggest that the FLA gene family may play an essential role in regulating the secondary growth of woody plants. It also provides a reference for further studies on the regulation of secondary growth and seed hair development by FLA genes in poplar and willow. [ABSTRACT FROM AUTHOR]

(A) The structural maps of pBI121-LeBAHD1/LeSAT1-eGFP overexpression vector and pYLCRISPR/Cas9-LeBAHD1/LeSAT1 knockout vectors. However, not all members of the BAHD acyltransferase gene family could be screened out from the I L. erythrorhizon i genome by only using AAT/SAT amino acid sequences as a query. Identification and Characterization of LeBAHD Genes in L. erythrorhizon Genome In order to have a comprehensive understanding of the characteristics and functions of BAHD family genes in I L. erythrorhizon i , 104 members were initially screened out from the I L. erythrorhizon i genome based on sequences homology and the conserved BAHD superfamily feature domain (Pfam: PF02458). [Extracted from the article]

Salix is a dioecious plant. Research on the molecular regulation mechanism of male and female inflorescence differentiation and development is necessary to analyze sex differentiation in the willow and the underlying mechanisms of unisexual flower development. However, at present, there are no reference genes suitable for stable expression in the process of willow inflorescence development. In this study, Salix suchowensis was used as the research material, nine candidate reference genes (α-TUB1, α-TUB2, ACT, H2A, DnaJ, CDC2, GAPDH, TIP41, β-TUB) were selected, and qRT-PCR technology was used to detect the expression of each candidate reference gene in female and male flowers at different developmental stages and using five algorithms (geNorm, Normfinder, Delta Ct, BestKeeper, and RefFinder) to comprehensively evaluate the stability of candidate reference genes. The results showed that ACT and DnaJ were stably expressed in all samples and could be used as reference genes. In addition, the reliability of the screening results was further verified via an expression pattern analysis of the CFS gene that encodes flower specific transcription factor in different samples. The stable reference genes selected in this study provide the basis for future research on the expression analysis of functional genes related to the development of male and female flowers of S. suchowensis. [ABSTRACT FROM AUTHOR]

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) have certain similarities with messenger RNAs (mRNAs) and are associated with numerous important biological processes, thereby demanding methods to distinguish them. Based on machine learning algorithms, a variety of methods are developed to identify lncRNAs, providing significant basic data support for subsequent studies. However, many tools lack certain scalability, versatility and balance, and some tools rely on genome sequence and annotation. In this paper, we propose a convenient and accurate tool "PreLnc", which uses high-confidence lncRNA and mRNA transcripts to build prediction models through feature selection and classifiers. The false discovery rate (FDR) adjusted p-value and Z-value were used for analyzing the tri-nucleotide composition of transcripts of different species. Conclusions can be drawn from the experiment that there were significant differences in RNA transcripts among plants, which may be related to evolutionary conservation and the fact that plants are under evolutionary pressure for a longer time than animals. Combining with the Pearson correlation coefficient, we use the incremental feature selection (IFS) method and the comparison of multiple classifiers to build the model. Finally, the balanced random forest was used to construct the classifier, and PreLnc obtained 91.09% accuracy for 349,186 transcripts of animals and plants. In addition, by comparing standard performance measurements, PreLnc performed better than other prediction tools. [ABSTRACT FROM AUTHOR]

Sex-related differences in sex ratio, growth, and herbivory are widely documented in many dioecious plants. The common pattern is for males to grow faster than females and to be less well-defended against herbivores, but Salix is an exception. To study sex-related differences in the patterns of resource allocation for growth and defense in willows, we conducted a large-scale field experiment to investigate the flowering sex ratio, mortality, growth traits, insect herbivory and content of defensive substances in three Salix populations comprising two species. Results demonstrate that the two Salix suchowensis Cheng populations have a female bias in the sex ratio, whereas no bias is found in the S. triandra L. population. Male individuals in the S. suchowensis populations have significantly higher mortality rates than females. However, the mortality rate of S. triandra population has no gender difference. This finding may be one of the explanations for the difference in sex ratio between the two species. The females are larger in height, ground diameter, and biomass, and have a higher nutritional quality (N concentration) than males in both species. Nevertheless, slow-growing males have a higher concentration of the defense chemical (total phenol) and lower degrees of insect herbivory than females. Additionally, biomass is positively correlated with herbivory and negatively correlated with defense in the two willow species. It is concluded that the degrees of herbivory would have a great influence on resource allocation for growth and defense. Meanwhile, it also provides important implications for understanding the evolution of dioecy. [ABSTRACT FROM AUTHOR]

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate. [ABSTRACT FROM AUTHOR]

Pinus massoniana and P. hwangshanensis are two conifer species located in southern China, which are of both economic and ornamental value. Around the middle and lower reaches of the Yangtze River, P. massoniana occurs mainly at altitudes below 700 m, while P. hwangshanensis can be found above 900 m. At altitudes where the distribution of both pines overlaps, a natural introgression hybrid exists, which we will further refer to as the Z pine. This pine has a morphological character that shares attributes of both P. massoniana and P. hwangshanensis. However, compared to the other two pines, its reproductive structure, the pinecone, has an ultra-low ripening rate with seeds that germinate poorly. In this study, we aimed to find the reason for the impaired cone maturation by comparing transcriptome libraries of P. massoniana and Z pine cones at seven successive growth stages. After sequencing and assembly, we obtained unigenes and then annotated them against NCBI's non-redundant nucleotide and protein sequences, Swiss-Prot, Clusters of Orthologous Groups, Gene Ontology and KEGG Orthology databases. Gene expression levels were estimated and differentially expressed genes (DEGs) of the two pines were mined and analyzed. We found that several of them indeed relate to reproductive process. At every growth stage, these genes are expressed at a higher level in P. massoniana than in the Z pine. These data provide insight into understanding which molecular mechanisms are altered between P. massoniana and the Z pine that might cause changes in the reproductive process. [ABSTRACT FROM AUTHOR]

Shrub willows are widely planted for landscaping, soil remediation, and biomass production, due to their rapid growth rates. Identification of regulatory genes in wood formation would provide clues for genetic engineering of willows for improved growth traits on marginal lands. Here, we conducted an expression quantitative trait locus (eQTL) analysis, using a full sibling F 1 population of Salix suchowensis , to explore the genetic mechanisms underlying wood formation. Based on variants identified from simplified genome sequencing and gene expression data from RNA sequencing, 16,487 eQTL blocks controlling 5505 genes were identified, including 2148 cis-eQTLs and 16,480 trans-eQTLs. eQTL hotspots were identified, based on eQTL frequency in genomic windows, revealing one hotspot controlling genes involved in wood formation regulation. Regulatory networks were further constructed, resulting in the identification of key regulatory genes, including three transcription factors ( JAZ1 , HAT22 , MYB36 ) and CLV1 , BAM1 , CYCB2;4 , CDKB2;1 , associated with the proliferation and differentiation activity of cambium cells. The enrichment of genes in plant hormone pathways indicates their critical roles in the regulation of wood formation. Our analyses provide a significant groundwork for a comprehensive understanding of the regulatory network of wood formation in S . suchowensis .