Characterization of the physiological parameters, effective components, and transcriptional profiles of Polygonum multiflorum Thunb. Under pH stress.

Polygonum multiflorum Thunb. is a traditional Chinese medicine with extensive distribution and robust adaptability, but comprehensive research on its acid and alkali resistance is presently lacking. This study aimed to analyze the effects of 5 months of continuous pH stress on the physiological and photosynthetic parameters of P. multiflorum, and the content of effective components. Results revealed that pH stress significantly influenced the normal growth, physiological functions, and photosynthetic indicators of P. multiflorum. At soil pH 4.5, the tubers of P. multiflorum exhibited the highest levels of 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-d-glucoside (THSG) and total anthraquinones at 5.41% and 0.38%, respectively. However, increased soil pH significantly reduced the content of THSG and total anthraquinones. Reference-free transcriptome analysis was further conducted on P. multiflorum treated at pH 4.5 and 9.5, generating a total of 47,305 unigenes with an N50 of 2118 bp, of which 31,058 (65.65%) were annotated. Additionally, 2472 differentially expressed genes (DEGs) were identified. Among them, 17 DEGs associated with the biosynthesis of THSG and anthraquinones were screened. A comprehensive analysis of differential gene expression and effective component content demonstrated a significant positive correlation between the content of effective components and the 14 DEGs' expression but a negative correlation with soil pH. This study highlighted the influence of varying soil pH values on the effective component content of P. multiflorum. Specific acidic conditions proved beneficial for the synthesis and accumulation of THSG and total anthraquinones in P. multiflorum, thereby enhancing the quality of the medicinal material.

Co-expression network analysis of genes and networks associated with wheat pistillody.

Crop male sterility has great value in theoretical research and breeding application. HTS-1, whose stamens transformed into pistils or pistil-like structures, is an important male sterility material selecting from Chinese Spring three-pistil (CSTP) wheat. However the molecular mechanism of pistillody development in HTS-1 remains a mystery. RNA-seq data of 11 wheat tissues were obtained from the National Center for Biotechnology Information (NCBI), including the stamens of CSTP and the pistils and pistillodic stamen of HTS-1. The Salmon program was utilized to quantify the gene expression levels of the 11 wheat tissues; and gene quantification results were normalized by transcripts per million (TPM). In total, 58,576 genes were used to construct block-wise network by co-expression networks analysis (WGCNA) R package. We obtained all of modules significantly associated with the 11 wheat tissues. AgriGO V2.0 was used to do Gene Ontology (GO) enrichment analysis; and genes and transcription factors (TFs) in these significant modules about wheat pistillody development were identified from GO enrichment results. Basic local alignment search tool (BLAST) was used to align HTS-1 proteins with the published pistillody-related proteins and TFs. Genes about wheat pistillody development were analyzed and validated by qRT-PCR. The MEturquoise, MEsaddlebrown, MEplum, MEcoral1, MElightsteelblue1, and MEdarkslateblue modules were significantly corelated to pistillodic stamen (correlation p < 0.05). Moreover, 206 genes related to carpel development (GO:0048440) or gynoecium development (GO:0048467) were identified only in the MEturquoise module by Gene Ontology (GO) analysis, and 42 of 206 genes were hub genes in MEturquoise module. qRT-PCR results showed that 38 of the 42 hub genes had highly expressed in pistils and pistillodic stamens than in stamens. A total of 15 pistillody development-related proteins were validated by BLAST. Transcription factors (TFs) were also analyzed in the MEturquoise module, and 618 TFs were identified. In total, 56 TFs from 11 families were considered to regulate the development of pistillodic stamen. The co-expression network showed that six of HB and three of BES1 genes were identified in 42 hub genes. This indicated that TFs played important roles in wheat pistillody development. In addition, there were 11 of ethylene-related genes connected with TFs or hub genes, suggesting the important roles of ethylene-related genes in pistillody development. These results provide important insights into the molecular interactions underlying pistillody development.

The bZIP Transcription Factor Family in Adzuki Bean (Vigna Angularis): Genome-Wide Identification, Evolution, and Expression Under Abiotic Stress During the Bud Stage.

Adzuki bean (Vigna angularis) is an important dietary legume crop that was first cultivated and domesticated in Asia. Currently, little is known concerning the evolution and expression patterns of the basic leucine zipper (bZIP) family transcription factors in the adzuki bean. Through the PFAM search, 72 bZIP members of adzuki bean (VabZIP) were identified from the reference genome. Most of them were located on 11 chromosomes and seven on an unknown chromosome. A comprehensive analysis, including evolutionary, motifs, gene structure, cis-elements, and collinearity was performed to identify VabZIP members. The subcellular localization results showed VabZIPs might locate on the nuclear. Quantitative real-time PCR (qRT-PCR) analysis of the relative expression of VabZIPs in different tissues at the bud stage revealed that VabZIPs had a tissue-specific expression pattern, and its expression was influenced by abiotic stress. These characteristics of VabZIPs provide insights for future research aimed at developing interventions to improve abiotic stress resistance.

Resequencing of 683 common bean genotypes identifies yield component trait associations across a north-south cline.

We conducted a large-scale genome-wide association study evaluation of 683 common bean accessions, including landraces and breeding lines, grown over 3 years and in four environments across China, ranging in latitude from 18.23° to 45.75° N, with different planting dates and abiotic or biotic stresses. A total of 505 loci were associated with yield components, of which seed size, flowering time and harvest maturity traits were stable across years and environments. Some loci aligned with candidate genes controlling these traits. Yield components were observed to have strong associations with a gene-rich region on the long arm of chromosome 1. Manipulation of seed size, through selection of seed length versus seed width and height, was deemed possible, providing a genome-based means to select for important yield components. This study shows that evaluation of large germplasm collections across north-south geographic clines is useful in the detection of marker associations that determine grain yield in pulses.

RicetissueTFDB: A Genome-Wide Identification of Tissue-Specific Transcription Factors in Rice.

Transcription factors (TFs) regulate plant gene expression in different tissues. To investigate TF genes in rice ( L.), a genome-wide TF identification was conducted with the japonica rice genome. This study identified 3078 putative TFs in 59 families. The TF number of the top 10 TF families accounted for 58% of the 3078 rice TFs. The three largest TF families were the myeloblastosis (MYB) superfamily, basic helix-loop-helix (bHLH), and far-red-impaired response (FAR1), which contained 413, 228, and 210 TF members, respectively. The expression profiles of the 3078 TF genes were surveyed with the RNA sequencing (RNA-seq) data of 13 rice tissue types. Based on these expression profiles, we validated 1087 TFs expressed in 13 rice tissue types, which accounted for 35.32% of the 3078 putative TFs. We further analyzed the tissue-specific TFs in rice. In total, 28, 14, 14, 10, 9, 5, 5, 4, 3, 3, 2, 11, and 1 tissue-specific TF sequences were identified in the dry seed, pistil, spikelet, aleurone, anther, ovules, embryo 25 d after pollination (DAP), seed 5 DAP, root, leaf, seed 10 DAP, shoot, and endosperm 25 DAP, respectively. Moreover, we constructed RicetissueTFDB (), a comprehensive and public rice TF database that integrates tissue expression characters, genomic location, and Gene Ontology (GO) terms for each TF. The RicetissueTFDB database will facilitate the identification of target TFs and the functional studies about rice TFs.

TaEPFL1, an EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) secreted peptide gene, is required for stamen development in wheat.

Members of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family play diverse roles in plant growth and development, including the guidance of inflorescence architecture and pedicel length. In this work, we identified and characterized the EFPL gene TaEPFL1 from the wheat pistillody mutant HTS-1. Sequence alignment and phylogenetic analysis indicated that TaEPFL1 belongs to the EPFL1 gene. Quantitative real-time RT-PCR analysis showed that the TaEPFL1 gene is expressed at an abnormally high level in pistillody stamens compared with that in pistils and stamens. Heterologous expression of the TaEPFL1 gene in Arabidopsis caused shortened filaments and pedicels and might reduce the level of AtACO2 gene expression. These results suggest that TaEPFL1 plays an important role in the development of stamen and that overexpression of TaEPFL1 results in abnormal stamens. We deduced that the overexpression of the TaEPFL1 gene may contribute to the homeotic transformation of stamens into pistils or pistil-like structures in wheat. These data offer insights into the molecular mechanism of pistillody mutation in wheat.

High-density genetic map construction and mapping of the homologous transformation sterility gene (hts) in wheat using GBS markers.

BACKGROUND: Homologous transformation sterility-1 (HTS-1) is a novel wheat mutant that exhibits pistillody, the transformation of stamens into pistils or pistil-like structures. More extreme phenotypes of this mutation can have six pistils or pistil-like structures without any stamens in a floret. Thus, HTS-1 is highly valuable for studies of wheat hybrid breeding and flower development. Previous studies have shown that two major genes (Pis1 and hts) control pistillody in HTS-1. The Pis1 gene controls the three-pistil trait in the three-pistil wheat mutant and has been mapped on chromosome 2D, but the hts gene has not been mapped or identified. To do so, we crossed HTS-1 with CM28TP (three-pistil mutant) and constructed a high-density linkage map with the F2 population (200 individuals). RESULTS: The map covered 2779.96 cM, and the genetic distance per chromosome ranged from 37.59 cM to 318.95 cM. The average distance between markers was 1.04 cM. We then mapped hts between GBS-SNP markers 4A_109 and 4A_119, separated by 2.0 cM and 5.2 Mb. To find the candidate genes, the hts region was enlarged to 7.2 Mb, encompassing 752 protein-coding genes. We identified TaWin1 as a possible candidate gene after comparing the 752 genes with 206 common differentially expressed genes between pistillody stamens (PS) versus normal stamens (S) and pistils (P) versus S. Real-time PCR indicated that TaWin1 was highly expressed in HTS-1 during the pistil-and-stamen-differentiating stage, at levels approximately 120 times greater than those in CM28TP. Further analysis indicated that TaWin1 was mainly expressed in HTS-1 PS, supporting its status as a candidate gene of hts. Thus, TaWin1 overexpression probably leads to the transformation of stamens into pistils in wheat. CONCLUSIONS: The results of this study provide a foundation for further research on stamen and pistil development, with implications for wheat-hybrid breeding programs.

Development of a high-density linkage map and mapping of the three-pistil gene (Pis1) in wheat using GBS markers.

BACKGROUND: The wheat mutant line three-pistil (TP) exhibits three pistils per floret. As TP normally has two or three seeds in each of the florets on the same spike, there is the possibility of increasing the number of grains per spike. Therefore, TP is a highly valuable mutant for breeding and for the study of floral development in wheat. To map the three-pistil gene (Pis1), genotyping-by-sequencing single-nucleotide polymorphism (GBS-SNP) data from an F2 mapping population (CM28 × CM28TP) was used to construct a genetic map that is of significant value. RESULTS: In the present study, a high-density genetic map of wheat containing 2917 GBS-SNP markers was constructed. Twenty-one linkage groups were resolved, with a total length of 2371.40 cM. The individual chromosomes range from 2.64 cM to 454.55 cM with an average marker density of 0.81 cM. The Pis1 gene was mapped using this high-resolution map, and two flanking SNP markers tightly linked to the gene, M70 and M71, were identified. The Pis1 is 3.00 cM from M70 and 1.10 cM from M71. In bread wheat genome, M70 and M71 were found to delimit a physical distance of 3.40 Mb, which encompasses 127 protein-coding genes. To validate the GBS-generated genotypic data and to eliminate missing marker data in the Pis1 region, five Kompetitive Allele-Specific PCR (KASP) assays were designed from corresponding GBS sequences, which harbor SNPs that surround Pis1. Three KASP-SNP markers, KM70, KM71, and KM75, were remapped to the Pis1 gene region. CONCLUSIONS: This work not only lays the foundation for the map-based cloning of Pis1 but can also serve as a valuable tool for studying marker-trait association of important traits and marker-assisted breeding in wheat.

Molecular cloning, characterization and expression of WAG-2 alternative splicing transcripts in developing spikes of Aegilops tauschii.

WAG-2 is a C-class MADS-box gene, which is orthologous to AGAMOUS (AG) in Arabidopsis. The AG group C-class MADS-box genes are involved in stamen and pistil identity. In this study, two WAG-2 transcripts, namely, WAG-2f and WAG- 2g, were isolated and characterized from Aegilops tauschii. The open reading frames of WAG-2f and WAG-2g were 825 and 822 bp, respectively, encoding 275 and 274 amino acid residues. BLAST searches of partial WAG-2 genomic sequence against the draft sequence of Ae. tauschii genome database revealed the complex structure of WAG-2 gene, which consisted of seven exons and six introns. The WAG-2f and WAG-2g cDNAs were two alternative splicing transcripts. The alternative splicing events were produced by an alternative 5' splice site. The expression level of WAG-2f transcript, which was extremely weak in young spikes of floret primordium formation stage, increased as the spikes developed. The highest expression was observed in the spikes at the anther separation stage. Low expression levels of WAG-2f were also detected at the tetrad stage. The WAG- 2g transcript was expressed at all four stages of spike development but at a relatively low level. The expression pattern of the two transcripts was distinctly different during floral development, thereby suggesting a functional divergence.

Potential of marker selection to increase prediction accuracy of genomic selection in soybean (Glycine max L.).

Genomic selection is a promising molecular breeding strategy enhancing genetic gain per unit time. The objectives of our study were to (1) explore the prediction accuracy of genomic selection for plant height and yield per plant in soybean [Glycine max (L.) Merr.], (2) discuss the relationship between prediction accuracy and numbers of markers, and (3) evaluate the effect of marker preselection based on different methods on the prediction accuracy. Our study is based on a population of 235 soybean varieties which were evaluated for plant height and yield per plant at multiple locations and genotyped by 5361 single nucleotide polymorphism markers. We applied ridge regression best linear unbiased prediction coupled with fivefold cross-validations and evaluated three strategies of marker preselection. For plant height, marker density and marker preselection procedure impacted prediction accuracy only marginally. In contrast, for grain yield, prediction accuracy based on markers selected with a haplotype block analyses-based approach increased by approximately 4 % compared with random or equidistant marker sampling. Thus, applying marker preselection based on haplotype blocks is an interesting option for a cost-efficient implementation of genomic selection for grain yield in soybean breeding.

Pistillody mutant reveals key insights into stamen and pistil development in wheat (Triticum aestivum L.).

BACKGROUND: The pistillody mutant wheat (Triticum aestivum L.) plant HTS-1 exhibits homeotic transformation of stamens into pistils or pistil-like structures. Unlike common wheat varieties, HTS-1 produces three to six pistils per floret, potentially increasing the yield. Thus, HTS-1 is highly valuable in the study of floral development in wheat. In this study, we conducted RNA sequencing of the transcriptomes of the pistillody stamen (PS) and the pistil (P) from HTS-1 plants, and the stamen (S) from the non-pistillody control variety Chinese Spring TP to gain insights into pistil and stamen development in wheat. RESULTS: Approximately 40 Gb of processed reads were obtained from PS, P, and S. De novo assembly yielded 121,210 putative unigenes, with a mean length of 695 bp. Among these high-quality unigenes, 59,199 (48.84%) had at least one significant match with an existing gene model. A total of 23, 263, and 553 differentially expressed genes were identified in PS vs. P, PS vs. S, and P vs. S, respectively, with differences in expression greater than five-fold. Among the differentially expressed genes, 206 were highly correlated with stamen and pistil development. These genes include WM27B, DL, YAB1, YABBY4, WM 5, CER 1, and WBLH1, which have been implicated in flower development. The expression patterns of 25 differentially expressed genes were confirmed through quantitative real-time reverse transcription PCR. CONCLUSIONS: Analysis of this transcriptome resource enabled us to characterize gene expression profiles, examine differential gene expression, and produce a candidate gene list related to wheat stamen and pistil development. This work is significant for the development of genomic resources for wheat, and provides important insights into the molecular mechanisms of wheat stamen and pistil development.

Cloning and characterization of endo-ß-1,4-glucanase genes in the common wheat line three pistils.

In this work, we report the cloning and characterization of endo-ß-1,4-glucanase (EGase) genes (TaEG) in the common wheat line three pistils. Three TaEG homoeologous genes (TaEG-4A, TaEG-4B and TaEG-4D) were isolated and found to be located on chromosomes 4AL, 4BS and 4DS, respectively. The three genes showed high conservation of their coding nucleotide sequences and 3 untranslated region. The putative TaEG protein had a molecular mass of 69 kDa, a theoretical pI of 9.39 and a transmembrane domain of 74-96 amino acids in the N-terminus that anchored the protein to the membrane. The genome sequences of TaEG-4A, TaEG-4B and TaEG-4D contained six exons and five introns. All of the introns, except for intron IV, varied in length and sequence composition. Phylogenetic analysis revealed that TaEG was most closely related to rice (Oryza sativa) OsGLU1. The TaEG transcript levels increased significantly during the subsidiary pistil primordium differentiation phase (spike size ∼7-10 mm) in Chuanmai 28 TP (CM28TP). These data provide a basis for future research into the function of TaEG and offer insights into the molecular mechanism of the three pistils mutation in wheat.

Identification of differentially expressed genes in three-pistil mutation in wheat using annealing control primer system.

The common wheat line three-pistil (TP) is a valuable mutant for wheat breeding. The TP mutation has normal spike morphology; however, it only produces three pistils per floret. Therefore, it has potential to increase the grain number per spike. In order to determine the underlying molecular mechanism, an annealing control primer system was used to identify the different expressed genes in three-pistil mutation. Using 120 arbitrary ACP primers, we identified three differentially expressed genes in young spikes between two near-isogenic lines (i.e., Chuanmai 28 TP and Chinese Spring TP) and their recurrent parents. We tentatively designated the three differentially expressed genes as DETP-1, DETP-2, and DETP-3. DETP-1 showed similar function with maize cytoplasmic membrane protein, which is involved in cell division in bacteria. DETP-3 is homologous to maize endo-1, 4-beta-glucanase (EGases), which is associated with plant development, cell wall loosening, stem flowering, and root expansion. DETP-2 showed no significant hit with any sequence found in the database and translates unknown protein. These genes would likely play an important role in determining the three pistils trait in wheat.

Nucleotide diversity and molecular evolution of the WAG-2 gene in common wheat (Triticum aestivum L) and its relatives.

In this work, we examined the genetic diversity and evolution of the WAG-2 gene based on new WAG-2 alleles isolated from wheat and its relatives. Only single nucleotide polymorphisms (SNP) and no insertions and deletions (indels) were found in exon sequences of WAG-2 from different species. More SNPs and indels occurred in introns than in exons. For exons, exons+introns and introns, the nucleotide polymorphism π decreased from diploid and tetraploid genotypes to hexaploid genotypes. This finding indicated that the diversity of WAG-2 in diploids was greater than in hexaploids because of the strong selection pressure on the latter. All dn/ds ratios were < 1.0, indicating that WAG-2 belongs to a conserved gene affected by negative selection. Thirty-nine of the 57 particular SNPs and eight of the 10 indels were detected in diploid species. The degree of divergence in intron length among WAG-2 clones and phylogenetic tree topology suggested the existence of three homoeologs in the A, B or D genome of common wheat. Wheat AG-like genes were divided into WAG-1 and WAG-2 clades. The latter clade contained WAG-2, OsMADS3 and ZMM2 genes, indicating functional homoeology among them.

Nucleotide diversity and molecular evolution of the WAG-2 gene in common wheat (Triticum aestivum L) and its relatives

In this work, we examined the genetic diversity and evolution of the WAG-2 gene based on new WAG-2 alleles isolated from wheat and its relatives. Only single nucleotide polymorphisms (SNP) and no insertions and deletions (indels) were found in exon sequences of WAG-2 from different species. More SNPs and indels occurred in introns than in exons. For exons, exons+introns and introns, the nucleotide polymorphism Π decreased from diploid and tetraploid genotypes to hexaploid genotypes. This finding indicated that the diversity of WAG-2 in diploids was greater than in hexaploids because of the strong selection pressure on the latter. All dn/ds ratios were < 1.0, indicating that WAG-2 belongs to a conserved gene affected by negative selection. Thirty-nine of the 57 particular SNPs and eight of the 10 indels were detected in diploid species. The degree of divergence in intron length among WAG-2 clones and phylogenetic tree topology suggested the existence of three homoeologs in the A, B or D genome of common wheat. Wheat AG-like genes were divided into WAG-1 and WAG-2 clades. The latter clade contained WAG-2, OsMADS3 and ZMM2 genes, indicating functional homoeology among them.

[The dynamic change of total alkaloids content from Pinellia ternate].

OBJECTIVE: To determine the alkaloids content of Pinellia ternate in different growing time for finding out the best harvest time. METHODS: The alkaloid was extracted by chloroform from Pinellia ternate and tested at 417 nm with the spectroscopy by acid dye. The result was analyzed in One-Way ANOVA. RESULTS: The calibration curve of alkaloid was linear( r = 0.9989). The recovery rate was perfect (100.1%). The alkaloids content in different growing time had very significant difference ( F = 12.789, P < 0.01). CONCLUSION: This method is simple and quick. The alkaloids content in whole seedling stage is higher than that in other growing stages, while the mean total alkaloids output per plant is the highest in intensive seedling-downfallen stage which is considered as the best harvest time.

Characterization of the common wheat (Triticum aestivum L.) mutation line producing three pistils in a floret.

In a normal wheat (Triticum ssp.L.) spike, one floret carries only one pistil that will further develop into one grain after fertilization. The cultivated common wheat (T. aestivum L.) mutation line Three Pistils (TP) carried three pistils in a floret. Although one or two of the pistils died out before seed set in some florets, there were exist many florets that set three seeds. Normally, it was observed that there were one to three seeds in different florets of the same spike. Therefore, this mutation trait could raise considerably the number of grains per spike. The weight of 100 grains in three seeds set florets was lower than that of in one seed set florets. But three seeds set florets were significantly to surpass the one seed set florets in grain(s) weight per floret. Based on these results, the three pistils trait was suggested to be an interesting germplasm resource. Localisation of the gene controlling the three pistils trait was carried out by the method of crossing TP with the Chinese Spring disomic substitutions. F2 population segregation analysis revealed that only the 5B F2 population did not show homogeneity to control population. chi2-test analysis indicated that 5B F2 population, and only this population, was deviated from the Mendelian segregation ratio (3:1). As a conclusion, the gene for three pistils trait was located on chromosome 5B. According to the Recommended rules for gene symbolization in wheat, the name of the dominant gene for three pistils trait in the line TP was suggested as Pis1.

[Study on optimum extraction conditions of alkaloids from Pinellia ternate].

The optimum extraction conditions of alkaloids from Pinellia ternate (Thunb.) Breit were studied by orthogonal test. The results showed that the highest extraction rate of the alkaloids could be obtained by smashing the material in 60 (sieve number) of fragmentation and socking the material in 2.575 mol/L ammonia water, extracting alkaloids with 18 times as much chlorolform at room temperature for 25 hours. The highest extraction rate of alkaloids was 0.0817%.

[Survey of Pinellia ternata and its adulterants].

The tubers of Pinellia ternata (Thunb.) Breit. distributed widely in China, have been used in traditional medicine. Recently, P. ternata is deficient in herb market and its adulterants are used. This article mainly summarizes some researches on P. ternata and its adulterants from four aspects, including pharmacognostics, chemical component, pharmacology and toxicity. Also some suggestions are presented about the study prospect of P. ternata.