Genome-wide identification and characterization of SRLK gene family reveal their roles in self-incompatibility of Erigeron breviscapus.

Self-incompatibility (SI) is a reproductive protection mechanism that plants acquired during evolution to prevent self-recession. As the female determinant of SI specificity, SRK has been shown to be the only recognized gene on the stigma and plays important roles in SI response. Asteraceae is the largest family of dicotyledonous plants, many of which exhibit self-incompatibility. However, systematic studies on SRK gene family in Asteraceae are still limited due to lack of high-quality genomic data. In this study, we performed the first systematic genome-wide identification of S-locus receptor like kinases (SRLKs) in the self-incompatible Asteraceae species, Erigeron breviscapus, which is also a widely used perennial medicinal plant endemic to China.52 SRLK genes were identified in the E. breviscapus genome. Structural analysis revealed that the EbSRLK proteins in E. breviscapus are conserved. SRLK proteins from E. breviscapus and other SI plants are clustered into 7 clades, and the majority of the EbSRLK proteins are distributed in Clade I. Chromosomal and duplication analyses indicate that 65% of the EbSRLK genes belong to tandem repeats and could be divided into six tandem gene clusters. Gene expression patterns obtained in E. breviscapus multiple-tissue RNA-Seq data revealed differential temporal and spatial features of EbSRLK genes. Among these, two EbSRLK genes having high expression levels in tongue flowers were cloned. Subcellular localization assay demonstrated that both of their fused proteins are localized on the plasma membrane. All these results indicated that EbSRLK genes possibly involved in SI response in E. breviscapus. This comprehensive genome-wide study of the SRLK gene family in E. breviscapus provides valuable information for understanding the mechanism of SSI in Asteraceae.

Genetic Diversity and Association Mapping of Grain-Size Traits in Rice Landraces from the Honghe Hani Rice Terraces System in Yunnan Province.

The Honghe Hani Rice Terraces System (HHRTS) of Yunnan Province is an important agricultural and cultural heritage landscape. Until now, a large number of local rice landraces have been planted. Mining excellent genes contained in these landraces provides a reference for variety improvement and new variety breeding. In this study, 96 rice landraces collected from the Hani terraces were planted in Honghe Mengzi, Yunnan Province, in 2013, 2014, 2015, and 2021, and five major grain traits were measured and analyzed. The genomic variation of 96 rice landraces was scanned by 201 simple sequence repeat (SSR) markers. The genetic diversity, population structure, and genetic relationships of the natural population were analyzed. The mixed linear model (MLM) method of the TASSEL software was used to analyze the associations between markers and traits. A total of 936 alleles were amplified by 201 pairs of SSR primers. The average number of observed alleles (Na), the effective number of alleles (Ne), Shannon's information index (I), heterozygosity (H), and the polymorphism information content (PIC) per marker were 4.66, 2.71, 1.08, 0.15, and 0.55, respectively. Ninety-six landraces were divided into two groups by population structure, clustering, and principal component analysis, and indica rice was the main group. The coefficients of variation of the five traits ranged from 6.80 to 15.24%, and their broad heritabilities were more than 70%. In addition, there were positive correlations among the same grain traits between different years. Through MLM analysis, 2, 36, 7, 7, and 4 SSR markers were significantly associated with grain length (GL), grain width (GW), grain thickness (GT), grain length-width ratio (LWR), and thousand-grain weight (TGW), respectively. The explanation rates of phenotypic variation were 16.31 (RM449, Chr. 1)-23.51% (RM316, Chr. 9), 10.84 (RM523, Chr. 3; RM161/RM305, Chr. 5)-43.01% (RM5496, Chr. 1), 11.98 (RM161/RM305, Chr. 5)-24.72% (RM275, Chr. 6), 12.68 (RM126, Chr. 8)-36.96% (RM5496, Chr. 1), and 17.65 (RM4499, Chr. 2)-26.32% (RM25, Chr. 8), respectively. The associated markers were distributed on 12 chromosomes of the genome.

Marsdenia tenacissima genome reveals calcium adaptation and tenacissoside biosynthesis.

Marsdenia tenacissima is a medicinal plant widely distributed in the calcium-rich karst regions of southwest China. However, the lack of a reference genome has hampered the implementation of molecular techniques in its breeding, pharmacology and domestication. We generated the chromosome-level genome assembly in Apocynaceae using combined SMRT sequencing and Hi-C. The genome length was 381.76 Mb, with 98.9% of it found on 11 chromosomes. The genome contained 222.63 Mb of repetitive sequences and 21 899 predicted gene models, with a contig N50 of 6.57 Mb. Phylogenetic analysis revealed that M. tenacissima diverged from Calotropis gigantea at least 13.43 million years ago. Comparative genomics showed that M. tenacissima underwent ancient shared whole-genome duplication. This event, together with tandem duplication, contributed to 70.71% of gene-family expansion. Both pseudogene analysis and selective pressure calculations suggested calcium-related adaptive evolution in the M. tenacissima genome. Calcium-induced differentially expressed genes (DEGs) were mainly enriched in cell-wall-related processes. Domains (e.g. Fasciclin and Amb_all) and cis-elements (e.g. MYB and MYC) frequently occurred in the coding and promoter regions of cell-wall DEGs, respectively, and the expression levels of these genes correlated significantly with those of calcium-signal-related transcription factors. Moreover, calcium addition increased tenacissoside I, G and H contents. The availability of this high-quality genome provides valuable genomic information for genetic breeding and molecular design, and lends insights into the calcium adaptation of M. tenacissima in karst areas.

Comparative physiological, transcriptomic, and WGCNA analyses reveal the key genes and regulatory pathways associated with drought tolerance in Tartary buckwheat.

Drought stress is one of the major abiotic stress factors that affect plant growth and crop productivity. Tartary buckwheat is a nutritionally balanced and flavonoid-rich pseudocereal crop and also has strong adaptability to different adverse environments including drought. However, little is known about its drought tolerance mechanism. In this study, we performed comparative physiological and transcriptomic analyses of two contrasting drought-resistant Tartary buckwheat genotypes under nature drought treatment in the reproductive stage. Under drought stress, the drought-tolerant genotype XZSN had significantly higher contents of relative water, proline, and soluble sugar, as well as lower relative electrolyte leakage in the leaves than the drought-susceptible LK3. A total of 5,058 (2,165 upregulated and 2,893 downregulated) and 5,182 (2,358 upregulated and 2,824 downregulated) potential drought-responsive genes were identified in XZSN and LK3 by transcriptome sequencing analysis, respectively. Among the potential drought-responsive genes of XZSN, 1,206 and 1,274 genes were identified to be potential positive and negative contributors for XZSN having higher drought resistance ability than LK3. Furthermore, 851 out of 1,206 positive drought-resistant genes were further identified to be the core drought-resistant genes of XZSN based on WGCNA analysis, and most of them were induced earlier and quicker by drought stress than those in LK3. Functional annotation of the 851 core drought-resistant genes found that a large number of stress-responsive genes were involved in TFs, abscisic acid (ABA) biosynthesis, signal transduction and response, non-ABA signal molecule biosynthesis, water holding, oxygen species scavenging, osmotic adjustment, cell damage prevention, and so on. Transcriptional regulatory network analyses identified the potential regulators of these drought-resistant functional genes and found that the HD-ZIP and MYB TFs might be the key downstream TFs of drought resistance in Tartary buckwheat. Taken together, these results indicated that the XZSN genotype was more drought-tolerant than the LK3 genotype as evidenced by triggering the rapid and dramatic transcriptional reprogramming of drought-resistant genes to reduce water loss, prevent cell damage, and so on. This research expands our current understanding of the drought tolerance mechanisms of Tartary buckwheat and provides important information for its further drought resistance research and variety breeding.

De novo transcriptome assembly, gene annotation, and EST-SSR marker development of an important medicinal and edible crop, Amomum tsaoko (Zingiberaceae).

BACKGROUND: Amomum tsaoko is a medicinal and food dual-use crop that belongs to the Zingiberaceae family. However, the lack of transcriptomic and genomic information has limited the understanding of the genetic basis of this species. Here, we performed transcriptome sequencing of samples from different A. tsaoko tissues, and identified and characterized the expressed sequence tag-simple sequence repeat (EST-SSR) markers. RESULTS: A total of 58,278,226 high-quality clean reads were obtained and de novo assembled to generate 146,911 unigenes with an N50 length of 2002 bp. A total of 128,174 unigenes were successfully annotated by searching seven protein databases, and 496 unigenes were identified as annotated as putative terpenoid biosynthesis-related genes. Furthermore, a total of 55,590 EST-SSR loci were detected, and 42,333 primer pairs were successfully designed. We randomly selected 80 primer pairs to validate their polymorphism in A. tsaoko; 18 of these primer pairs produced distinct, clear, and reproducible polymorphisms. A total of 98 bands and 96 polymorphic bands were amplified by 18 pairs of EST-SSR primers for the 72 A. tsaoko accessions. The Shannon's information index (I) ranged from 0.477 (AM208) to 1.701 (AM242) with an average of 1.183, and the polymorphism information content (PIC) ranged from 0.223 (AM208) to 0.779 (AM247) with an average of 0.580, indicating that these markers had a high level of polymorphism. Analysis of molecular variance (AMOVA) indicated relatively low genetic differentiation among the six A. tsaoko populations. Cross-species amplification showed that 14 of the 18 EST-SSR primer pairs have transferability between 11 Zingiberaceae species. CONCLUSIONS: Our study is the first to provide transcriptome data of this important medicinal and edible crop, and these newly developed EST-SSR markers are a very efficient tool for germplasm evaluation, genetic diversity, and molecular marker-assisted selection in A. tsaoko.

Integrated microRNA and transcriptome profiling reveal key miRNA-mRNA interaction pairs associated with seed development in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat seed development is an extremely complex process involving many gene regulatory pathways. MicroRNAs (miRNAs) have been identified as the important negative regulators of gene expression and performed crucial regulatory roles in various plant biological processes. However, whether miRNAs participate in Tartary buckwheat seed development remains unexplored. RESULTS: In this study, we first identified 26 miRNA biosynthesis genes in the Tartary buckwheat genome and described their phylogeny and expression profiling. Then we performed small RNA (sRNA) sequencing for Tartary buckwheat seeds at three developmental stages to identify the miRNAs associated with seed development. In total, 230 miRNAs, including 101 conserved and 129 novel miRNAs, were first identified in Tartary buckwheat, and 3268 target genes were successfully predicted. Among these miRNAs, 76 exhibited differential expression during seed development, and 1534 target genes which correspond to 74 differentially expressed miRNAs (DEMs) were identified. Based on integrated analysis of DEMs and their targets expression, 65 miRNA-mRNA interaction pairs (25 DEMs corresponding to 65 target genes) were identified that exhibited significantly opposite expression during Tartary buckwheat seed development, and 6 of the miRNA-mRNA pairs were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) and ligase-mediated rapid amplification of 5' cDNA ends (5'-RLM-RACE). Functional annotation of the 65 target mRNAs showed that 56 miRNA-mRNA interaction pairs major involved in cell differentiation and proliferation, cell elongation, hormones response, organogenesis, embryo and endosperm development, seed size, mineral elements transport, and flavonoid biosynthesis, which indicated that they are the key miRNA-mRNA pairs for Tartary buckwheat seed development. CONCLUSIONS: Our findings provided insights for the first time into miRNA-mediated regulatory pathways in Tartary buckwheat seed development and suggested that miRNAs play important role in Tartary buckwheat seed development. These findings will be help to study the roles and regulatory mechanism of miRNAs in Tartary buckwheat seed development.

Development of 23 novel microsatellite markers of Amomum tsao-ko (Zingiberaceae) based on restriction-site-associated DNA sequencing.

Amomum tsao-ko (Zingiberaceae) is a traditional Chinese medicine and condiment, and an important economic crop in the tropical forest of southwest China. However, few simple sequence repeat (SSR) markers are available in A. tsao-ko, which is hindering genetic research in this species. The aim of this study was to develop and characterize microsatellite markers for A. tsao-ko using restriction-site-associated DNA sequencing. A total of 115,482 microsatellites were identified using MISA software, and 13,411 SSR primer pairs were designed. 100 pairs of SSR primers were selected at random and used to evaluate polymorphisms among 4 A. tsao-ko samples. Finally, 23 pairs of SSR primers with clear bands and obvious polymorphism were selected for genetic diversity analysis of 72 A. tsao-ko accessions. The number of alleles and effective number of alleles per locus ranged from 2 to 6 and from 1.315 to 3.776, respectively. The observed heterozygosity ranged from 0.208 to 0.779, and the expected heterozygosity was from 0.239 to 0.735. The average values of the polymorphic information content were 0.454. Hardy-Weinberg equilibrium (HWE) analysis showed that 10 loci significantly deviated from HWE (P < 0.05). The pairwise FST and genetic distance values revealed low levels of genetic differentiation and high genetic similarity among six A. tsao-ko populations. These microsatellite markers developed will provide a valuable tool for further germplasm characterization, genetic diversity, and breeding studies in A. tsao-ko.

Genome-wide identification and expression profile of the MADS-box gene family in Erigeron breviscapus.

The MADS-box gene family encodes transcription factors with many biological functions that extensively regulate plant growth, development and reproduction. Erigeron breviscapus is a medicinal herb used widely in traditional Chinese medicine, and is believed to improve blood circulation and ameliorate platelet coagulation. In order to gain a detailed understanding of how transcription factor expression may regulate the growth of this potentially important medicinal plant, a genome-wide analysis of the MADS-box gene family of E. breviscapus is needed. In the present study, 44 MADS-box genes were identified in E. breviscapus and categorized into five subgroups (MIKC, Mα, Mß, Mγ and Mδ) according to their phylogenetic relationships with the Arabidopsis MADS-box genes. Additionally, the functional domain, subcellular location and motif compositions of the E. breviscapus MADS-box gene products were characterized. The expression levels for each of the E. breviscapus MADS-box (EbMADS) genes were analyzed in flower, leaf, stem and root organs, and showed that the majority of EbMADS genes were expressed in flowers. Meanwhile, some MADS genes were found to express high levels in leaf, stem and root, indicating that the MADS-box genes are involved in various aspects of the physiological and developmental processes of the E. breviscapus. The results from gene expression analysis under different pollination treatments revealed that the MADS-box genes were highly expressed after non-pollinated treatment. To the best of our knowledge, this study describes the first genome-wide analysis of the E. breviscapus MADS-box gene family, and the results provide valuable information for understanding of the classification, cloning and putative functions of the MADS-box family.

Determination of Pyrethroids in Paris polyphylla Sample by High-Performance Liquid Chromatography Using Ultrasound-Assisted Magnetic Solid-Phase Extraction.

A novel ultrasound-assisted magnetic solid-phase extraction (UA-MSPE) was developed for the separation/preconcentration of trace amounts of pyrethroids (fenpropathrin, fenvalerate, deltamethrin, and bifenthrin) in Paris polyphylla sample using carbon nanotubes based on Fe3O4 magnetic nanoparticles (Fe3O4@CNT MNPs), and high-performance liquid chromatography-UV is described. High recoveries of pyrethroids were obtained at a low MNPs concentration because sonication enhances the contact chances between magnetic nanoparticles and extractable analytes and promotes the extractability of the MSPE process. After the extraction, the adsorbent can be conveniently separated from the sample solution by an external magnet, and the adsorbed analytes were eluted from magnetic Fe3O4@CNT. The main factors influencing the extraction efficiency including the amount of the MNPs, the extraction time, the pH of sample solution, the sonicating time, and the desorption conditions were studied and optimized. Under the optimized experimental conditions, a good linearity was observed in the range of 1-100.0 ng mL-1 for all the analytes, with the correlation coefficients (r) ranging from 0.9962 to 0.9991. The limits of detection of the four pyrethroids are 0.53, 0.26, 0.47, and 0.67 ng mL-1, respectively. The recoveries of the method were in the range between 85.5% and 93.2%. This method is much faster and more effective than traditional SPE methods, and it is promising for the analysis of pyrethroids residues.

Molecular cloning and expression analysis of SRLK1 gene in self-incompatible Asteraceae species Erigeron breviscapus.

Based on the transcriptome data, using RACE techniques, we cloned the full-length EbSRLK1 gene in a medicinal, self-incompatible Asteraceae species, Erigeron breviscapus. Bioinformatics approaches were used to analyze the DNA and protein sequences, physical and chemical properties, and domains of the encoded protein. The full-length EbSRLK1 cDNA is 2891 base pairs (bp) with an open reading frame (ORF) of 2634 bp, which encodes the EbSRLK1 protein with 878 amino acids and an estimated molecular weight of 98.13 kD. The EbSRLK1 protein has the characteristic domain structure of S-locus receptor-like protein kinases, which contains one transmembrane domain but lacks the signal peptide. Quantitative real-time PCR (qRT-PCR) analysis showed that the EbSRLK1 gene is lowly expressed in roots, stems and leaves, but highly expressed in flowers, especially in flowers one day prior to opening. Western blot analysis showed that the EbSRLK1 protein is expressed in stems, leaves, and flowers, but is almost undetectable in roots. The EbSRLK1 protein expression is induced in self-pollinated but not in cross-pollinated E. breviscapus flowers. Cloning and expression analysis of EbSRLK1 lay a solid foundation for elucidating the role of EbSRLK1 in regulating self-incompatibility in E. breviscapus.

Unigene-based RNA-seq provides insights on drought stress responses in Marsdenia tenacissima.

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine, which often grows on the karst landform and the water conservation capacity of land is very poorly and drought occurrences frequently. We found M. tenacissima has strong drought resistance because of continuousdrought16 d, the leaves of M. tenacissima were fully curly and dying. But the leaves were fully almost recovering after re-watering 24h. The activity of SOD and POD were almost doubled under drought stress. The content of osmotic regulating substance proline and soluble sugar were three times than control group. But after re-watering, these indexes were declined rapidly. Three cDNA libraries of control, drought stress, and re-watering treatments were constructed. There were 43,129,228, 47,116,844, and 42,815,454 clean reads with Q20 values of 98.06, 98.04, and 97.88respectively.SRA accession number of raw data was PRJNA498187 on NCBI. A total of 8672, 6043, and 6537 differentially expressed genes (DEGs) were identified in control vs drought stress, control vs re-watering, and drought stress vs re-watering, respectively. In addition, 1039, 1016, and 980 transcription factors (TFs) were identified, respectively. Among them, 363, 267, and 299 TFs were identified as DEGs in drought stress, re-watering, and drought stress and re-watering, respectively. These differentially expressed TFs mainly belonged to the bHLH, bZIP, C2H2, ERF, MYB, MYB-related, and NAC families. A comparative analysis found that 1174 genes were up-regulated and 2344 were down-regulated under drought stress and this pattern was the opposite to that found after re-watering. Among the up-regulated genes, 64 genes were homologous to known functional genes that directly protect plants against drought stress. Furthermore, 44 protein kinases and 38 TFs with opposite expression patterns under drought stress and re-watering were identified, which are possibly candidate regulators for drought stress resistance in M. tenacissima. Our study is the first to characterize the M. tenacissima transcriptome in response to drought stress, and will serve as a useful resource for future studies on the functions of candidate protein kinases and TFs involved in M. tenacissima drought stress resistance.

[Molecular cloning and expression analysis of sucrose synthase gene from Dendrobium officinale].

OBJECTIVE: Clone of sucrose synthase of Dendribium officinale and expression analysis, to provide the theory basis for research the relationship between polysaccharide synthesis of D. officinale and sucrose synthase activity. METHOD: According to homologous sequence of sucrose synthase gene on GenBank, application the technology of RT-PCR and RACE, clone the full length of D. officinale. Target gene amplified with T vector was transformed into competent E. coli. BL21, IPTG induced expression, SDS-PAGE analysis. RESULT: A full length cDNA encoding sucrose synthase was isolated from the D. officinale, named DOSS1, the GenBank accession number is HQ856835, the cDNA is 2781 bp in length containing an open reading frame of 2424 bp encoding 807 amino acids with a predicted molecular mass of 92.3 x 10(3), the deduced amino acid sequence of D. officinale sucrose synthase shares 95% identity with Mokara yellow (AF530568); shares 90% identity with Oncidium goldiana (AF530567); shares more than 80% with other monocotyledonous plants. CONCLUSION: Cloned the sucrose synthase gene and induced an obvious band successfully.