Plant Molecular Biology Reporter最新文献

Mutations in the CER9 gene of Arabidopsis thaliana L. contribute to the amplification of the cuticular wax and consequently mitigation of water loss, thereby fortifying drought resilience. Recently, genes homologous to CER9, termed SUD1 genes, have been annotated in bread wheat (Triticum aestivum L.). However, no research has been done on these genes in T. aestivum. Hence, our study aimed to employ CRISPR/Cas technology to knock out the CER9/SUD1 gene orthologs in bread wheat. For this, five guide RNAs were meticulously chosen and merged into a singular vector. Delivery of the CRISPR/Cas components was arranged through Agrobacterium tumefaciens, utilized for transforming immature embryos of two agricultural spring bread wheat varieties: Taya and Sigma. Among the 13 transgenic plants procured, four manifested positivity for the reporter gene GFP and Cas9 gene. Notably, substantial deletions ranging from 284 to 398 bp within the CER9/SUD1 gene were discerned in these plants. Additionally, two of the edited plants exhibited an absence of CER9/SUD1 transcripts, while the other two displayed a noteworthy 5.4-fold reduction in CER9/SUD1 gene expression compared to the wild type. Intriguingly, the genome-edited plants of the T₁ generation showcased enhanced growth parameters compared to the wild type under both standard and drought conditions.

The waxy cuticle on plant surfaces, comprising a cutin polymer matrix and complex very long chain fatty acids, figure in protective barrier against water loss and environmental damage. In this study, we employed a F₂ population (comprising of 237 individual plants) constructed by the crossing of HX128 (female parent without cuticular wax) with HX027 (male parent with significant cuticular wax). Two extreme phenotypic bulks were constructed according to the variation of wax trait in F₂ population, and bulked segregant RNA-seq (BSR-seq) was implemented to decipher the molecular underpinnings of waxiness trait in durum wheat. The QTL related to waxiness was mapped between 0.8 and 23.4 Mb on chromosome 2B. According to the differential sequence information of target interval between the parents, polymorphic primers were designed to screen F₂ population. The QTL of waxiness trait was further narrowed down between 6.8 and 10.1 Mb using the composite interval mapping tool. The LOD value was 129.53, with phenotypic variance explained (PVE) up to 44.65%. The annotation and expression analysis of the genes in the QTL region entailed selection of 15 potential candidate genes. Among which, eleven new genes with four previously reported genes were found that seem to be a gene cluster mediating wax synthesis at W1 loci. This study provides important insights into fine mapping and cloning of genes involved in wax synthesis with improved efficacy.

The study of the association between polymorphism at the DNA level and the diversity of phenotypic traits is an essential tool in breeding programs. To identify informative microsatellite markers related to agronomic traits, this research including 25 bread wheat genotypes was carried out. The experiment was set up in a randomized complete block design with three replications in rainfed and irrigated conditions during two cropping seasons (2018–2020) in the cold Mediterranean climate of Iran. Variance analysis showed significant differences between genotypes for most of the traits. The 16 microsatellite primers out of 20 had considerable polymorphisms, and three markers, namely XCFD168-2D, XGWM350-7D, and XGWM136-1A, were introduced as the most significant markers for subsequent studies. Cluster analysis by the UPGMA method classified 25 wheat genotypes into four groups. Genotypes 1, 3, and 25 have the most significant genetic distance with genotypes 13, 7, and Pishgam. Association analysis by stepwise regression showed that in both years under rainfed conditions, the XGWM350 marker for 1000-grain weight, the XCFD5 marker for spike length, and the XGWM165 and XGWM70 markers for spike dry weight, and under irrigated conditions, the XGWM265 marker for grain yield exhibited significant associations. Also, the XGWM136 and XCFD5 were found to be common markers associated with agronomic traits for all the test environments. In addition, most of the markers were associated with 1000-grain weight, mitt penalty length, and spike grain weight in rainfed conditions and 1000-grain weight in irrigated conditions. After identifying molecular markers related to increased yield and drought tolerance, they can be used as selection criteria to accelerate wheat breeding programs. Also, these marker-trait associations can help wheat improvement programs through marker-assisted selection.

Graphical Abstract

Akebiae Fructus (AF) is the dry and near-ripe fruit of Akebia trifoliata (Thunb.) Koidz. Triterpenoid saponins are important characteristic components of AF, but their molecular biosynthetic pathway has not yet been identified. In this study, DNBSEQ was used to sequence the transcriptome of the five stages of AF from May to September. A total of 96.87 Gb of data were assembled, including 115,430 unigenes with an average sequence length of 1445 bp. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses for metabolic pathway enrichment identified 434 unigenes involved in two metabolic pathways related to triterpenoid saponin biosynthesis, of which 255 encoded 19 key enzymes in the triterpenoid saponin biosynthetic pathway. Sequence analyses and homology modeling of the key enzyme, β-amyrin synthase (β-AS), which is implicated in triterpenoid saponin biosynthesis, showed that it has a conserved substrate-binding domain. Differentially expressed genes (DEGs) were identified by comparing gene expression levels between September and other months, and multiple genes encoding key enzymes, such as β-AS, and the transcription factors (TFs) that are involved in triterpenoid saponin biosynthesis were further analyzed. The chemical constituents of triterpenoid saponins were identified via ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UHPLC-QE-MS), and triterpenoid saponin content in AF at different developmental periods was determined by high performance liquid chromatography (HPLC). Finally, the expression levels of some unigenes encoding key enzymes were fully verified using quantitative real-time polymerase chain reaction (qRT-PCR). Herein, we elucidate the biosynthetic pathway of triterpenoid saponins in AF and its key enzymes, laying a foundation for future investigations of AF biosynthesis regulation.

The medicinal Lippia alba and Petiveria alliacea, originating from Central and South America, exhibit a wide range of beneficial properties, including antimicrobial, antifungal, anti-inflammatory, antitumor, analgesic, and antibacterial effects. However, little is known about their population structure and genetic diversity, which may hinder the establishment of their cultivation in different regions of Colombia. In this study, we conducted a comprehensive analysis of the genetic diversity and population structure of 17 samples of L. alba from the departments of Tolima, Valle del Cauca, and Putumayo, as well as 31 samples of P. alliacea from the departments of Cundinamarca, Boyacá, Tolima, and Valle del Cauca. We employed restriction-site associated DNA sequencing (RADseq) with the enzyme PstI. We performed denovo_map and ref_map pipeline for L. alba and identified a total of 17,036 loci and 14,562 SNPs, respectively, revealing a genetic variation of 5.19% (FST of 0.05; p < 0.001) among its populations and two delimited genetic groups. For P. alliacea, in denovo_map our analysis discovered 6395 SNPs, indicating substantial genetic variation of 75% among the studied populations (FST = 0.75; p < 0.001), resulting in the delineation of four genetic groups. Our findings will contribute to providing valuable molecular data on the populations of these medicinal plants and provide evidence of the genetic flow existing among L. alba populations, while P. alliacea populations are more structured. We also performed a FDIST analysis; for L. alba using the results with reference, we identified 37 SNPs coding for proteins in biological, molecular, and cellular processes; and finally, we highlighted the gene SASPL_104284 involved in metabolic processes.

Exploring the resistance genes of citrus to Huanglongbing (HLB) is the foundation and key to disease resistance breeding. Based on comparative genomic transcriptome data, four pectin acetylesterase (PAE) genes responsive to Candidatus liberibacter asiaticus (CaLas) infection induction were screened, and a gene cloned with higher differential expression levels was identified, named CsPAE55. Bioinformatics analyses showed that CsPAE55 was conserved but had sequence differences compared with homologs. The subcellular localization results of tobacco indicated that CsPAE55 protein was mainly localized in the nucleus and cytoplasm. RT-qPCR analysis showed that the expression of CsPAE55 was related to variety tolerance, tissue site, and symptom development. In addition, we established virus vector-mediated infection systems in citrus, namely gene silencing systems mediated by virus-induced gene silencing (VIGS) and gene overexpression systems mediated by citrus leaf blotch virus (CLBV), and obtained CsPAE55 silencing and overexpression plants. And we established a stable transformation system mediated by Agrobacterium rhizogenes in citrus and obtained CsPAE55 silencing and overexpression citrus hairy roots. The analysis of hormone content and gene expression in CsPAE55 plants also indicated that overexpression of CsPAE55 regulated the transcriptional regulation of genes involved in systemic acquired resistance (SAR) response. Using Protein–Protein Interaction (PPI) to predict and screen for a citrus protein CsARF1 that may interact with CsPAE55, and preliminarily verifying its interaction with CsPAE55 protein through Yeast Two-hybrid (Y2H) and Bimolecular Fluorescent Complimentary (BIFC). In summary, our data provided theoretical basis and genetic resources for subsequent molecular breeding of citrus resistance to HLB disease.

Downy mildew (DM) disease, caused by obligate oomycete Peronospora plantaginis Underwood, is the single most damaging disease affecting the yield and quality of isabgol (Plantago ovata Forsk). The infection leads to characteristic ash-colored downy growth, and as the disease progresses, leaf tissue turns yellowish due to loss of chlorophyll, ultimately reducing the effective photosynthetic area. However, reports on the molecular mechanisms mediating host plant resistance are still unknown. In the present study, we conducted a comparative transcriptome between the resistant and susceptible genotypes of isabgol in response to DM infection. We identified significantly differentially expressed genes (DEGs) genes: 6928 in control (RU) vs. infected (RI) of DPO-185 (resistant DM) and 8779 in control (SU) vs. infected (SI) of DPO-14 (susceptible to DM). Putative genes encoding pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered immunity (ETI), cell wall degrading enzymes, phytohormone signalling, and phenylpropanoid biosynthesis pathways involved in host–pathogen interaction were identified in addition to the identification of 58 candidate resistance (R) genes enriched in response to DM infection. The expression of 11 genes involved in plant defense quantified using RT-qPCR indicated a significant difference in the expression pattern. This study for the first time provides a glimpse of transcriptional responses to the DM resistance in isabgol which can guide investigating the molecular mechanisms associated with plant defense and to develop control mechanisms accordingly for DM disease.

The present study focuses on the identification of variation in genetic diversity in some of the breeding populations using yield, yield component, and root morpho-physiological traits in Beta vulgaris L. Eighty genotypes from 8 different sugar beet populations were used under a randomized complete block design with five replicates at the Azad University research field during the cropping season of 2021–2022. Significant statistical differences were recorded among the populations that indicate the genetic difference of the studied sugar beet population. Mean comparison of analyzed data revealed, P191 to displayed better traits in terms of sugar content, nitrogen content, pure sugar content, extraction coefficient, and root dry matter. Similarly, population M224 exhibited better root number, root yield, leaf weight, and sugar yield. The dendrogram using UPGMA revealed that the population formed three distinct clusters, with the greatest genetic distance between M249 and the 191 populations. This distance is expected to create maximum heterosis through crossbreeding between these two populations. The results indicate that the number of roots significantly affects RY, LY, Na, and WSY, but does not have a significant correlation with other traits. RY is correlated with LY, Na, and WSY, while LY is inversely correlated with WSC, ECS, and DM, and directly correlated with Na. Principal component analysis based on varimax rotation revealed that three factors explained the total variance to an extent of 87.6%: the first factor named WSC explained 54.5%, the second factor, called RY, accounted for 22.8%, and the third factor contributed to 10.2% of the total variance. The biplot from the first two components showed populations similar to cluster analysis grouping. The findings of this research provide valuable insights into the breeding and improvement of sugar beet populations.

Backcross breeding is used to transfer an desirable trait from donor to recurrent parent (RP), and it takes six to seven generations for 99% recovery of the recurrent parent genome (RPG). Marker-assisted backcross breeding (MABB) was proposed to accelerate the recovery of the RPG. Over the period of time, MABB has evolved as per the available resources and time frame. One of the major modifications is the number of foreground-selected individuals which are subjected to background selection. This number varied widely and lacked consensus in different studies. Further, the major drawback of the existing method is that the DUS traits are recorded directly in the improved lines. Once the traits are fixed in the finished product, there is no going back to bring the corresponding traits of RP. The modified MABB method (DUS-MAS) can ensure higher recovery of DUS traits and RPG. A yield index has also been developed for the identification of high yielding individuals in backcross generation. In DUS-MAS, the foreground-positive individuals are selected first, and then individuals with a higher yield index are selected. Thereafter, the foreground and yield index positive individuals are selected for higher recovery of DUS traits. Five such individuals are subjected to marker-based background recovery of which only two with the highest recovery were advanced to the next generation. All these steps are to be followed in BC₁F₁, BC₂F₁, and BC₂F₂ generations. It substantially reduces the cost by reducing the use of laboratory chemicals by more than 75%.

Bergenia crassifolia (L.) Fritsch, of the family Bergenia Moench, has a broad distribution in China and has been used in Chinese medicine and Chinese gardens extensively. Approximately 544 million high-quality sequence reads and 75,115 unigenes were obtained, among which 40,102 (53.39%) were annotated in NR, 27,769 (36.97%) were annotated in Swiss-Prot, 27,266 (36.30%) were annotated in GO, 12,644 (16.83%) were annotated in KEGG, and 37,808 (50.33%) were annotated in eggNOG database. A total of 1,495 DEUs were obtained, and the expression patterns of these DEUs were gathered into eight categories, and the functions of genes in each categories were analyzed in KEGG pathway. The results may meet the needs for carbon–nitrogen metabolism, adaptation to climate change, and accumulation of secondary metabolites’ studies in B. crassifolia and its relatives.