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Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.

Winter dormancy is an important biological feature for tea plant to survive cold winters, and it also affects the economic output of tea plant, one of the few woody plants in the world whose leaves are harvested and one of the few non-conifer evergreen species with characterized dormancies. To discover the bud dormancy regulation mechanism of tea plant in winter, we analyzed the global gene expression profiles of axillary buds at the paradormancy, endodormancy, ecodormancy, and bud flush stages by RNA-Seq analysis. In total, 16,125 differentially expressed genes (DEGs) were identified among the different measured conditions. Gene set enrichment analysis was performed on the DEGs identified from each dormancy transition. Enriched gene ontology terms, gene sets and transcription factors were mainly associated with epigenetic mechanisms, phytohormone signaling pathways, and callose-related cellular communication regulation. Furthermore, differentially expressed transcription factors as well as chromatin- and phytohormone-associated genes were identified. GI-, CAL-, SVP-, PHYB-, SFR6-, LHY-, ZTL-, PIF4/6-, ABI4-, EIN3-, ETR1-, CCA1-, PIN3-, CDK-, and CO-related gene sets were enriched. Based on sequence homology analysis, we summarized the key genes with significant expression differences in poplar and tea plant. The major molecular pathways involved in tea plant dormancy regulation are consistent with those of poplar to a certain extent; however, the gene expression patterns varied. This study provides the global transcriptome profiles of overwintering buds at different dormancy stages and is meaningful for improving the understanding of bud dormancy in tea plant.

recently expanded its range northwards in China. It is unknown if the range expansion has a genetic and/or epigenetic basis, or merely an environmental basis due to a warming climate. To test these possibilities, we used an RNAseq approach with a common greenhouse design to examine gene expression in individuals from the northern edge and central portion of alligator weed range from China to determine if there were differences in their responses to cold temperatures. We hypothesized that if the recent range expansion was primarily environmental, we would observe few differences or only differences unrelated to low-temperature adaptations. We assembled over 75,000 genes of which over 65,000 had long open reading frames with similarity to sequences from arabidopsis. Differences in expression between northern and southern populations that were both exposed to low temperatures showed similar expression among genes in the C-REPEAT/DRE BINDING FACTOR (CBF) regulon. However, gene set and sub-network enrichment analysis indicated differences in the response of photosynthetic processes and oxidative stress responses were different between the two populations and we relate these differences to cold adaptation. The transcriptome differences in response to cold between the individuals from the two populations is consistent with adaptations potentiating or resulting from selection after expansion into colder environments and may indicate that genetic changes have accompanied the recent northward expansion of in China. However, we cannot rule out the possibility of epigenetic changes may have a role in this expansion.

DORMANCY ASSOCIATED MADS-BOX (DAM) genes are related to AGAMOUS-LIKE 24 and SHORT VEGETATIVE PHASE genes of arabidopsis and are differentially regulated coordinately with endodormancy induction and release in buds of several perennial plant species. DAM genes were first shown to directly impact endodormancy in peach where a deletion of a series of DAM resulted in loss of endodormancy induction. We have cloned and characterized several MADS box genes from the model perennial weed leafy spurge. Leafy spurge DAM genes are preferentially expressed in shoot tips and buds in response to cold temperatures and day length in a manner that is relative to the level of endodormancy induced by various environmental conditions. Over-expression of one DAM gene in arabidopsis delays flowering. Additionally, we show that at least one DAM gene is differentially regulated by chromatin remodeling. Comparisons of the DAM gene promoters between poplar and leafy spurge have identified several conserved sequences that may be important for their expression patterns in response to dormancy-inducing stimuli.

Expanding the southern range of herbaceous peony (Paeonia lactiflora Pall.) is a meaningful and worthwhile horticultural endeavor in the Northern Hemisphere. However, high temperatures in winter seriously hinder the bud dormancy release and flowering of peony in the more southern areas of subtropical and tropical regions. Resource introduction and hybridization can contribute to creating new cultivars with high adaptability in a warmer winter climate. In this study, three representative cultivars of P. lactiflora were screened for flowering capabilities and their annual growth cycles were observed to provide information needed for hybridization. Among these three cultivars, 'Hang Baishao' is the best adapted cultivar for southern growing regions and is unique in its ability to thrive in southern areas of N 30°00'. Pollen viability of 'Hang Baishao' was 55.60% based on five measuring methods, which makes it an excellent male parent in hybridization. Hybrid plants among these three cultivars grew well, but all of their flower buds aborted. Additionally, the ability of three growth regulators that advance the flowering of 'Hang Baishao' to promote an indoor cultivation strategy for improving peony application as a potted or cut-flower plant was tested. 5-azacytidine could impact the growth of 'Hang Baishao' and induce dwarfism and small flowers but not advance the flowering time. Gibberellin A3 promoted the sprouting and growth significantly, but all plants eventually withered. Chilling at 0-4°C for four weeks and irrigation with 300 mg/L humic acid was the optimal combination used to hasten flowering and ensure flowering quality simultaneously. These results can lay the foundation for future studies on the chilling requirement trait, bud dormancy release and key functional gene exploration of herbaceous peony. Additionally, this study can also provide guidance for expanding the range of economically important plants with the winter dormancy trait to the low-latitude regions.

Leafy spurge (Euphorbia esula L.) is a noxious perennial weed that produces underground adventitious buds, which are crucial for generating new vegetative shoots following periods of freezing temperatures or exposure to various control measures. It is also capable of flowering and producing seeds, but requires vernalization in some cases. DORMANCY ASSOCIATED MADS-BOX (DAM) genes have been proposed to play a direct role in the transition to winter-induced dormancy and maintenance through regulation of the FLOWERING LOCUS T (FT) gene, which also is likely involved in the vernalization process. To explore the regulation of FT and DAM during dormancy transitions in leafy spurge, the transcript accumulation of two previously cloned DAM splice variants and two different previously cloned FT genes was characterized. Under long-photoperiods (16 h light), both DAM and FT transcripts accumulate in a diurnal manner. Tissue specific expression patterns indicated the tissues with high DAM expression had low FT expression and vice versa. DAM expression is detected in leaves, stems, shoot tips, and crown buds. FT transcripts were detected mainly in leaves and flowers. Under dormancy inducing conditions, DAM and FT genes had an inverse expression pattern. Additionally, chromatin immunoprecipitation assays were performed using DAM-like protein specific antibodies to demonstrate that DAM or related proteins likely bind to cryptic and/or conserved CArG boxes in the promoter regions of FT genes isolated from endodormant crown buds. These results are consistent with the hypothesis that DAM proteins play a crucial role in leafy spurge dormancy transition and maintenance, potentially by negatively regulating the expression of FT.

Information concerning genes and signals regulating cold acclimation processes in plants is abundant; however, less is known about genes and signals regulating the deacclimation process. A population of primarily winter B. napus varieties was used to conduct a genome-wide association study and to compare the transcriptomes from two winter B. napus varieties showing time-dependent differences in response to cold acclimation and deacclimation treatments. These studies helped to identify loci, candidate genes, and signaling processes impacting deacclimation in B. napus. GWAS identified polymorphisms at five different loci associated with freezing tolerance following deacclimation. Local linkage decay rates near these polymorphisms identified 38 possible candidate genes. Several of these genes have been reported as differentially regulated by cold stress in arabidopsis (Arabidopsis thaliana), including a calcium-binding EF-hand family protein (encoded by BnaCnng10250D) that was also differentially expressed during deacclimation in this study. Thousands of other genes differentially expressed during the acclimation and deacclimation treatments implicated processes involving oxidative stress, photosynthesis, light-regulated diurnal responses, and growth regulation. Generally, responses observed during acclimation were reversed within one week of deacclimation. The primary differences between the two winter B. napus varieties with differential deacclimation responses involved protection from oxidative stress and the ability to maintain photosynthesis.

To survive winter, many perennial plants become endodormant, a state of suspended growth maintained even in favorable growing environments. To understand vegetative bud endodormancy, we collected paradormant, endodormant, and ecodormant axillary buds from Populus trees growing under natural conditions. Of 44,441 Populus gene models analyzed using NimbleGen microarrays, we found that 1,362 (3.1%) were differentially expressed among the three dormancy states, and 429 (1.0%) were differentially expressed during only one of the two dormancy transitions (FDR p-value < 0.05). Of all differentially expressed genes, 69% were down-regulated from paradormancy to endodormancy, which was expected given the lower metabolic activity associated with endodormancy. Dormancy transitions were accompanied by changes in genes associated with DNA methylation (via RNA-directed DNA methylation) and histone modifications (via Polycomb Repressive Complex 2), confirming and extending knowledge of chromatin modifications as major features of dormancy transitions. Among the chromatin-associated genes, two genes similar to SPT (SUPPRESSOR OF TY) were strongly up-regulated during endodormancy. Transcription factor genes and gene sets that were atypically up-regulated during endodormancy include a gene that seems to encode a trihelix transcription factor and genes associated with proteins involved in responses to ethylene, cold, and other abiotic stresses. These latter transcription factors include ETHYLENE INSENSITIVE 3 (EIN3), ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN (EBP), ETHYLENE RESPONSE FACTOR (ERF), ZINC FINGER PROTEIN 10 (ZAT10), ZAT12, and WRKY DNA-binding domain proteins. Analyses of phytohormone-associated genes suggest important changes in responses to ethylene, auxin, and brassinosteroids occur during endodormancy. We found weaker evidence for changes in genes associated with salicylic acid and jasmonic acid, and little evidence for important changes in genes associated with gibberellins, abscisic acid, and cytokinin. We identified 315 upstream sequence motifs associated with eight patterns of gene expression, including novel motifs and motifs associated with the circadian clock and responses to photoperiod, cold, dehydration, and ABA. Analogies between flowering and endodormancy suggest important roles for genes similar to SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL), DORMANCY ASSOCIATED MADS-BOX (DAM), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1).

Flowering time is an important agronomic trait for canola breeders, as it provides growers with options for minimizing exposure to heat stress during flowering and to more effectively utilize soil moisture. Plants have evolved various systems to control seasonal rhythms in reproductive phenology including an internal circadian clock that responds to environmental signals. In this study, we used canola cultivar ‘Westar’ as a recurrent parent and canola cultivar ‘Surpass 400’ as the donor parent to generate a chromosome segment substitution line (CSSL) and to map a flowering time locus on chromosome A10 using molecular marker-assisted selection. This CSSL contains an introgressed 4.6 mega-bases (Mb) segment (between 13 and 17.6 Mb) of Surpass 400, which substantially delayed flowering compared with Westar. To map flowering time gene(s) within this locus, eight introgression lines (ILs) were developed carrying a series of different lengths of introgressed chromosome A10 segments using five co-dominant polymorphic markers located at 13.5, 14.0, 14.5, 15.0, 15.5, and 16.0 Mb. Eight ILs were crossed with Westar reciprocally and flowering time of resultant 16 F1 hybrids and parents were evaluated in a greenhouse (2021 and 2022). Four ILs (IL005, IL017, IL035, and IL013) showed delayed flowering compared to Westar (P < 0.0001), and their reciprocal crosses displayed a phenotype intermediate in flowering time of both homozygote parents. These results indicated that flowering time is partial or incomplete dominance, and the flowering time locus mapped within a 1 Mb region between two co-dominant polymorphic markers at 14.5–15.5 Mb on chromosome A10. The flowering time locus was delineated to be between 14.60 and 15.5 Mb based on genotypic data at the crossover site, and candidate genes within this region are associated with flowering time in canola and/or Arabidopsis. The co-dominant markers identified on chromosome A10 should be useful for marker assisted selection in breeding programs but will need to be validated to other breeding populations or germplasm accessions of canola.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread infectious disease that is currently a major cause of economic losses in pig production. In Hungary, a National PRRS Eradication Program has been introduced to attain a more efficient, economic, and competitive international market position. The program has been also approved by the EU, but the resulting legal obligations have imposed a burden on Hungarian producers to comply with EU competition rules. The implementation of the program has been carried out by the veterinary authorities with the consent of, continuous support from and monitoring conducted by organisations within the pig sector as well as a scientific committee. The PRRS eradication program in Hungary was based on a regional territorial principle and was compulsory for all pig holdings within the regions. In Hungary, large fattening farms operate as all-in/all-out or continuous flow systems. Large-scale breeding herds are predominantly farrow-to-finish types. Although its significance has decreased in recent decades, 20% of the Hungarian pig population is still kept on small (backyard) farms (<100 animals). All PRRSV-infected large-scale farms had to develop a unit-adapted eradication plan, including external and internal biosecurity measures, vaccinations, etc. It was crucial to render each fattening unit free of the disease, as fattening units play a significant role in spreading the virus within the country. The eradication efforts mainly implemented were depopulation–repopulation methods, but on some farms a testing and removal method has been used. As the eradication progressed over the years, the introduction of infected fattening pigs was restricted. Thanks to these measures, Hungarian large-scale fattening farms became PRRSV-free by the end of 2018. The PRRSV-free status of small-scale herds was achieved by the end of 2015 and was maintained between 2016 and 2021. By 31 December 2021, all breeding pigs in large-scale farms in Hungary were free of wild-type PRRS virus. By 31 March 2022, the total pig population of the country, including all backyard farms and fattening units, achieved PRRSV-free status. The future goal is to ensure and maintain the PRRSV-free status of Hungary via strict import regulations of live animals combined with the continuous and thorough screening of incoming and resident herds for the presence of the virus.