Identification of Seven Key Structural Genes in the Anthocyanin Biosynthesis Pathway in Sepals of Hydrangea macrophylla.

Under specific cultivation conditions, the sepal color of Hydrangea macrophylla (H. macrophylla) changes from red to blue due to the complexation of aluminum ions (Al3+), delphinidin 3-glucoside, and copigments. However, this phenomenon cannot occur in all cultivars despite the presence of sufficient Al3+ and copigments. To explore the mechanism of sepal bluing in H. macrophylla, there is an urgent need to study the molecular regulation of the anthocyanin biosynthesis pathway. However, the key structural genes, other than CHS, regulating anthocyanin biosynthesis in the sepals of H. macrophylla have not been identified. In this study, based on full-length transcriptome data from H.macrophylla 'Bailmer', the key structural genes regulating anthocyanin biosynthesis in the sepals of H. macrophylla were isolated and investigated. Ultimately, seven key structural genes, HmCHS1, HmCHI, HmF3H1, HmF3'H1, HmF3'5'H, HmDFR2, and HmANS3, were demonstrated to show high expression levels in colored sepals. The expression levels of these seven genes increased gradually with the development of sepals and were highest in the full-bloom stage. The trend of gene expression was consistent with the trend of anthocyanin contents. It was concluded that the seven selected genes were involved in anthocyanin biosynthesis in the sepals of H. macrophylla. The full-length sequence data have been deposited into the NCBI Sequence Read Archive (SRA) with accession number PRJNA849710. This study lays a good foundation for the further elucidation of the molecular mechanism of sepal coloration in H. macrophylla.

The identification of key candidate genes mediating yellow seedling lethality in a Lilium regale mutant.

Leaf color mutants are ideal materials for exploring plant photosynthesis mechanisms, chlorophyll biosynthetic pathways and chloroplast development. The yellow seedling lethal mutant lrysl1 was discovered from self-bred progenies of Lilium regale; however, the mechanism of leaf color mutation remains unclear. In this study, the ultrastructural and physiological features and de novo RNA-Seq data of a L. regale leaf color mutant and wild-type L. regale were investigated. Genetic analysis indicated that the characteristics of the lrysl1 mutant were controlled by a recessive nuclear gene. The chlorophyll a, chlorophyll b and carotenoid contents in the mutant leaves were lower than those in the wild-type leaves. Furthermore, the contents of the chlorophyll precursors aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (ProtoIX), Mg-protoporphyrin IX (Mg-ProtoIX), and protochlorophyll (Pchl) decreased significantly in mutant leaves. Transcriptome data from the mutant and wild type showed that a total of 892 differentially expressed genes were obtained, of which 668 and 224 were upregulated genes and downregulated genes in the mutant, respectively. Almost all genes in the photosynthesis pathway and chlorophyll biosynthetic pathway were downregulated in the mutant, which corroborated the differences in the physiological features mentioned above. Further research indicated that the chloroplasts of the mutant leaves exhibited an abnormal morphology and distribution and that the expression of a gene related to chloroplast development was downregulated. It was concluded that abnormal chloroplast development was the main cause of leaf color mutation in the mutant lrysl1 and that LrGLK was a gene related to chloroplast development in L. regale. This research provides a foundation for further research on the mechanism by which LrGLK regulates chloroplast development in L. regale.

QTLs position of some important ornamental traits in recently developed OO lily population.

Lilium L. is a perennial ornamental bulbous species, belonging to Liliaceae family, which consists of about 100 species. One of the most important hybrids in Lilium L. is the Oriental hybrid lily. Different cross combinations have been done in the lily family such as AA (Asiatic × Asiatic), AL (Asiatic × Longiflorum), and OT (Oriental × Trumpet). The OO (Oriental × Oriental) combination is a new one. SSR and AFLP markers were used to overlap each other and the genetic linkage groups were created according to the haploid number of lily chromosomes (12 linkage groups). In this experiment, the final F1 population, which creates a genetic linkage group, was 100 individuals. For map construction, JOINMAP 4.0 software by treating segregation data of markers as a CP (out breeder full-sib family) model was used. After evaluation of ornamental traits, MapQTL 4.0 software was also used to find possible QTLs on these linkage maps. A total of 940 primers were tested and the best ones, which were 172 primer pairs (96 AFLP and 76 SSR markers), were used for map construction and the total of 616 loci (465 loci for AFLP marker and 151 loci for SSR marker) were scored. The entire mapped length was 2144.2 cM. 8 QTLs were obtained for flower number which is an important trait in lily. Each QTL locus explained the phenotypic variation of 2.4-89.5%. The highest amount of LOD (35.21) was found in LG-F1P2 for FN4 QTL. For leaf number, one-QTL was mapped with LOD of 7.08 between 2 markers on the LG-M10 of maternal maps. The QTL for petal length was placed on the LG-F1P2 of the F1 hybrid maps on the E-CGC/M-CGC-4 primer combination. The petal width QTLs also were mapped on the E-CGC/M-CGC-4. Qualitative locus named LN was mapped on the LG-M10 of the maternal maps. PW2 QTL was also localized on the LG-F4 of the paternal maps. In this experiment, 5 QTLs also were mapped for spot number in all F1 hybrids and paternal and maternal maps, and spot size. Moreover, one QTL with the length of 51 cM was measured on the LG-M8 of the maternal maps. Plant height QTL with the LOD of 12.54 was mapped on the primer combination of E-CGC/M-CGC-4 on the LG-F1P2 of the F1 hybrid maps.

Selection of reference genes for quantitative real-time PCR analysis of photosynthesis-related genes expression in Lilium regale.

Photosynthesis is closely related to the growth of plants. A stable reference gene is fundamental for studies of the molecular mechanism of photosynthesis in Lilium regale. Therefore, it is very important to select a suitable reference gene for qRT-PCR analysis on genes of photosynthetic system, chlorophyll biosynthetic pathway and chloroplast development in Lilium regale. Three kinds of tissues, leaves and bulbs (abnormal leaves) of tissue culture plantlets and cotyledons of seedlings of the wild-type and mutant Lilium regale were selected as materials for qRT-PCR. Six housekeeping genes were selected as candidate genes from transcriptome sequencing data of the wild-type and yellow seedling lethal mutant of Lilium regale. Finally, the expression stability of six candidate reference genes was analyzed using geNorm, NormFinder, and BestKeeper software, the comparative ∆Ct method, and the RefFinder program. The results showed that LrActin2 was the best reference gene for qRT-PCR analysis of photosynthesis-related genes expression in leaves of tissue culture plantlets and seedlings of Lilium regale. This study provided useful data for further research on molecular mechanism of photosynthesis in the Lilium.

Transcriptome Analysis Identifies Key Candidate Genes Mediating Purple Ovary Coloration in Asiatic Hybrid Lilies.

Lily tepals have a short lifespan. Once the tepals senesce, the ornamental value of the flower is lost. Some cultivars have attractive purple ovaries and fruits which greatly enhance the ornamental value of Asiatic hybrid lilies. However, little is known about the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries. To investigate the transcriptional network that governs purple ovary coloration in Asiatic hybrid lilies, we obtained transcriptome data from green ovaries (S1) and purple ovaries (S2) of Asiatic "Tiny Padhye". Comparative transcriptome analysis revealed 4228 differentially expressed genes. Differential expression analysis revealed that ten unigenes including four CHS genes, one CHI gene, one F3H gene, one F3'H gene, one DFR gene, one UFGT gene, and one 3RT gene were significantly up-regulated in purple ovaries. One MYB gene, LhMYB12-Lat, was identified as a key transcription factor determining the distribution of anthocyanins in Asiatic hybrid lily ovaries. Further qPCR results showed unigenes related to anthocyanin biosynthesis were highly expressed in purple ovaries of three purple-ovaried Asiatic hybrid lilies at stages 2 and 3, while they showed an extremely low level of expression in ovaries of three green-ovaried Asiatic hybrid lilies during all developmental stages. In addition, shading treatment significantly decreased pigment accumulation by suppressing the expression of several unigenes related to anthocyanin biosynthesis in ovaries of Asiatic "Tiny Padhye". Lastly, a total of 15,048 Simple Sequence Repeats (SSRs) were identified in 13,710 sequences, and primer pairs for SSRs were designed. The results could further our understanding of the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries.

Enhancement of micropollutant biotransformation by adding manganese sand in constructed wetlands.

Recent investigations have shown that the addition of manganese (Mn) sand to constructed wetlands (i.e., Mn-amended CWs) can improve the performance of organic micropollutants (MPs) removal. In addition to the direct oxidation and adsorption of Mn oxides, the indirect role of Mn oxides in MP biotransformation is crucial to the removal of MPs but has seldom been referred to. Herein, we constructed lab-scale CWs with or without the addition of natural Mn sand (∼35% Mn oxides) to decipher the influence of Mn oxides on the biotransformation of the six selected MPs which commonly existed in the wastewater. The experimental results showed that the addition of Mn sand to CWs can improve the removal of MPs (8.48% atrazine, 13.16% atenolol, and 6.27% sulfamethoxazole [pairwise Wilcoxon test p < 0.05]). Combining the detection of transformation products and metagenomic sequencing, we found that the enhanced removal of atrazine in the Mn-amended CWs was mainly due to the bioaugmented hydroxylation process. The enrichment of biotransformation-related genes and associated microbes of atenolol and sulfamethoxazole in Mn-amended CWs indicated that the addition of Mn sand to CWs can strengthen the biotransformation of MPs. Furthermore, we found that these MP-biodegrading microbes were widely present in the full-scale CWs. Overall, our research provides fundamental information and insights for further application of Mn-amended CWs in MP removal.

Construction and analysis of a high-density genetic linkage map in cabbage (Brassica oleracea L. var. capitata).

BACKGROUND: Brassica oleracea encompass a family of vegetables and cabbage that are among the most widely cultivated crops. In 2009, the B. oleracea Genome Sequencing Project was launched using next generation sequencing technology. None of the available maps were detailed enough to anchor the sequence scaffolds for the Genome Sequencing Project. This report describes the development of a large number of SSR and SNP markers from the whole genome shotgun sequence data of B. oleracea, and the construction of a high-density genetic linkage map using a double haploid mapping population. RESULTS: The B. oleracea high-density genetic linkage map that was constructed includes 1,227 markers in nine linkage groups spanning a total of 1197.9 cM with an average of 0.98 cM between adjacent loci. There were 602 SSR markers and 625 SNP markers on the map. The chromosome with the highest number of markers (186) was C03, and the chromosome with smallest number of markers (99) was C09. CONCLUSIONS: This first high-density map allowed the assembled scaffolds to be anchored to pseudochromosomes. The map also provides useful information for positional cloning, molecular breeding, and integration of information of genes and traits in B. oleracea. All the markers on the map will be transferable and could be used for the construction of other genetic maps.

Fine mapping of the major QTLs for biochemical variation of sulforaphane in broccoli florets using a DH population.

Glucoraphanin is a major secondary metabolite found in Brassicaceae vegetables, especially broccoli, and its degradation product sulforaphane plays an essential role in anticancer. The fine mapping of sulforaphane metabolism quantitative trait loci (QTLs) in broccoli florets is necessary for future marker-assisted selection strategies. In this study, we utilized a doubled haploid population consisting of 176 lines derived from two inbred lines (86,101 and 90,196) with significant differences in sulforaphane content, coupled with extensive genotypic and phenotypic data from two independent environments. A linkage map consisting of 438 simple sequence repeats markers was constructed, covering a length of 1168.26 cM. A total of 18 QTLs for sulforaphane metabolism in broccoli florets were detected, 10 were detected in 2017, and the other 8 were detected in 2018. The LOD values of all QTLs ranged from 3.06 to 14.47, explaining 1.74-7.03% of the biochemical variation between two years. Finally, 6 QTLs (qSF-C3-1, qSF-C3-2, qSF-C3-3, qSF-C3-5, qSF-C3-6 and qSF-C7) were stably detected in more than one environment, each accounting for 4.54-7.03% of the phenotypic variation explained (PVE) and a total of 30.88-34.86% of PVE. Our study provides new insights into sulforaphane metabolism in broccoli florets and marker-assisted selection breeding in Brassica oleracea crops.

Tobacco rattle virus-induced PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH) gene silencing in Solanum pseudocapsicum L.

Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. The present study constitutes the first application of VIGS in S. pseudocapsicum, which has great ornamental and pharmaceutical value, using tobacco rattle virus (TRV) vectors. Two marker genes, PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH), were used to test the VIGS system in S. pseudocapsicum. The photobleaching and yellow-leaf phenotypes of the silenced plants were shown to significantly correlate with the down-regulation of endogenous SpPDS and SpChlH, respectively (P ≤ 0.05). Moreover, the parameters potentially affecting the efficiency of VIGS in S. pseudocapsicum, including the Agrobacterium strain and the inoculation method (leaf syringe-infiltration, sprout vacuum-infiltration and seed vacuum-infiltration), were compared. The optimized VIGS parameters were the leaf syringe-infiltration method, the Agrobacterium strain GV3101 and the growth of agro-inoculated plants at 25°. With these parameters, the silencing efficiency of SpPDS and SpChlH could reach approximately 50% in S. pseudocapsicum. Additionally, the suitability of various reference genes was screened by RT-qPCR using three candidate genes, and the results demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) can serve as a suitable reference for assessing the gene expression levels of VIGS systems in S. pseudocapsicum. The proven application of VIGS in S. pseudocapsicum and the characterization of a suitable reference gene in the present work will expedite the functional characterization of novel genes in S. pseudocapsicum.

Spatiotemporal Transcriptome Analysis Provides Insights into Bicolor Tepal Development in Lilium "Tiny Padhye".

The bicolor Asiatic hybrid lily cultivar "Tiny Padhye" is an attractive variety because of its unique color pattern. During its bicolor tepal development, the upper tepals undergo a rapid color change from green to white, while the tepal bases change from green to purple. However, the molecular mechanisms underlying these changes remain largely uncharacterized. To systematically investigate the dynamics of the lily bicolor tepal transcriptome during development, we generated 15 RNA-seq libraries from the upper tepals (S2-U) and basal tepals (S1-D, S2-D, S3-D, and S4-D) of Lilium "Tiny Padhye." Utilizing the Illumina platform, a total of 295,787 unigenes were obtained from 713.12 million high-quality paired-end reads. A total of 16,182 unigenes were identified as differentially expressed genes during tepal development. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (61 unigenes), and chlorophyll metabolic pathway (106 unigenes) were identified. Further analyses showed that most anthocyanin biosynthesis genes were transcribed coordinately in the tepal bases, but not in the upper tepals, suggesting that the bicolor trait of "Tiny Padhye" tepals is caused by the transcriptional regulation of anthocyanin biosynthetic genes. Meanwhile, the high expression level of chlorophyll degradation genes and low expression level of chlorophyll biosynthetic genes resulted in the absence of chlorophylls from "Tiny Padhye" tepals after flowering. Transcription factors putatively involved in the anthocyanin biosynthetic pathway and chlorophyll metabolism in lilies were identified using a weighted gene co-expression network analysis and their possible roles in lily bicolor tepal development were discussed. In conclusion, these extensive transcriptome data provide a platform for elucidating the molecular mechanisms of bicolor tepals in lilies and provide a basis for similar research in other closely related species.

Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.).

Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. To date, several reliable reference gene identifications have been reported in Lilium spp., but none has been obtained for lily tepals at different developmental stages. In this study, ten candidate reference genes were selected and evaluated for their expression stability in Lilium 'Tiny Padhye' during the process of bicolor tepal development. The expression stability of these candidates was evaluated by three software programs (geNorm, NormFinder, and BestKeeper) and the comparative ΔCt method, and comprehensive stability rankings were generated by RefFinder. As a result, TIP41-like family gene (TIP41) and actin (ACT) were the best combination of reference genes for tepals at different developmental stages; TIP41 and F-box family gene (F-box) for tepals under shading treatment; ACT, actin11 (ACT11), and elongation factor 1-α (EF1-α) for different tissues; and ACT, TIP41, and ACT11 for all samples. The selected optimal reference genes were further verified by analyzing the expression levels of flavonoid 3'-hydroxylase (LhF3'H) and anthocyanidin 3-O-glucosyltransfersae (LhUFGT) in tepals at different developmental stages. This study provides useful information for gene expression characterization in lilies under different experimental conditions, and can serve as a basis for similar research in other closely related species.

Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium.

Aerial bulbils are an important propagative organ, playing an important role in population expansion. However, the detailed gene regulatory patterns and molecular mechanism underlying bulbil formation remain unclear. Triploid Lilium lancifolium, which develops many aerial bulbils on the leaf axils of middle-upper stem, is a useful species for investigating bulbil formation. To investigate the mechanism of bulbil formation in triploid L. lancifolium, we performed histological and transcriptomic analyses using samples of leaf axils located in the upper and lower stem of triploid L. lancifolium during bulbil formation. Histological results indicated that the bulbils of triploid L. lancifolium are derived from axillary meristems that initiate de novo from cells on the adaxial side of the petiole base. Transcriptomic analysis generated ~650 million high-quality reads and 11,871 differentially expressed genes (DEGs). Functional analysis showed that the DEGs were significantly enriched in starch and sucrose metabolism and plant hormone signal transduction. Starch synthesis and accumulation likely promoted the initiation of upper bulbils in triploid L. lancifolium. Hormone-associated pathways exhibited distinct patterns of change in each sample. Auxin likely promoted the initiation of bulbils and then inhibited further bulbil formation. High biosynthesis and low degradation of cytokinin might have led to bulbil formation in the upper leaf axil. The present study achieved a global transcriptomic analysis focused on gene expression changes and pathways' enrichment during upper bulbil formation in triploid L. lancifolium, laying a solid foundation for future molecular studies on bulbil formation.

Chromosome Doubling of Microspore-Derived Plants from Cabbage (Brassica oleracea var. capitata L.) and Broccoli (Brassica oleracea var. italica L.).

Chromosome doubling of microspore-derived plants is an important factor in the practical application of microspore culture technology because breeding programs require a large number of genetically stable, homozygous doubled haploid plants with a high level of fertility. In the present paper, 29 populations of microspore-derived plantlets from cabbage (Brassica oleracea var. capitata) and broccoli (Brassica oleracea var. italica) were used to study the ploidy level and spontaneous chromosome doubling of these populations, the artificial chromosome doubling induced by colchicine, and the influence of tissue culture duration on the chromosomal ploidy of the microspore-derived regenerants. Spontaneous chromosome doubling occurred randomly and was genotype dependent. In the plant populations derived from microspores, there were haploids, diploids, and even a low frequency of polyploids and mixed-ploidy plantlets. The total spontaneous doubling in the 14 cabbage populations ranged from 0 to 76.9%, compared with 52.2 to 100% in the 15 broccoli populations. To improve the rate of chromosome doubling, an efficient and reliable artificial chromosome doubling protocol (i.e., the immersion of haploid plantlet roots in a colchicine solution) was developed for cabbage and broccoli microspore-derived haploids. The optimal chromosome doubling of the haploids was obtained with a solution of 0.2% colchicine for 9-12 h or 0.4% colchicine for 3-9 h for cabbage and 0.05% colchicine for 6-12 h for broccoli. This protocol produced chromosome doubling in over 50% of the haploid genotypes for most of the populations derived from cabbage and broccoli. Notably, after 1 or more years in tissue culture, the chromosomes of the haploids were doubled, and most of the haploids turned into doubled haploid or mixed-ploidy plants. This is the first report indicating that tissue culture duration can change the chromosomal ploidy of microspore-derived regenerants.