Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile.

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

The backs of our eyes are lined with retinal pigment epithelial cells (or RPE cells for short). These cells provide nutrition to surrounding cells and contain a pigment called melanin that absorbs excess light that might interfere with vision. By doing so, they support the cells that receive light to enable vision. However, with age, RPE cells can become damaged and less able to support other cells. This can lead to a disease called age-related macular degeneration, which can cause blindness. One potential way to treat this disease is to transplant healthy RPE cells into eyes that have lost them. These healthy cells can be grown in the laboratory from human pluripotent stem cells, which have the capacity to turn into various specialist cells. Stem cell-derived RPE cells growing in a dish contain varying amounts of melanin, resulting in some being darker than others. This raised the question of whether pigment levels affect the function of RPE cells. However, it was difficult to compare single cells containing various amounts of pigment as most previous studies only analyzed large numbers of RPE cells mixed together. Nakai-Futatsugi et al. overcame this hurdle using a technique called Automated Live imaging and cell Picking System (also known as ALPS). More than 2300 stem cell-derived RPE cells were photographed individually and the color of each cell was recorded. The gene expression of each cell was then measured to investigate whether certain genes being switched on or off affects pigment levels and cell function. Analysis did not find a consistent pattern of gene expression underlying the pigmentation of RPE cells. Even gene expression related to the production of melanin was only slightly linked to the color of the cells. These findings suggests that the RPE cell color fluctuates and is not primarily determined by which genes are switched on or off. Future experiments are required to determine whether the findings are the same for RPE cells grown naturally in the eyes and whether different pigment levels affect their capacity to protect the rest of the eye.

Integrative analysis of transcriptome and target metabolites uncovering flavonoid biosynthesis regulation of changing petal colors in Nymphaea 'Feitian 2'.

BACKGROUND: Nymphaea (waterlily) is known for its rich colors and role as an important aquatic ornamental plant globally. Nymphaea atrans and some hybrids, including N. 'Feitian 2,' are more appealing due to the gradual color change of their petals at different flower developmental stages. The petals of N. 'Feitian 2' gradually change color from light blue-purple to deep rose-red throughout flowering. The mechanism of the phenomenon remains unclear. RESULTS: In this work, flavonoids in the petals of N. 'Feitian 2' at six flowering stages were examined to identify the influence of flavonoid components on flower color changes. Additionally, six cDNA libraries of N. 'Feitian 2' over two blooming stages were developed, and the transcriptome was sequenced to identify the molecular mechanism governing petal color changes. As a result, 18 flavonoid metabolites were identified, including five anthocyanins and 13 flavonols. Anthocyanin accumulation during flower development is the primary driver of petal color change. A total of 12 differentially expressed genes (DEGs) in the flavonoid biosynthesis pathway were uncovered, and these DEGs were significantly positively correlated with anthocyanin accumulation. Six structural genes were ultimately focused on, as their expression levels varied significantly across different flowering stages. Moreover, 104 differentially expressed transcription factors (TFs) were uncovered, and three MYBs associated with flavonoid biosynthesis were screened. The RT-qPCR results were generally aligned with high-throughput sequencing results. CONCLUSIONS: This research offers a foundation to clarify the mechanisms underlying changes in the petal color of waterlilies.

Comparative transcriptomic and metabolomic profiles reveal fruit peel color variation in two red pomegranate cultivars.

Pomegranate (Punica granatum L.) which belongs to family Lythraceae, is one of the most important fruit crops of many tropical and subtropical regions. A high variability in fruit color is observed among different pomegranate accessions, which arises from the qualitative and quantitative differences in anthocyanins. However, the mechanism of fruit color variation is still not fully elucidated. In the present study, we investigated the red color mutation between a red-skinned pomegranate 'Hongbaoshi' and a purple-red-skinned cultivar 'Moshiliu', by using transcriptomic and metabolomic approaches. A total of 51 anthocyanins were identified from fruit peels, among which 3-glucoside and 3,5-diglucoside of cyanidin (Cy), delphinidin (Dp), and pelargonidin (Pg) were dominant. High proportion of Pg in early stages of 'Hongbaoshi' but high Dp in late stages of 'Moshiliu' were characterized. The unique high levels of Cy and Dp anthocyanins accumulating from early developmental stages accounted for the purple-red phenotype of 'Moshiliu'. Transcriptomic analysis revealed an early down-regulated and late up-regulated of anthocyanin-related structure genes in 'Moshiliu' compared with 'Hongbaoshi'. Alao, ANR was specially expressed in 'Hongbaoshi', with extremely low expression levels in 'Moshiliu'. For transcription factors R2R3-MYB, the profiles demonstrated a much higher transcription levels of three subgroup (SG) 5 MYBs and a sharp decrease in expression of SG6 MYB LOC116202527 in high-anthocyanin 'Moshiliu'. SG4 MYBs exhibited two entirely different patterns, LOC116203744 and LOC116212505 were down-regulated whereas LOC116205515 and LOC116212778 were up-regulated in 'Moshiliu' pomegranate. The results indicate that specific SG members of the MYB family might promote the peel coloration in different manners and play important roles in color mutation in pomegranate.

Polymorphism in the aggressive mimicry lure of the parasitic freshwater mussel Lampsilis fasciola.

Unionoid freshwater mussels (Bivalvia: Unionidae) are free-living apart from a brief, obligately parasitic, larval stage that infects fish hosts, and gravid female mussels have evolved a spectrum of strategies to infect fish hosts with their larvae. In many North American species, this involves displaying a mantle lure: a pigmented fleshy extension that acts as an aggressive mimic of a host fish prey, thereby eliciting a feeding response that results in host infection. The mantle lure of Lampsilis fasciola is of particular interest because it is apparently polymorphic, with two distinct primary lure phenotypes. One, described as "darter-like", has "eyespots", a mottled body coloration, prominent marginal extensions, and a distinct "tail". The other, described as "worm-like", lacks those features and has an orange and black coloration. We investigated this phenomenon using genomics, captive rearing, biogeographic, and behavioral analyses. Within-brood lure variation and within-population phylogenomic (ddRAD-seq) analyses of individuals bearing different lures confirmed that this phenomenon is a true polymorphism. The relative abundance of the two morphs appears stable over ecological timeframes: the ratio of the two lure phenotypes in a River Raisin (MI) population in 2017 was consistent with that of museum samples collected at the same site six decades earlier. Within the River Raisin, four main "darter-like" lure motifs visually approximated four co-occurring darter species (Etheostoma blennioides, E. exile, E. microperca, and Percina maculata), and the "worm-like" lure resembled a widespread common leech, Macrobdella decora. Darters and leeches are typical prey of Micropterus dolomieui (smallmouth bass), the primary fish host of L. fasciola. In situ field recordings of the L. fasciola "darter" and "leech" lure display behaviors, and the lure display of co-occurring congener L. cardium, were captured. Despite having putative models in distinct phyla, both L. fasciola lure morphs have largely similar display behaviors that differ significantly from that of sympatric L. cardium individuals. Some minor differences in the behavior between the two L. fasciola morphs were observed, but we found no clear evidence for a behavioral component of the polymorphism given the criteria measured. Discovery of discrete within-brood inheritance of the lure polymorphism implies potential control by a single genetic locus and identifies L. fasciola as a promising study system to identify regulatory genes controlling a key adaptive trait of freshwater mussels.

Genetic tapestry of Capsicum fruit colors: a comparative analysis of four cultivated species.

KEY MESSAGE: Genome-wide association study of color spaces across the four cultivated Capsicum spp. revealed a shared set of genes influencing fruit color, suggesting mechanisms and pathways across Capsicum species are conserved during the speciation. Notably, Cytochrome P450 of the carotenoid pathway, MYB transcription factor, and pentatricopeptide repeat-containing protein are the major genes responsible for fruit color variation across the Capsicum species. Peppers (Capsicum spp.) rank among the most widely consumed spices globally. Fruit color, serving as a determinant for use in food colorants and cosmeceuticals and an indicator of nutritional contents, significantly influences market quality and price. Cultivated Capsicum species display extensive phenotypic diversity, especially in fruit coloration. Our study leveraged the genetic variance within four Capsicum species (Capsicum baccatum, Capsicum chinense, Capsicum frutescens, and Capsicum annuum) to elucidate the genetic mechanisms driving color variation in peppers and related Solanaceae species. We analyzed color metrics and chromatic attributes (Red, Green, Blue, L*, a*, b*, Luminosity, Hue, and Chroma) on samples cultivated over six years (2015-2021). We resolved genomic regions associated with fruit color diversity through the sets of SNPs obtained from Genotyping by Sequencing (GBS) and genome-wide association study (GWAS) with a Multi-Locus Mixed Linear Model (MLMM). Significant SNPs with FDR correction were identified, within the Cytochrome P450, MYB-related genes, Pentatricopeptide repeat proteins, and ABC transporter family were the most common among the four species, indicating comparative evolution of fruit colors. We further validated the role of a pentatricopeptide repeat-containing protein (Chr01:31,205,460) and a cytochrome P450 enzyme (Chr08:45,351,919) via competitive allele-specific PCR (KASP) genotyping. Our findings advance the understanding of the genetic underpinnings of Capsicum fruit coloration, with developed KASP assays holding potential for applications in crop breeding and aligning with consumer preferences. This study provides a cornerstone for future research into exploiting Capsicum's diverse fruit color variation.

Sequence and epigenetic variations of R2R3-MYB transcription factors determine the diversity of taproot skin and flesh colors in different cultivated types of radish (Raphanus sativus L.).

KEY MESSAGE: This study found that three paralogous R2R3-MYB transcription factors exhibit functional divergence among different subspecies and cultivated types in radish. Cultivated radish taproots exhibit a wide range of color variations due to unique anthocyanin accumulation patterns in various tissues. This study investigated the universal principles of taproot color regulation that developed during domestication of different subspecies and cultivated types. The key candidate genes RsMYB1 and RsMYB2, which control anthocyanin accumulation in radish taproots, were identified using bulked segregant analysis in two genetic populations. We introduced the RsMYB1-RsF3'H-RsMYB1Met genetic model to elucidate the complex and unstable genetic regulation of taproot flesh color in Xinlimei radish. Furthermore, we analyzed the expression patterns of three R2R3-MYB transcription factors in lines with different taproot colors and investigated the relationship between RsMYB haplotypes and anthocyanin accumulation in a natural population of 56 germplasms. The results revealed that three paralogous RsMYBs underwent functional divergence during radish domestication, with RsMYB1 regulating the red flesh of Xinlimei radish, and RsMYB2 and RsMYB3 regulating the red skin of East Asian big long radish (R. sativus var. hortensis) and European small radish (R. sativus var. sativus), respectively. Moreover, RsMYB1-H1, RsMYB2-H10, and RsMYB3-H6 were identified as the primary haplotypes exerting regulatory functions on anthocyanin synthesis. These findings provide an understanding of the genetic mechanisms regulating anthocyanin synthesis in radish and offer a potential strategy for early prediction of color variations in breeding programs.

Study on the causes of changes in colour during Hibiscus syriacus flowering based on transcriptome and metabolome analyses.

BACKGROUND: The flower colour of H. syriacus 'Qiansiban' transitions from fuchsia to pink-purple and finally to pale purple, thereby enhancing the ornamental value of the cultivars. However, the molecular mechanism underlying this change in flower colour in H. syriacus has not been elucidated. In this study, the transcriptomic data of H. syriacus 'Qiansiban' at five developmental stages were analysed to investigate the impact of flavonoid components on flower colour variation. Additionally, five cDNA libraries were constructed from H. syriacus 'Qiansiban' during critical blooming stages, and the transcriptomes were sequenced to investigate the molecular mechanisms underlying changes in flower colouration. RESULTS: High-performance liquid chromatography‒mass spectrometry detected five anthocyanins in H. syriacus 'Qiansiban', with malvaccin-3-O-glucoside being the predominant compound in the flowers of H. syriacus at different stages, followed by petunigenin-3-O-glucoside. The levels of these five anthocyanins exhibited gradual declines throughout the flowering process. In terms of the composition and profile of flavonoids and flavonols, a total of seven flavonoids were identified: quercetin-3-glucoside, luteolin-7-O-glucoside, Santianol-7-O-glucoside, kaempferol-O-hexosyl-C-hexarbonoside, apigenin-C-diglucoside, luteolin-3,7-diglucoside, and apigenin-7-O-rutinoside. A total of 2,702 DEGs were identified based on the selected reference genome. Based on the enrichment analysis of differentially expressed genes, we identified 9 structural genes (PAL, CHS, FLS, DRF, ANS, CHI, F3H, F3'5'H, and UFGT) and 7 transcription factors (3 MYB, 4 bHLH) associated with flavonoid biosynthesis. The qRT‒PCR results were in good agreement with the high-throughput sequencing data. CONCLUSION: This study will establish a fundamental basis for elucidating the mechanisms underlying alterations in the flower pigmentation of H. syriacus.

Transcriptome analysis of sexual dimorphism in dorsal down coloration in goslings.

BACKGROUND: In day-old Hungarian white goose goslings, there is a noticeable difference in dorsal down coloration between males and females, with females having darker dorsal plumage and males having lighter plumage. The ability to autosex day-old goslings based on their dorsal down coloration is important for managing them efficiently and planning their nutrition in the poultry industry. The aim of this study was to determine the biological and genetic factors underlying this difference in dorsal down colorationthrough histological analysis, biochemical assays, transcriptomic profiling, and q‒PCR analysis. RESULTS: Tissue analysis and biochemical assays revealed that compared with males, 17-day-old embryos and day-old goslings of female geese exhibited a greater density of melanin-containing feather follicles and a greater melanin concentration in these follicles during development. Both female and male goslings had lower melanin concentrations in their dorsal skin compared to 17-day-old embryos. Transcriptome analysis identified a set of differentially expressed genes (DEGs) (MC1R, TYR, TYRP1, DCT and MITF) associated with melanogenesis pathways that were downregulated or silenced specifically in the dorsal skin of day-old goslings compared to 17-day-old embryos, affecting melanin synthesis in feather follicles. Additionally, two key genes (MC1R and MITF) associated with feather coloration showed differences between males and females, with females having higher expression levels correlated with increased melanin synthesis and darker plumage. CONCLUSION: The expression of multiple melanogenesis genes determines melanin synthesis in goose feather follicles. The dorsal down coloration of day-old Hungarian white goose goslings shows sexual dimorphism, likely due to differences in the expression of the MC1R and MITF genes between males and females. These results could help us better understand why male and female goslings exhibit different plumage patterns.

Transcriptomic and metabolomic analyses reveal CmMYB308 as a key regulator in the pink flower color variation of 'Dante Purple' chrysanthemum.

KEY MESSAGE: CmMYB308 was identified as a key regulator in chrysanthemum flower color variation from purple to pink by conducting transcriptome and metabolome analysis. CmMYB308 can inhibit anthocyanin biosynthesis by suppressing the expression of CmPAL, CmC4H, and Cm4CL. Flower color variation is a widespread natural occurrence that plays a significant role in floral breeding. We discovered a variation in the flower of the chrysanthemum cultivar 'Dante Purple' (abbreviated as 'DP'), where the flower color shifted from purple to pink. We successfully propagated these pink flowers through tissue culture and designated them as DPM. By conducting transcriptome and metabolome analysis, we identified a reduction in the expression of critical genes involved in anthocyanin biosynthesis-CmPAL, CmC4H, and Cm4CL-in the DPM. This downregulation led to an accumulation of phenylalanine and cinnamic acid within the general phenylpropanoid pathway (GPP), which prevented their conversion into cyanidin and cyanidin 3-glucoside. As a result, the flowers turned pink. Additional transformation and biochemical experiments confirmed that the upregulation of CmMYB308 gene expression in the DPM directly suppressed CmPAL-1 and CmC4H genes, which indirectly affected Cm4CL-3 expression and ultimately inhibited anthocyanin biosynthesis in the DPM. This study offers a preliminary insight into the molecular mechanism underlying chrysanthemum flower color mutation, paving the way for genetic improvements in chrysanthemum flower color breeding.

Transcriptomic Analysis of Green Leaf Plants and White-Green Leaf Mutants in Haworthia cooperi var. pilifera.

Haworthia cooperi var. pilifera is a succulent plant with ornamental value. The white-green leaf mutant (wl) showed a significant difference in leaf color from the wild-type plant (WT). In this study, we integrated the transcriptomes of wl and WT plants to screen differentially expressed genes related to leaf color variation. The results of transcriptome analysis showed that 84,163 unigenes were obtained after de novo assembly and the NR database annotated the largest number of unigenes, which accounted for 57.13%, followed by NT (43.02%), GO (39.84%), Swiss-Prot (39.25%), KEGG (36.06%), and COG (24.88%). Our finding showed that 2586 genes were differentially expressed in the two samples, including 1996 down-regulated genes and 590 up-regulated genes. GO analysis predicted that these differentially expressed genes (DEGs) participate in 12 cellular components, 20 biological processes, and 13 molecular function terms and KEGG analysis showed that metabolic pathways, plant-pathogen interaction, glycerophospholipid metabolism, endocytosis, plant hormone signal transduction, and ether lipid metabolism were enriched among all identified pathways. Through functional enrichment analysis of DEGs, we found that they were involved in chloroplast division and the biosynthesis of plant pigments, including chlorophyll, carotenoids, anthocyanin, and transcription factor families, which might be related to the formation mechanism of leaf color. Taken together, these results present insights into the difference in gene expression characteristics in leaves between WT and wl mutants and provide a new insight for breeding colorful leaf phenotypes in succulent plants.

Fine Mapping of Candidate Gene Controlling Anthocyanin Biosynthesis for Purple Peel in Solanum melongena L.

Fruit color is an intuitive quality of horticultural crops that can be used as an evaluation criterion for fruit ripening and is an important factor affecting consumers' purchase choices. In this study, a genetic population from the cross of green peel 'Qidong' and purple peel '8 guo' revealed that the purple to green color of eggplant peel is dominant and controlled by a pair of alleles. Bulked segregant analysis (BSA), SNP haplotyping, and fine genetic mapping delimited candidate genes to a 350 kb region of eggplant chromosome 10 flanked by markers KA2381 and CA8828. One ANS gene (EGP22363) was predicted to be a candidate gene based on gene annotation and sequence alignment of the 350-kb region. Sequence analysis revealed that a single base mutation of 'T' to 'C' on the exon green peel, which caused hydrophobicity to become hydrophilic serine, led to a change in the three-level spatial structure. Additionally, EGP22363 was more highly expressed in purple peels than in green peels. Collectively, EGP22363 is a strong candidate gene for anthocyanin biosynthesis in purple eggplant peels. These results provide important information for molecular marker-assisted selection in eggplants, and a basis for analyzing the regulatory pathways responsible for anthocyanin biosynthesis in eggplants.

Clpf encodes pentatricopeptide repeat protein (PPR5) and regulates pink flesh color in watermelon (Citrullus lanatus L.).

KEY MESSAGE: The gene controlling pink flesh in watermelon was finely mapped to a 55.26-kb region on chromosome 6. The prime candidate gene, Cla97C06G122120 (ClPPR5), was identified through forward genetics. Carotenoids offer numerous health benefits; while, they cannot be synthesized by the human body. Watermelon stands out as one of the richest sources of carotenoids. In this study, genetic generations derived from parental lines W15-059 (red flesh) and JQ13-3 (pink flesh) revealed the presence of the recessive gene Clpf responsible for the pink flesh (pf) trait in watermelon. Comparative analysis of pigment components and microstructure indicated that the disparity in flesh color between the parental lines primarily stemmed from variations in lycopene content, as well as differences in chromoplast number and size. Subsequent bulk segregant analysis (BSA-seq) and genetic mapping successfully narrowed down the Clpf locus to a 55.26-kb region on chromosome 6, harboring two candidate genes. Through sequence comparison and gene expression analysis, Cla97C06G122120 (annotated as a pentatricopeptide repeat, PPR) was predicted as the prime candidate gene related to pink flesh trait. To further investigate the role of the PPR gene, its homologous gene in tomato was silenced using a virus-induced system. The resulting silenced fruit lines displayed diminished carotenoid accumulation compared with the wild-type, indicating the potential regulatory function of the PPR gene in pigment accumulation. This study significantly contributes to our understanding of the forward genetics underlying watermelon flesh traits, particularly in relation to carotenoid accumulation. The findings lay essential groundwork for elucidating mechanisms governing pigment synthesis and deposition in watermelon flesh, thereby providing valuable insights for future breeding strategies aimed at enhancing fruit quality and nutritional value.

The Transcription Factor MiMYB8 Suppresses Peel Coloration in Postharvest 'Guifei' Mango in Response to High Concentration of Exogenous Ethylene by Negatively Modulating MiPAL1.

Anthocyanin accumulation is regulated by specific genes during fruit ripening. Currently, peel coloration of mango fruit in response to exogenous ethylene and the underlying molecular mechanism remain largely unknown. The role of MiMYB8 on suppressing peel coloration in postharvest 'Guifei' mango was investigated by physiology detection, RNA-seq, qRT-PCR, bioinformatics analysis, yeast one-hybrid, dual-luciferase reporter assay, and transient overexpression. Results showed that compared with the control, low concentration of exogenous ethylene (ETH, 500 mg·L-1) significantly promoted peel coloration of mango fruit (cv. Guifei). However, a higher concentration of ETH (1000 mg·L-1) suppressed color transformation, which is associated with higher chlorophyll content, lower a* value, anthocyanin content, and phenylalanine ammonia-lyase (PAL) activity of mango fruit. M. indica myeloblastosis8 MiMYB8 and MiPAL1 were differentially expressed during storage. MiMYB8 was highly similar to those found in other plant species related to anthocyanin biosynthesis and was located in the nucleus. MiMYB8 suppressed the transcription of MiPAL1 by binding directly to its promoter. Transient overexpression of MiMYB8 in tobacco leaves and mango fruit inhibited anthocyanin accumulation by decreasing PAL activity and down-regulating the gene expression. Our observations suggest that MiMYB8 may act as repressor of anthocyanin synthesis by negatively modulating the MiPAL gene during ripening of mango fruit, which provides us with a theoretical basis for the scientific use of exogenous ethylene in practice.

Insights into the mechanism of color formation of the freshwater prawn (Macrobrachium rosenbergii) revealed by de novo assembly transcriptome analysis.

Body color is an important visual indicator of crustacean quality and plays a major role in consumer acceptability, perceived quality, and the market price of crustaceans. The freshwater prawn (Macrobrachium rosenbergii) has two distinct phenotypic variations, characterized by dark blue and light yellow body colors. However, the underlying mechanisms regulating the body color of M. rosenbergii remain unclear. In this study, the composition of shell color parameters and pigment cells of raw and cooked dark blue and light yellow M. rosenbergii was investigated and the mechanisms associated with body color were elucidated by transcriptome analysis. The results showed significant differences in the raw shells of the dark blue and light yellow M. rosenbergii (L: 26.20 ± 0.53 vs. 29.25 ± 0.45; a: -0.88 ± 0.19 vs. 0.35 ± 0.18; b: 1.73 ± 0.20 vs. 3.46 ± 0.37; dE: 70.33 ± 0.53 vs. 67.34 ± 0.45, respectively, p = 0.000) as well as the cooked shells (L: 58.14 ± 0.81 vs. 55.78 ± 0.55; a: 19.30 ± 0.56 vs. 16.42 ± 0.40; b: 23.60 ± 0.66 vs. 20.30 ± 0.40, respectively, p < 0.05). Transcriptome differential gene analysis obtained 39.02 Gb of raw data and 158,026 unigenes. Comprehensive searches of the SwissProt, Nr, KEGG, Pfam, and KOG databases resulted in successful annotations of 23,902 (33 %), 40,436 (25.59 %), 32,015 (20.26 %), 26,139 (16.54 %), and 22,155 (14.02 %) proteins, respectively. By KEGG pathway analysis, numerous differentially expressed genes were related to pigmentation-related pathways (MAPK signaling pathway, Wnt signaling pathway, melanin production, tyrosine metabolism, and cell-cell communication process). Candidate DEGs that may be involved in body color included apolipoprotein D, crustacyanin, cytochrome P450, and tyrosinase, as verified by quantitative real-time PCR. The results of this study provide useful references to further elucidate the molecular mechanisms of color formation of M. rosenbergii and other crustaceans.

Identification of pigmentation genes in skin, muscle and tail of a Thai-flag variety of Siamese fighting fish Betta splendens.

Pigmentation genes expressed in skin, body muscle and tail of Thai-flag compared with Blue, White and Red varieties of Siamese fighting fish Betta splendens were identified. In total, 22,919 new unigenes were found. Pearson correlation and PCA analysis revealed that expression profiles of genes in muscle, skin and tail across solid color variety were similar. In contrast, those in skin and red tail part of Thai-flag were closely related but they showed different expression profiles with the white tail part. Moreover, 21,347-64,965 SNPs were identified in exonic regions of identified genes. In total, 28,899 genes were differentially expressed between paired comparisons of libraries where 13,907 genes (48.12 %) were upregulated and 14,992 genes (51.88 %) were downregulated. DEGs between paired libraries were 106-5775 genes relative to the compared libraries (56-2982 and 50-2782 for upregulated and downregulated DEGs). Interestingly, 432 pigmentation genes of B. splendens were found. Of these, 297 DEGs showed differential expression between varieties. Many DEGs in melanogenesis (Bsmcr1r, Bsmcr5r, and Bsslc2a15b), tyrosine metabolism (Bstyr, Bstyrp1b and Bsdct), stripe repressor (BsAsip1 and BsAsip2b), pteridine (Bsgch2) and carotenoid (BsBco2) biosynthesis were downregulated in the Thai-flag compared with solid color varieties. Expression of Bsbco1l, Bsfrem2b, Bskcnj13, Bszic2a and Bspah in skin, muscle and tail of Thai-flag, Blue, Red and White varieties was analyzed by qRT-PCR and revealed differential expression between fish varieties and showed anatomical tissue-preferred expression patterns in the same fish variety. The information could be applied to assist genetic-based development of new B. splendens varieties in the future.

Identification and functional marker development of SbPLSH1 conferring purple leaf sheath in sorghum.

KEY MESSAGE: We identified a SbPLSH1gene conferring purple leaf sheath in sorghum (sorghumbicolor(L.) Moench)and developed a functional markerfor it. The purple leaf sheath of sorghum, a trait mostly related to anthocyanin deposition, is a visually distinguishable morphological marker widely used to evaluate the purity of crop hybrids. We aimed to dissect the genetic mechanism for leaf sheath color to mine the genes regulating this trait. In this study, two F2 populations were constructed by crossing a purple leaf sheath inbred line (Gaoliangzhe) with two green leaf sheath inbred lines (BTx623 and Silimei). Based on the results of bulked-segregant analysis sequencing, bulk-segregant RNA sequencing, and map-based cloning, SbPLSH1 (Sobic.006G175700), which encodes a bHLH transcription factor on chromosome 6, was identified as the candidate gene for purple leaf sheath in sorghum. Genetic analysis demonstrated that overexpression of SbPLSH1 in Arabidopsis resulted in anthocyanin deposition and purple petiole, while two single-nucleotide polymorphism (SNP) variants on the exon 6 resulted in loss of function. Further haplotype analysis revealed that there were two missense mutations and one cis-acting element mutation in SbPLSH1, which are closely associated with leaf sheath color in sorghum. Based on the variations, a functional marker (LSC4-2) for marker-assisted selection was developed, which has a broad-spectrum capability of distinguishing leaf sheath color in natural variants. In summary, this study lays a foundation for analyzing the genetic mechanism for sorghum leaf sheath color.

Molecular evolution of Phytocyanin gene and analysis of expression at different coloring periods in apple (Malus domestica).

BACKGROUND: PC (phytocyanin) is a class of copper-containing electron transfer proteins closely related to plant photosynthesis, abiotic stress responses growth and development in plants, and regulation of the expression of some flavonoids and phenylpropanoids, etc., however, compared with other plants, the PC gene family has not been systematically characterized in apple. RESULTS: A total of 59 MdPC gene members unevenly distributed across 12 chromosomes were identified at the genome-wide level. The proteins of the MdPC family were classified into four subfamilies based on differences in copper binding sites and glycosylation sites: Apple Early nodulin-like proteins (MdENODLs), Apple Uclacyanin-like proteins (MdUCLs), Apple Stellacyanin-like proteins (MdSCLs), and Apple Plantacyanin-like proteins (MdPLCLs). Some MdPC members with similar gene structures and conserved motifs belong to the same group or subfamily. The internal collinearity analysis revealed 14 collinearity gene pairs among members of the apple MdPC gene. Interspecific collinearity analysis showed that apple had 31 and 35 homologous gene pairs with strawberry and grape, respectively. Selection pressure analysis indicated that the MdPC gene was under purifying selection. Prediction of protein interactions showed that MdPC family members interacted strongly with the Nad3 protein. GO annotation results indicated that the MdPC gene also regulated the biosynthesis of phenylpropanoids. Chip data analysis showed that (MdSCL3, MdSCL7 and MdENODL27) were highly expressed in mature fruits and peels. Many cis-regulatory elements related to light response, phytohormones, abiotic stresses and flavonoid biosynthetic genes regulation were identified 2000 bp upstream of the promoter of the MdPC gene, and qRT-PCR results showed that gene members in Group IV (MdSCL1/3, MdENODL27) were up-regulated at all five stages of apple coloring, but the highest expression was observed at the DAF13 (day after fruit bag removal) stage. The gene members in Group II (MdUCL9, MdPLCL3) showed down-regulated or lower expression in the first four stages of apple coloring but up-regulated and highest expression in the DAF 21 stage. CONCLUSION: Herein, one objective of these findings is to provide valuable information for understanding the structure, molecular evolution, and expression pattern of the MdPC gene, another major objective in this study was designed to lay the groundwork for further research on the molecular mechanism of PC gene regulation of apple fruit coloration.

Evolution repeats itself in replicate long-term studies in the wild.

The extent to which evolution is repeatable remains debated. Here, we study changes over time in the frequency of cryptic color-pattern morphs in 10 replicate long-term field studies of a stick insect, each spanning at least a decade (across 30 years of total data). We find predictable "up-and-down" fluctuations in stripe frequency in all populations, representing repeatable evolutionary dynamics based on standing genetic variation. A field experiment demonstrates that these fluctuations involve negative frequency-dependent natural selection (NFDS). These fluctuations rely on demographic and selective variability that pushes populations away from equilibrium, such that they can reliably move back toward it via NFDS. Last, we show that the origin of new cryptic forms is associated with multiple structural genomic variants such that which mutations arise affects evolution at larger temporal scales. Thus, evolution from existing variation is predictable and repeatable, but mutation adds complexity even for traits evolving deterministically under natural selection.

Complex petal spot formation in the Beetle Daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paralogous GdMYBSG6 transcription factors.

Gorteria diffusa has elaborate petal spots that attract pollinators through sexual deception, but how G. diffusa controls spot development is largely unknown. Here, we investigate how pigmentation is regulated during spot formation. We determined the anthocyanin composition of G. diffusa petals and combined gene expression analysis with protein interaction assays to characterise R2R3-MYBs that likely regulate pigment production in G. diffusa petal spots. We found that cyanidin 3-glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three subgroup 6 R2R3-MYB transcription factors (GdMYBSG6-1,2,3) that likely activate the production of spot pigmentation. These genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. Interaction assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes. We demonstrate that the elaboration of complex spots in G. diffusa begins with the accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three paralogous R2R3-MYB transcription factors. Our results indicate that the functional diversification of these GdMYBSG6s involved changes in the spatial control of their transcription, and modification of the duration of GdMYBSG6 gene expression contributes towards floral variation within the species.

Promoter replication of grape MYB transcription factor is associated with a new red flesh phenotype.

KEY MESSAGE: In our study, we discovered a fragment duplication autoregulation mechanism in 'ZS-HY', which may be the reason for the phenotype of red foliage and red flesh in grapes. In grapes, MYBA1 and MYBA2 are the main genetic factors responsible for skin coloration which are located at the color loci on chromosome 2, but the exact genes responsible for color have not been identified in the flesh. We used a new teinturier grape germplasm 'ZhongShan-HongYu' (ZS-HY) which accumulate anthocyanin both in skin and flesh as experimental materials. All tissues of 'ZS-HY' contained cyanidin 3-O-(6″-p-coumaroyl glucoside), and pelargonidins were detected in skin, flesh, and tendril. Through gene expression analysis at different stage of flesh, significant differences in the expression levels of VvMYBA1 were found. Gene amplification analysis showed that the VvMYBA1 promoter is composed of two alleles, VvMYBA1a and 'VvMYBA1c-like'. An insertion of a 408 bp repetitive fragment was detected in the allele 'VvMYBA1c-like'. In this process, we found the 408 bp repetitive fragment was co-segregated with red flesh and foliage phenotype. Our results revealed that the 408 bp fragment replication insertion in promoter of 'VvMYBA1c-like' was the target of its protein, and the number of repeat fragments was related to the increase of trans-activation of VvMYBA1 protein. The activation of promoter by VvMYBA1 was enhanced by the addition of VvMYC1. In addition, VvMYBA1 interacted with VvMYC1 to promote the expression of VvGT1 and VvGST4 genes in 'ZS-HY'. The discovery of this mutation event provides new insights into the regulation of VvMYBA1 on anthocyanin accumulation in red-fleshed grape, which is of great significance for molecular breeding of red-fleshed table grapes.