Comparative Transcriptome Analysis between Two Potato Cultivars in Tuber Induction to Reveal Associated Genes with Anthocyanin Accumulation.

Anthocyanins are generally accumulated within a few layers, including the epidermal cells of leaves and stems in plants. Solanum tuberosum cv. 'Jayoung' (hereafter, JY) is known to accumulate anthocyanin both in inner tissues and skins. We discovered that anthocyanin accumulation in the inner tissues of JY was almost diminished (more than 95% was decreased) in tuber induction condition. To investigate the transcriptomic mechanism of anthocyanin accumulation in JY flesh, which can be modulated by growth condition, we performed mRNA sequencing with white-colored flesh tissue of Solanum tuberosum cv. 'Atlantic' (hereafter, 'Daeseo', DS) grown under canonical growth conditions, a JY flesh sample grown under canonical growth conditions, and a JY flesh sample grown under tuber induction conditions. We could identify 36 common DEGs (differentially expressed genes) in JY flesh from canonical growth conditions that showed JY-specifically increased or decreased expression level. These genes were enriched with flavonoid biosynthetic process terms in GO analysis, as well as gene set enrichment analysis (GSEA) analysis. Further in silico analysis on expression levels of anthocyanin biosynthetic genes including rate-limiting genes such as StCHS and StCHI followed by RT-PCR and qRT-PCR analysis showed a strong positive correlation with the observed phenotypes. Finally, we identified StWRKY44 from 36 common DEGs as a possible regulator of anthocyanin accumulation, which was further supported by network analysis. In conclusion, we identified StWRKY44 as a putative regulator of tuber-induction-dependent anthocyanin accumulation.

Intron-retained radish (Raphanus sativus L.) RsMYB1 transcripts found in colored-taproot lines enhance anthocyanin accumulation in transgenic Arabidopsis plants.

KEY MESSAGE: Overexpression of the naturally occurring intron-retained (IR) forms of radish RsMYB1 and RsTT8 transcripts in Arabidopsis causes a substantial increase in anthocyanin accumulation. The production of anthocyanins in plants is tightly controlled by the MYB-bHLH-WD40 (MBW) complex. In this study, analysis of four radish (Raphanus sativus L.) inbred lines with different colored taproots revealed that regulatory genes of anthocyanin biosynthesis, RsMYB1 and RsTT8, produce three transcripts, one completely spliced and two intron retention (IR1 and IR2) forms. Transcripts RsMYB1-IR1 and RsMYB1-IR2 retained the 1st (380 nt) and 2nd (149 nt) introns, respectively; RsTT8-IR1 retained the 4th intron (113 nt); RsTT8-IR2 retained both the 3rd (128 nt) and 4th introns. Levels of most IR forms were substantially low in radish samples, but the RsTT8-IR2 level was higher than RsTT8 in red skin/red flesh (RsRf) root. Since all IR forms contained a stop codon within the intron, they were predicted to encode truncated proteins with defective interaction domains, resulting in the inability to form the MBW complex in vivo. However, tobacco leaves transiently co-expressing RsMYB1-IRs and RsTT8-IRs showed substantially higher anthocyanin accumulation than those co-expressing their spliced forms. Consistently, co-expression of constructs encoding truncated proteins with spliced or IR forms of their interaction partner in tobacco leaves did not result in anthocyanin accumulation. Compared with RsMYB1, the overexpression of RsMYB1-IRs in Arabidopsis pap1 mutant increased anthocyanin accumulation by > sevenfold and upregulated the expression of Arabidopsis flavonoid biosynthesis genes including AtTT8. Our results suggest that the stable co-expression of RsMYB1-IRs in fruit trees and vegetable crops could be used to increase their anthocyanin contents.

Molecular analysis of anthocyanin biosynthesis-related genes reveal BoTT8 associated with purple hypocotyl of broccoli (Brassica oleracea var. italica L.).

Broccoli (Brassica oleracea var. italica L.) is a highly nutritious vegetable that typically forms pure green or purple florets. However, green broccoli florets sometimes accumulate slight purplish pigmentation in response environmental factors, decreasing their market value. In the present study, we aimed to develop molecular markers to distinguish broccoli genotypes as pure green or purplish floret color at the early seedling stage. Anthocyanins are known to be involved in the purple pigmentation in plants. The purplish broccoli lines were shown to accumulate purple pigmentation in the hypocotyls of very young seedlings; therefore, the expression profiles of the structural and regulatory genes of anthocyanin biosynthesis were analyzed in the hypocotyls using qRT-PCR. BoPAL, BoDFR, BoMYB114, BoTT8, BoMYC1.1, BoMYC1.2, and BoTTG1 were identified as putative candidate genes responsible for the purple hypocotyl color. BoTT8 was much more highly expressed in the purple than green hypocotyls; therefore, it was cloned and sequenced from various broccoli lines, revealing SNP and InDel variations between these genotypes. We tested four SNPs (G > A; A > T; G > C; T > G) in the first three exons and a 14-bp InDel (ATATTTATATATAT) in the BoTT8 promoter in 51 broccoli genotypes, and we found these genetic variations could distinguish the green lines, purple lines, and F1 hybrids. These novel molecular markers could be useful in broccoli breeding programs to develop a true green or purple broccoli cultivar.

Genome-Wide Analysis of Glycoside Hydrolase Family 1 ß-glucosidase Genes in Brassica rapa and Their Potential Role in Pollen Development.

Glycoside hydrolase family 1 (GH1) ß-glucosidases (BGLUs) are encoded by a large number of genes, and are involved in many developmental processes and stress responses in plants. Due to their importance in plant growth and development, genome-wide analyses have been conducted in model plants (Arabidopsis and rice) and maize, but not in Brassica species, which are important vegetable crops. In this study, we systematically analyzed B. rapa BGLUs (BrBGLUs), and demonstrated the involvement of several genes in pollen development. Sixty-four BrBGLUs were identified in Brassica databases, which were anchored onto 10 chromosomes, with 10 tandem duplications. Phylogenetic analysis revealed that 64 genes were classified into 10 subgroups, and each subgroup had relatively conserved intron/exon structures. Clustering with Arabidopsis BGLUs (AtBGLUs) facilitated the identification of several important subgroups for flavonoid metabolism, the production of glucosinolates, the regulation of abscisic acid (ABA) levels, and other defense-related compounds. At least six BrBGLUs might be involved in pollen development. The expression of BrBGLU10/AtBGLU20, the analysis of co-expressed genes, and the examination of knocked down Arabidopsis plants strongly suggests that BrBGLU10/AtBGLU20 has an indispensable function in pollen development. The results that are obtained from this study may provide valuable information for the further understanding of ß-glucosidase function and Brassica breeding, for nutraceuticals-rich Brassica crops.

Overexpression of Acyl-ACP Thioesterases, CpFatB4 and CpFatB5, Induce Distinct Gene Expression Reprogramming in Developing Seeds of Brassica napus.

We examined the substrate preference of Cuphea paucipetala acyl-ACP thioesterases, CpFatB4 and CpFatB5, and gene expression changes associated with the modification of lipid composition in the seed, using Brassica napus transgenic plants overexpressing CpFatB4 or CpFatB5 under the control of a seed-specific promoter. CpFatB4 seeds contained a higher level of total saturated fatty acid (FA) content, with 4.3 times increase in 16:0 palmitic acid, whereas CpFatB5 seeds showed approximately 3% accumulation of 10:0 and 12:0 medium-chain FAs, and a small increase in other saturated FAs, resulting in higher levels of total saturated FAs. RNA-Seq analysis using entire developing pods at 8, 25, and 45 days after flowering (DAF) showed up-regulation of genes for ß-ketoacyl-acyl carrier protein synthase I/II, stearoyl-ACP desaturase, oleate desaturase, and linoleate desaturase, which could increase unsaturated FAs and possibly compensate for the increase in 16:0 palmitic acid at 45 DAF in CpFatB4 transgenic plants. In CpFatB5 transgenic plants, many putative chloroplast- or mitochondria-encoded genes were identified as differentially expressed. Our results report comprehensive gene expression changes induced by alterations of seed FA composition and reveal potential targets for further genetic modifications.

Purple Brassica oleracea var. capitata F. rubra is due to the loss of BoMYBL2-1 expression.

BACKGROUND: Water-soluble anthocyanin pigments are important ingredients in health-improving supplements and valuable for the food industry. Although great attention has been paid to the breeding and production of crops containing high levels of anthocyanin, genetic variation in red or purple cabbages (Brassica oleracea var. capitata F. rubra) has not yet been characterized at the molecular level. In this study, we identified the mechanism responsible for the establishment of purple color in cabbages. RESULTS: BoMYBL2-1 is one of the regulatory genes in the anthocyanin biosynthesis pathway in cabbages. It is a repressor whose expression is inversely correlated to anthocyanin synthesis and is not detectable in purple cabbages. Sequence analysis of purple cabbages revealed that most lacked BoMYBL2-1 coding sequences, although a few had a substitution in the region of the promoter 347 bp upstream of the gene that was associated with an absence of BoMYBL2-1 expression. Lack of transcriptional activity of the substitution-containing promoter was confirmed using transgenic Arabidopsis plants transformed with promoter::GUS fusion constructs. The finding that the defect in BoMYBL2-1 expression was solely responsible for purple coloration in cabbages was further demonstrated using genomic PCR and RT-PCR analyses of many other structural and regulatory genes in anthocyanin biosynthesis. Molecular markers for purple cabbages were developed and validated using 69 cabbage lines. CONCLUSION: Expression of BoMYBL2-1 was inversely correlated to anthocyanin content, and purple color in cabbages resulted from a loss of BoMYBL2-1 expression, caused by either the promoter substitution or deletion of the gene. This is the first report of molecular markers that distinguish purple cabbages. Such markers will be useful for the production of intraspecific and interspecific hybrids for functional foods, and for industrial purposes requiring high anthocyanin content.

Genome-Wide Identification and Characterization of Warming-Related Genes in Brassica rapa ssp. pekinensis.

For sustainable crop cultivation in the face of global warming, it is important to unravel the genetic mechanisms underlying plant adaptation to a warming climate and apply this information to breeding. Thermomorphogenesis and ambient temperature signaling pathways have been well studied in model plants, but little information is available for vegetable crops. Here, we investigated genes responsive to warming conditions from two Brassica rapa inbred lines with different geographic origins: subtropical (Kenshin) and temperate (Chiifu). Genes in Gene Ontology categories "response to heat", "heat acclimation", "response to light intensity", "response to oxidative stress", and "response to temperature stimulus" were upregulated under warming treatment in both lines, but genes involved in "response to auxin stimulus" were upregulated only in Kenshin under both warming and minor-warming conditions. We identified 16 putative high temperature (HT) adaptation-related genes, including 10 heat-shock response genes, 2 transcription factor genes, 1 splicing factor gene, and 3 others. BrPIF4, BrROF2, and BrMPSR1 are candidate genes that might function in HT adaptation. Auxin response, alternative splicing of BrHSFA2, and heat shock memory appear to be indispensable for HT adaptation in B. rapa. These results lay the foundation for molecular breeding and marker development to improve warming tolerance in B. rapa.

Natural variation in CIRCADIAN CLOCK ASSOCIATED 1 is associated with flowering time in Brassica rapa.

Flowering time is a very important agronomic trait and the development of molecular markers associated with this trait can facilitate crop breeding. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a core oscillator component of circadian rhythms that affect metabolic pathways in plants, has been implicated in flowering time control in species of Brassica. CCA1 gene sequences from three Brassica rapa inbred lines, showing either early flowering or late flowering phenotypes, were analyzed and a high level of sequence variation was identified, especially within the fourth intron. Using this information, three PCR primer sets were designed and tested using various inbred lines of B. rapa. The usage of InDel markers was further validated by evaluation of flowering time and high resolution melting (HRM) analysis. Both methods, PCR and HRM, validated the use of newly developed markers. Additional sequence analyses of Brassica plants with diploid (AA, BB, or CC) and allotetraploid genomes further confirmed a large number of sequence polymorphisms in the CCA1 gene, including insertions/deletions in the fourth intron. Our results demonstrated that sequence variations in CCA1 can be used to develop valuable trait-related molecular markers for Brassica crop breeding.

Expression of Genes Related to Phenylpropanoid Biosynthesis in Different Organs of Ixeris dentata var. albiflora.

Members of the genus Ixeris have long been used in traditional medicines as stomachics, sedatives, and diuretics. Phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate: coenzyme-A (CoA) ligase (4CL), chalcone synthase (CHS), and dihydroflavonol 4-reductase (DFR) are important enzymes in the phenylpropanoid pathway. In this study, we analyzed seven genes from Ixeris dentata var. albiflora that are involved in phenylpropanoid biosynthesis, using an Illumina/Solexa HiSeq 2000 platform. The amino acid sequence alignments for IdPALs, IdC4H, Id4CLs, IdCHS, and IdDFR showed high identity to sequences from other plants. We also investigated transcript levels using quantitative real-time PCR, and analyzed the accumulation of phenylpropanoids in different organs of I. dentata var. albiflora using high-performance liquid chromatography. The transcript levels of IdC4H, Id4CL1, IdCHS, and IdDFR were highest in the leaf. The catechin, chlorogenic acid, ferulic acid, and quercetin contents were also highest in the leaf. We suggest that expression of IdC4H, Id4CL1, IdCHS, and IdDFR is associated with the accumulation of phenylpropanoids. Our results may provide baseline information for elucidating the mechanism of phenylpropanoid biosynthesis in different organs of I. dentata var. albiflora.

Molecular Cloning and Characterization of Carotenoid Pathway Genes and Carotenoid Content in Ixeris dentata var. albiflora.

Ixeris dentata var. albiflora is considered as a potential therapeutic agent against mithridatism, calculous, indigestion, pneumonia, hepatitis, and tumors as well as good seasoned vegetable in Far East countries. Phytoene synthase (PSY), phytoene desaturase (PDS) ξ-carotene desaturase (ZDS), lycopene ß-cyclase (LCYB), lycopene ε-cyclase (LCYE), ε-ring carotene hydroxylase (CHXB), and zeaxanthin epoxidase (ZDS) are vital enzymes in the carotenoid biosynthesis pathway. We have examined these seven genes from I. dentata that are participated in carotenoid biosynthesis utilizing an Illumina/Solexa HiSeq 2000 platform. In silico analysis of the seven deduced amino acid sequences were revealed its closest homology with other Asteracea plants. Further, we explored transcript levels and carotenoid accumulation in various organs of I. dentata using quantitative real time PCR and high-performance liquid chromatography, respectively. The highest transcript levels were noticed in the leaf for all the genes while minimal levels were noticed in the root. The maximal carotenoid accumulation was also detected in the leaf. We proposed that these genes expressions are associated with the accumulation of carotenoids. Our findings may suggest the fundamental clues to unravel the molecular insights of carotenoid biosynthesis in various organs of I. dentata.

Intracellular Ca(2+) and K(+) concentration in Brassica oleracea leaf induces differential expression of transporter and stress-related genes.

BACKGROUND: One of the most important members of the genus Brassica, cabbage, requires a relatively high level of calcium for normal growth (Plant Cell Environ 7: 397-405, 1984; Plant Physiol 60: 854-856, 1977). Localized Ca(2+) deficiency in cabbage leaves causes tip-burn, bringing about serious economic losses (Euphytica 9:203-208, 1960; Ann Bot 43:363-372, 1979; Sci Hortic 14:131-138, 1981). Although it has been known that the occurrence of tip-burn is related to Ca(2+) deficiency, there is limited information on the underlying mechanisms of tip-burn or the relationship between Ca(2+) and tip-burn incidence. To obtain more information on the genetic control of tip-burn symptoms, we focused on the identification of genes differentially expressed in response to increasing intracellular Ca(2+) and K(+) concentrations in B. oleracea lines derived from tip-burn susceptible, tip-burn resistant cabbages (B. oleracea var. capitata), and kale (B. oleracea var. acephala). RESULTS: We compared the levels of major macronutrient cations, including Ca(2+) and K(+), in three leaf segments, the leaf apex (LA), middle of leaf (LM), and leaf base (LB), of tip-burn susceptible, tip-burn resistant cabbages, and kale. Ca(2+) and K(+) concentrations were highest in kale, followed by tip-burn resistant and then tip-burn susceptible cabbages. These cations generally accumulated to a greater extent in the LB than in the LA. Transcriptome analysis identified 58,096 loci as putative non-redundant genes in the three leaf segments of the three B. oleracea lines and showed significant changes in expression of 27,876 loci based on Ca(2+) and K(+) levels. Among these, 1844 loci were identified as tip-burn related phenotype-specific genes. Tip-burn resistant cabbage and kale-specific genes were largely related to stress and transport activity based on GO annotation. Tip-burn resistant cabbage and kale plants showed phenotypes clearly indicative of heat-shock, freezing, and drought stress tolerance compared to tip-burn susceptible cabbages, demonstrating a correlation between intracellular Ca(2+) and K(+) concentrations and tolerance of abiotic stress with differential gene expression. We selected 165 genes that were up- or down-regulated in response to increasing Ca(2+) and K(+) concentrations in the three leaf segments of the three plant lines. Gene ontology enrichment analysis indicated that these genes participated in regulatory metabolic processes or stress responses. CONCLUSIONS: Our results indicate that the genes involved in regulatory metabolic processes or stress responses were differentially expressed in response to increasing Ca(2+) and K(+) concentrations in the B. oleracea leaf. Our transcriptome data and the genes identified may serve as a starting point for understanding the mechanisms underlying essential macronutrient deficiencies in plants, as well as the features of tip-burn in cabbage and other Brassica species.

GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions.

Genome-wide identification and characterization of MADS-box family genes related to organ development and stress resistance in Brassica rapa.

BACKGROUND: MADS-box transcription factors (TFs) are important in floral organ specification as well as several other aspects of plant growth and development. Studies on stress resistance-related functions of MADS-box genes are very limited and no such functional studies in Brassica rapa have been reported. To gain insight into this gene family and to elucidate their roles in organ development and stress resistance, we performed genome-wide identification, characterization and expression analysis of MADS-box genes in B. rapa. RESULTS: Whole-genome survey of B. rapa revealed 167 MADS-box genes, which were categorized into type I (Mα, Mß and Mγ) and type II (MIKC(c) and MIKC*) based on phylogeny, protein motif structure and exon-intron organization. Expression analysis of 89 MIKC(c) and 11 MIKC* genes was then carried out. In addition to those with floral and vegetative tissue expression, we identified MADS-box genes with constitutive expression patterns at different stages of flower development. More importantly, from a low temperature-treated whole-genome microarray data set, 19 BrMADS genes were found to show variable transcript abundance in two contrasting inbred lines of B. rapa. Among these, 13 BrMADS genes were further validated and their differential expression was monitored in response to cold stress in the same two lines via qPCR expression analysis. Additionally, the set of 19 BrMADS genes was analyzed under drought and salt stress, and 8 and 6 genes were found to be induced by drought and salt, respectively. CONCLUSION: The extensive annotation and transcriptome profiling reported in this study will be useful for understanding the involvement of MADS-box genes in stress resistance in addition to their growth and developmental functions, which ultimately provides the basis for functional characterization and exploitation of the candidate genes for genetic engineering of B. rapa.

Anthocyanin biosynthesis for cold and freezing stress tolerance and desirable color in Brassica rapa.

Flavonoids are divided into several structural classes, including anthocyanins, which provide flower and leaf colors and other derivatives that play diverse roles in plant development and interactions with the environment. This study characterized four anthocyanidin synthase (ANS) genes of Brassica rapa, a structural gene of the anthocyanin biosynthetic pathway, and investigated their association with pigment formation, cold and freezing tolerance in B. rapa. Sequences of these genes were analyzed and compared with similar gene sequences from other species, and a high degree of homology with their respective functions was found. Organ-specific expression analysis revealed that these genes were only expressed in the colored portion of leaves of different lines of B. rapa. Conversely, B. rapa anthocyanidin synthase (BrANS) genes also showed responses to cold and freezing stress treatment in B. rapa. BrANSs were also shown to be regulated by two transcription factors, BrMYB2-2 and BrTT8, contrasting with anthocyanin accumulation and cold stress. Thus, the above results suggest the association of these genes with anthocyanin biosynthesis and cold and freezing stress tolerance and might be useful resources for development of cold-resistant Brassica crops with desirable colors as well.

Molecular characterization of the Brassica rapa auxin-repressed, superfamily genes, BrARP1 and BrDRM1.

Two auxin-repressed superfamily genes, auxin-repressed protein 1 (ARP1) and dormancy-associated protein 1 (DRM1), are highly expressed in both the dormant buds and non-growing tissues of several plant species. To further identify the function of these proteins in Chinese cabbage (Brassica rapa L. ssp. pekinensis), we examined comprehensive expression patterns of BrARP1 and BrDRM1 under various developmental and stress conditions. We also examined these same genes in transgenic Arabidopsis plants. Both genes were expressed in all tissues tested, but their levels were highest in mature tissues accompanied by low levels of the growth-associated marker, B. rapa ribosomal protein 27. Expression of both genes was induced by abiotic stresses, such as chilling, heat shock, and salt treatment. Overexpression of either BrARP1 or BrDRM1 in Arabidopsis causes a reduction in vegetative growth and seed productivity, without affecting morphology. The lengths of petioles and siliques were greatly reduced. Simultaneous expression of both genes showed an additive effect on the growth suppression, resulting in significant reduction in plant size. Knock-out of Arabidopsis ARP1, DRM1, or both, neither affected growth rate nor final size. Results suggest BrARP1 and BrDRM1 are either involved in growth arrest, or stop growth, possibly from inhibition of either cell elongation or cell expansion, thereby creating a "growth brake".

Rapid divergence of repetitive DNAs in Brassica relatives.

Centromeric, subtelomeric, and telomeric repetitive DNAs were characterized in Brassica species and the related Raphanus sativus and Arabidopsis thaliana. In general, rapid divergence of the repeats was found. The centromeric tandem satellite repeats were differentially distributed in the species studied, suggesting that centromeric repeats have diverged during the evolution of the A/C and B genome lineages. Sequence analysis of centromeric repeats suggested rapid evolution. Pericentromere-associated retrotransposons were identified and showed divergence during the evolution of the lineages as centromeric repeats. A novel subtelomeric tandem repeat from B. nigra was found to be conserved across the diploid Brassica genomes; however, this sequence was not identified in the related species. In contrast to previous studies, interstitial telomere-like repeats were identified in the pericentromeres of Brassica chromosomes, and these repeats may be associated with genomic stability. These results provide insight into genome evolution during polyploidization in Brassica and divergence within the Brassicaceae.

Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences.

Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n = 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumber.

Alleles disrupting LBD37-like gene by an 136 bp insertion show different distributions between green and purple cabbages (Brassica oleracea var. capitata).

BACKGROUND: In Arabidopsis thaliana (Arabidopsis), clade IIb lateral organ boundary domain (LBD) 37, 38, and 39 proteins negatively regulate anthocyanin biosynthesis and affect nitrogen responses. OBJECTIVE: To investigate the possible role of LBD genes in anthocyanin accumulations among green and purple cabbages (Brassica oleracea var. capitata), we determined sequence variations and expression levels of cabbage homologs of Arabidopsis LBD37, 38, and 39. METHODS: DNA and mRNA sequences of BoLBD37, BoLBD37L (BoLBD37-like), BoLBD38, BoLBD38L (BoLBD38-like), and BoLBD39 gene in cabbage were determined. Allelic variations of BoLBD37L alleles in cabbages, resulting from insertions, were validated by genomic DNA PCR. Gene expressions were examined by semi-quantitative reverse transcription (RT-PCR) or quantitative RT-PCR. RESULTS: Based on the expression analyses, BoLBD37L with two alleles, BoLBD37L-G and BoLBD37L-P, was selected as a candidate gene important for differential anthocyanin accumulation. BoLBD37L-P contains an 136 base pair insertion in the 2nd exon, producing two splicing variants encoding truncated proteins. Most purple cabbage lines were found to have BoLBD37L-P allele as homozygotes or heterozygotes, and only two out of sixty-four purple cabbages were identified as BoLBD37L-G homozygotes. Expression analyses of anthocyanin biosynthesis genes and their upstream regulators, including BoLBD37L, suggest that truncated proteins encoded by splicing variants of BoLBD37L-P may disrupt the BoLBD37L function as repressor. CONCLUSION: Difference in the C-terminal region of BoLBD37L-G and BolBD37L-P may affect the expression of downstream genes, BoMYB114L and BoTT8, resulting in differential anthocyanin accumulation.

Development of Molecular Markers for Predicting Radish (Raphanus sativus) Flesh Color Based on Polymorphisms in the RsTT8 Gene.

Red radish (Raphanus sativus L.) cultivars are a rich source of health-promoting anthocyanins and are considered a potential source of natural colorants used in the cosmetic industry. However, the development of red radish cultivars via conventional breeding is very difficult, given the unusual inheritance of the anthocyanin accumulation trait in radishes. Therefore, molecular markers linked with radish color are needed to facilitate radish breeding. Here, we characterized the RsTT8 gene isolated from four radish genotypes with different skin and flesh colors. Sequence analysis of RsTT8 revealed a large number of polymorphisms, including insertion/deletions (InDels), single nucleotide polymorphisms (SNPs), and simple sequence repeats (SSRs), between the red-fleshed and white-fleshed radish cultivars. To develop molecular markers on the basis of these polymorphisms for discriminating between radish genotypes with different colored flesh tissues, we designed four primer sets specific to the RsTT8 promoter, InDel, SSR, and WD40/acidic domain (WD/AD), and tested these primers on a diverse collection of radish lines. Except for the SSR-specific primer set, all primer sets successfully discriminated between red-fleshed and white-fleshed radish lines. Thus, we developed three molecular markers that can be efficiently used for breeding red-fleshed radish cultivars.

Molecular characterization of Arabidopsis thaliana LSH1 and LSH2 genes.

BACKGROUND: Arabidopsis thaliana genome encodes ten DUF640 (short for domain of unknown function 640)/ALOG (short for Arabidopsis LSH1 and Oryza G1) proteins, also known as light-dependent short hypocotyl (LSH) proteins. While some of the LSH genes regulate organ boundary determination and shade avoidance response, the function of most of these genes remains largely unknown. OBJECTIVE: In this study, we aimed to characterize the function of AtLSH1 and AtLSH2 in Arabidopsis. METHODS: We overexpressed AtLSH1 and AtLSH2 (with or without the FLAG tag) in Arabidopsis Col-0 plants under the control of the 35S promoter. We also generated knockout or knockdown lines of these genes by miRNA-induced gene silencing (MIGS). We conducted intensive phenotypic analysis of these transgenic lines, and finally performed RNA-seq analysis of two AtLSH2 overexpression (OX) lines. RESULTS: Although AtLSH1 and AtLSH2 amino acid sequences showed high similarly, AtLSH2-OX lines showed much higher levels of their transcripts than those of AtLSH1-OX lines. Additionally, overexpression of AtLSH1 and AtLSH2 greatly inhibited hypocotyl elongation in a light-independent manner, and reduced both vegetative and reproductive growth. However, knockout or knockdown of both these AtLSH genes did not affect plant phenotype. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) identified by RNA-seq revealed enrichment of the GO term 'response to stimulus', included phytohormone-responsive genes; however, genes responsible for the abnormal phenotypes of AtLSH2-OX lines could not be identified. CONCLUSION: Although our data revealed no close association between light and phytohormone signaling components, overexpression of AtLSH1 and AtLSH2 greatly reduced vegetative and reproductive growth of Arabidopsis plants. This property could be used to generate new plants by regulating expression of AtLSH1 and AtLSH2.