Evaluation and validation of reference genes in Cymbidium faberi for real-time quantitative PCR.

For all organs at all Cymbidium faberi stages, ACT, UBQ3 and GAPDH can be selected as reference genes. For organs of the vegetative stage, UBQ2 and UBQ3 can be chosen for analysis of normalized gene expression. For the bud stage, ACT and UBQ3 can be used for analysis of gene expression. For the full blossom stage, ACT, UBQ3 and UBQ2 can be introduced into relative gene expression analysis. For vegetative organs, UBQ2 and ACT can be used as reference genes. For reproductive organs, ACT, UBQ3 and UBQ2 can be used as a reference for data processing. CfAG1 gene expression is more consistent when UBQ3, GAPDH and ACT are used as reference genes.

The Origin and Evolution of Chromosomal Reciprocal Translocation in Quasipaa boulengeri (Anura, Dicroglossidae).

Chromosomal rearrangements have long fascinated evolutionary biologists for being widely implicated in causing genetic differentiation. Suppressed recombination has been demonstrated in various species with inversion; however, there is controversy over whether such recombination suppression would facilitate divergence in reciprocal translocation with reduced fitness. In this study, we used the spiny frog, Quasipaa boulengeri, whose western Sichuan Basin populations exhibit translocation polymorphisms, to test whether the genetic markers on translocated (rearranged) or normal chromosomes have driven this genetic differentiation. We also investigated its overall genetic structure and the possibility of chromosomal fixation. Whole-chromosome painting and genetic structure clustering suggested a single origin of the translocation polymorphisms, and high-throughput sequencing of rearranged chromosomes isolated many markers with known localizations on chromosomes. Using these markers, distinct patterns of gene flow were found between rearranged and normal chromosomes. Genetic differentiation was only found in the translocated chromosomes, not in normal chromosomes or the mitochondrial genome. Hybrid unfitness cannot explain the genetic differentiation, as then the differentiation would be observed throughout the whole genome. Our results suggest that suppressed recombination drives genetic differentiation into a balanced chromosomal polymorphism. Mapping to a reference genome, we found that the region of genetic differentiation covered a wide range of translocated chromosomes, not only in the vicinity of chromosomal breakpoints. Our results imply that the suppressed recombination region could be extended by accumulation of repetitive sequences or capture of alleles that are adapted to the local environment, following the spread and/or fixation of chromosomal rearrangement.

Sex chromosomal dimorphisms narrated by X-chromosome translocation in a spiny frog (Quasipaa boulengeri).

BACKGROUND: In the general model of sex chromosome evolution for diploid dioecious organisms, the Y (or W) chromosome is derived, while the homogametic sex presumably represents the ancestral condition. However, in the frog species Quasipaa boulengeri, heteromorphisms caused by a translocation between chromosomes 1 and 6 are not related to sex, because the same heteromorphic chromosomes are found both in males and females at the cytological level. To confirm whether those heteromorphisms are unrelated to sex, a sex-linked locus was mapped at the chromosomal level and sequenced to identify any haplotype difference between sexes. RESULTS: Chromosome 1 was assigned to the sex chromosome pair by mapping the sex-linked locus. X-chromosome translocation was demonstrated and confirmed by the karyotypes of the progeny. Translocation heteromorphisms were involved in normal and translocated X chromosomes in the rearranged populations. Based on phylogenetic inference using both male and female sex-linked haplotypes, recombination was suppressed not only between the Y and normal X chromosomes, respectively the Y and translocated X chromosomes, but also between the normal and translocated X chromosomes. Both males and females shared not only the same translocation heteromorphisms but also the X chromosomal dimorphisms in this frog. CONCLUSIONS: The reverse of the typical situation, in which the X is derived and the Y has remained unchanged, is known to be very rare. In the present study, X-chromosome translocation has been known to cause sex chromosomal dimorphisms. The X chromosome has gone processes of genetic differentiation and/or structural changes by chance, which may facilitate sex chromosome differentiation. These sex chromosomal dimorphisms presenting in both sexes may represent the early stages of sex chromosome differentiation and aid in understanding sex chromosome evolution.

Suppressed Recombination of Sex Chromosomes Is Not Caused by Chromosomal Reciprocal Translocation in Spiny Frog (Quasipaa boulengeri).

Chromosome rearrangements (CRs) are perceived to be related to sex chromosome evolution, but it is a matter of controversy whether CRs are the initial causative mechanism of suppressed recombination for sex differentiation. The early stages of sex chromosome evolution in amphibians may represent intermediate states of differentiation, and if so, they potentially shed light on the ultimate cause of suppressed recombination and the role of CRs in sex chromosome differentiation. In this paper, we showed that sex determination differs among 16 populations of spiny frog (Quasipaa boulengeri), in which individuals have normal and rearranged chromosomes caused by reciprocal translocation. In eastern areas, without translocation, genetic differentiation between sexes was relatively low, suggesting unrestricted recombination. In comparison, in western populations that have both normal and translocated chromosomes, a male-heterogametic system and lack of X-Y recombination were identified by male-specific alleles and heterozygote excess. However, such genetic differentiation between sexes in western populations was not directly related to karyotypes, as it was found in individuals with both normal and translocated karyotypes. In the western Sichuan Basin, male-specific and translocation-specific allelic frequency distributions suggested that recombination of sex-differentiation ceased in all populations, but recombination suppression caused by translocation did not exist in some populations. Combined with phylogenetic inference, this indicated that the establishment of sex-linkage had taken place independently of reciprocal translocation, and translocation was not the ultimate cause of sex chromosome differentiation. Furthermore, comparison of the genetic diversity of alleles on Y chromosomes, X chromosomes, and autosomes in western populations showed a reduction of effective population size on sex chromosomes, which may be caused by reciprocal translocation. It indicates that, although it is not the ultimate cause of recombination suppression, reciprocal translocation may enhance sex chromosome differentiation.

Microsatellites mapping for non-model species with chromosomal rearrangement: a case study in the frog Quasipaa boulengeri (Anura: Dicroglossidae).

Gene mapping is an important resource for understanding the evolution of genes and cytogenetics. Model species with a known genetic map or genome sequence allow for the selection of genetic markers on a desired chromosome, while it is hard to locate these markers on chromosomes of non-model species without such references. A frog species, Quasipaa boulengeri, shows chromosomal rearrangement polymorphisms, making itself a fascinating model for chromosomal speciation mediated by suppressed recombination. However, no markers have been located on its rearranged chromosomes. We present a complete protocol to map microsatellites based on mechanical microdissection and chromosome amplification techniques. Following this protocol, we mapped 71 microsatellites of Q. boulengeri at the chromosome level. In total, eight loci were assigned to rearranged chromosomes, and the other 63 loci might attach to other chromosomes. These microsatellites could be used to compare the gene flow and verify the chromosomal suppressed recombination hypothesis in Q. boulengeri. This integrated protocol could be effectively used to map genes to chromosomes for non-model species.

PpCBF3 from Cold-Tolerant Kentucky Bluegrass Involved in Freezing Tolerance Associated with Up-Regulation of Cold-Related Genes in Transgenic Arabidopsis thaliana.

Dehydration-Responsive Element Binding proteins (DREB)/C-repeat (CRT) Binding Factors (CBF) have been identified as transcriptional activators during plant responses to cold stress. The objective of this study was to determine the physiological roles of a CBF gene isolated from a cold-tolerant perennial grass species, Kentucky bluegrass (Poa pratensis L.), which designated as PpCBF3, in regulating plant tolerance to freezing stress. Transient transformation of Arabidopsis thaliana mesophyll protoplast with PpCBF3-eGFP fused protein showed that PpCBF3 was localized to the nucleus. RT-PCR analysis showed that PpCBF3 was specifically induced by cold stress (4°C) but not by drought stress [induced by 20% polyethylene glycol 6000 solution (PEG-6000)] or salt stress (150 mM NaCl). Transgenic Arabidopsis overexpressing PpCBF3 showed significant improvement in freezing (-20°C) tolerance demonstrated by a lower percentage of chlorotic leaves, lower cellular electrolyte leakage (EL) and H2O2 and O2.- content, and higher chlorophyll content and photochemical efficiency compared to the wild type. Relative mRNA expression level analysis by qRT-PCR indicated that the improved freezing tolerance of transgenic Arabidopsis plants overexpressing PpCBF3 was conferred by sustained activation of downstream cold responsive (COR) genes. Other interesting phenotypic changes in the PpCBF3-transgenic Arabidopsis plants included late flowering and slow growth or 'dwarfism', both of which are desirable phenotypic traits for perennial turfgrasses. Therefore, PpCBF3 has potential to be used in genetic engineering for improvement of turfgrass freezing tolerance and other desirable traits.

Differential protein expression in Phalaenopsis under low temperature.

A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

Evaluation of internal control for gene expression in Phalaenopsis by quantitative real-time PCR.

The selection of appropriate reference genes is one of the most important steps to obtain reliable results for normalizing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of MADS-box gene in Phalaenopsis. In this study, we cloned 12 candidate reference genes including 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), cytoskeletal structural protein actin (ACT1, ACT2, ACT3, ACT4, ACT5), ubiquitin protein (UBQ1 and UBQ2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the cytoskeletal structural proteins α-tubulin (TUA) and ß-tubulin (TUB) in Phalaenopsis and evaluated their expression reliability. The expression of these candidate reference genes was analyzed using geNorm and normFinder software packages; the results showed that ACT2 and ACT4 were the highest stability reference genes for all experiment sets based on normFinder, followed by ACT1 or ACT3, while ACT3 and ACT4 were the highest stability reference genes for most experiment sets based on geNorm, then TUB or others. Taken together, Actin genes were the higher stability reference genes for all tissues at total developmental stages, and similar results came from analysis by normFinder. According to geNorm analysis, ACT3 and ACT4 were the most stable reference genes for all tissues tested and tissues at reproductive stages; TUB and ACT5 or ACT4 were the most stable reference genes for vegetative tissues or roots. The most stable reference genes for all vegetative tissues and only leaves were ACT4 and ACT5, ACT2 and ACT3, respectively; ACT1 and ACT3 were the most stable genes and sufficient for reliable normalization of flower tissues. While EF1α, UBQ1, UBQ2, and GAPDH were found to be unsuitable as a reference gene in our analysis for flower tissues, total tissues, and reproductive stages; UBQ2 and 18S were identified as the least stable reference genes for vegetative tissues at different stages, different tissues at vegetative stages; TUA and 18S were the least reliable reference genes for the samples from roots at all developmental stages. This is the first systematic report on the selection of reference genes in Phalaenopsis, and these data will facilitate future work on gene expression in orchid.

Molecular cloning and spatiotemporal expression of an APETALA1/FRUITFULL-like MADS-box gene from the orchid (Cymbidium faberi).

In order to identify genes involved in floral transition and development of the orchid species, a full-length APETALA1/FRUITFULL-like (AP1/FUL-like) MADS box cDNA was cloned from Cymbidium faberi (C. faberi) sepals and designated as C. faberi APETALA1-like (CfAP11], JQ031272.1). The deduced amino acid sequence of CfAP11 shared 84% homology with a member of the AP1/FUL-like group of MADS box genes (AY927238.1, Dendrobium thyrsiflorum FUL-like MADS box protein 3 mRNA). Phylogenetic analysis shows that CfAP11 belonged to the AP1/FUL transcription factor subfamily. Bioinformatics analysis shows that the deduced protein had a MADS domain and a relatively conservative K region. The secondary structure of CfAP11 mainly consisted of alpha helices (58.97%), and the three-dimensional structure of the protein was similar to that of homologues in Roza hybrida, Oryza sativa and Narcissus tazetta. Real-time quantitative PCR (qRT-PCR) results reveal low levels of its mRNA in roots, lower levels in leaves during reproductive period than vegetative period, and higher levels in pedicels at full-blossom stage than at bud stage. These results suggest that CfAP11 is involved in floral induction and floral development. Additionally, we observed higher levels of CfAP11 expression in pedicels and ovaries than in other tissues during full-blossom stage, which suggests that CfAP11 may also be involved in fruit formation in certain mechanism.

[Spatiotemporal expression patterns of three vernalization genes in wheat].

To identify spatiotemporal expression patterns of vernalization genes in common wheat, we analyzed expression characteristics of several vernalization genes (VRN1, VRN2 and VRN3) in the wheat cultivars 'Chinese spring' and 'Luohan 2' by RT-PCR. The VRN1 gene was expressed at different levels in the leaves and roots at the 3-leaf stage, stems, flag leaves at the grain-filling stage, anthers, ovules, and developing seeds in 'Chinese spring'. Expression of VRN1 increased before flowering date, then decreased after flowering time. Expression of VRN1 was not detected in dry seeds or seeds germination. Expression patterns of VRN1 in 'Luohan 2' were similar to those in 'Chinese spring', except that it was not expressed in roots or in the leaves at the 3-leaf stage in 'Luohan 2'. Expression of VRN2 was only detected in the leaves at the 3-leaf stage and in the embryo buds during seeds germination. The Spatiotemporal expression of VRN3 was similar to that of VRN1, except that VRN3 was not expressed in roots. These results improved our understanding of the molecular regulation of vernalization genes in common wheat.