How to perform RT-qPCR accurately in plant species?: a case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

Background: Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. Results: An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Conclusions: Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway

Asymmetric somatic plant hybridization : status and applications

To create asymmetric somatic hybrids, the genome of the so-called donor protoplast is fragmented prior to protoplast fusion. As a result, only a limited amount of the donor genome is transferred to the fusion product. This technique can circumvent some commonly observed problems related to symmetric fusion and offers a practical breeding tool for asexual hybridization. Genomes are typically fragmented by irradiation, microprotoplast production or application of metabolic inhibitors such as iodoacetamide. Irradiation and microprotoplast production fragment the nuclear genome, whereas iodoacetamide inactivates the cytoplasmic genome. It can therefore be used to introduce cytoplasmic male ste- rility, an important practical application. For hybrid verification and genome characterization, molecular markers and cytogenetic techniques are applied. This review highlights and discusses progress made during the last decade in sper- matophytes asymmetric protoplast fusion

Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol protein complexes

Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

To develop an efficient protocol for shoot regeneration of root chicory (Cichorium intybus var. sativum), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l-1) plus 6-Benzylaminopurine (6-BAP) (1 mg l-1), indole-3-acetic acid (IAA) (0.01 mg l-1) plus 6-BAP (1.0 mg l-1), and IAA (0.5 mg l-1) plus (0.5 mg l-1) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l-1) plus 6-BAP (0.5 mg l-1). MS medium containing indole-3-butylric acid IBA (1 mgl-1) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 RifR (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l-1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes

Differential expression of fructan 1-exohydrolase genes involved in inulin biodegradation in chicory (Cichorium intybus) cultivars

Fructan 1-exohydrolase (1-FEH; FEH) enzymes are involved in inulin degradation in the roots of chicory. rehgiH FEH expression in cold temperatures can decrease the quality and the quantity of the inulin. This is the case at the end of the growing season and during cold storage. Little is known at molecular level whether the expression levels of fructan 1-exohydrolase genes vary among chicory cultivars, especially during cold storage of roots. Real-time RT-PCR is the most sensitive method for the detection of low abundance mRNAs. Quantitative real time PCR (RT-qPCR) analysis of the FEH mRNAs (including FEHI and FEHII) in cold stored roots of three chicory cultivars was studied. RT-qPCR results showed variable levels of FEHII and FEHI expression in different hgitonehis of the chicory cultivars studied. Moreover, longer cold storage of roots significantly induced expression levels of FEHII in some chicory phenotypes. These results could be used in breeding programmes to increase inulin content in root chicory by selecting genotypes based on their FEH expression profiles

Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported.</p> <p>Results</p> <p>Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (<it>NADHD</it>), actin (<it>ACT</it>), β-tubulin (<it>TUB</it>), glyceraldehyde-3-phosphate-dehydrogenase (<it>GADPH</it>), histone H3 (<it>H3</it>), elongation factor 1-alpha (<it>EF</it>) and 18S rRNA (<it>rRNA</it>) were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. <it>ACT</it>, <it>EF</it>, and <it>rRNA </it>were the most stable genes as identified by the three different analysis methods. In addition, the use of <it>ACT, EF </it>and <it>GAPDH </it>as reference genes was illustrated by analysing 1-<it>FEHII </it>(<it>FEHII</it>) expression in chicory root and leaf tissues. These analyses revealed the biological variation in <it>FEHII </it>transcript expression among the tissues studied, and between individual plants.</p> <p>Conclusions</p> <p>geNorm, NormFinder, and BestKeeper analyses indicated that <it>ACT</it>, <it>EF </it>and <it>rRNA </it>had the highest expression stability across leaf and root tissues, while <it>GAPDH </it>and <it>NADHD </it>showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as <it>ACT </it>and <it>EF </it>allows accurate normalisation of gene expression in chicory leaf and root tissues.</p

Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

To develop an efficient protocol for shoot regeneration of root chicory (Cichorium intybus var. sativum), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l-1) plus 6-Benzylaminopurine (6-BAP) (1 mg l-1), indole-3-acetic acid (IAA) (0.01 mg l-1) plus 6-BAP (1.0 mg l-1), and IAA (0.5 mg l-1) plus (0.5 mg l-1) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l-1) plus 6-BAP (0.5 mg l-1). MS medium containing indole-3-butylric acid IBA (1 mgl-1) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 RifR (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l-1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes.Keywords: Chicory, regeneration, transformation, 1-Naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), 6-Benzylaminopurine (6-BAP

Multipoint-likelihood maximization mapping on 4 segregating populations to achieve an integrated framework map for QTL analysis in pot azalea (Rhododendron simsii hybrids)

<p>Abstract</p> <p>Background</p> <p>Azalea (<it>Rhododendron simsii </it>hybrids) is the most important flowering pot plant produced in Belgium, being exported world-wide. In the breeding program, flower color is the main feature for selection, only in later stages cultivation related plant quality traits are evaluated. As a result, plants with attractive flowering are kept too long in the breeding cycle. The inheritance of flower color has been well studied; information on the heritability of cultivation related quality traits is lacking. For this purpose, QTL mapping in diverse genetic backgrounds appeared to be a must and therefore 4 mapping populations were made and analyzed.</p> <p>Results</p> <p>An integrated framework map on four individual linkage maps in <it>Rhododendron simsii </it>hybrids was constructed. For genotyping, mainly dominant scored AFLP (on average 364 per population) and MYB-based markers (15) were combined with co-dominant SSR (23) and EST markers (12). Linkage groups were estimated in JoinMap. A consensus grouping for the 4 mapping populations was made and applied in each individual mapping population. Finally, 16 stable linkage groups were set for the 4 populations; the azalea chromosome number being 13. A combination of regression mapping (JoinMap) and multipoint-likelihood maximization (Carthagène) enabled the construction of 4 maps and their alignment. A large portion of loci (43%) was common to at least two populations and could therefore serve as bridging markers. The different steps taken for map optimization and integration into a reference framework map for QTL mapping are discussed.</p> <p>Conclusions</p> <p>This is the first map of azalea up to our knowledge. AFLP and SSR markers are used as a reference backbone and functional markers (EST and MYB) were added as candidate genes for QTL analysis. The alignment of the 4 maps on the basis of framework markers will facilitate in turn the alignment of QTL regions detected in each of the populations. The approach we took is thoroughly different than the recently published integrated maps and well-suited for mapping in a non-model crop.</p

Towards an optimal sampling strategy for assessing genetic variation within and among white clover (Trifolium repens L.) cultivars using AFLP

Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci) from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He) and Shannon diversity index (I) were greatly underestimated, whereas with 20, more than 95% of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation

L'amélioration des plantes et la biotechnologie végétale appliquée

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