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      Copy number variation of a gene cluster encoding endopolygalacturonase mediates flesh texture and stone adhesion in peach

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          Copy number variation at the F-M locus plays a driving role in flesh texture diversification in peach.

          Abstract

          Texture is an important attribute affecting consumer perception of fruit quality. Peach melting flesh and flesh adhesion to stone (endocarp) are simply inherited and controlled by the F-M locus on linkage group (LG) 4. Here, we report that two genes encoding endopolygalacturonase (endoPG) in the F-M locus, designated PpendoPGF and PpendoPGM, are associated with the melting flesh and stone adhesion traits. PpendoPGM controls melting flesh while PpendoPGF has pleiotropic effects on both melting flesh and stone adhesion. The F-M locus has three allelic copy number variants of endoPG, H 1 ( PpendoPGF and PpendoPGM), H 2 ( PpendoPGM), and H 3 (null). The H 2 haplotype represents the ancestral one while the H 1 and H 3 haplotypes are two variants due to duplication and deletion of PpendoPGM, respectively. Accessions with H 1H 1, H 1H 2, or H 1H 3 genotypes show the freestone or semi-freestone and melting flesh phenotype, while both H 2H 2 and H 2H 3 accessions have the clingstone and melting flesh phenotype. The H 3H 3 accessions have the clingstone and non-melting flesh phenotype. Our study not only demonstrates a driving role of gene copy number variations in flesh texture diversification in fruit trees, but also provides a useful diagnostic tool for early seedling selection in peach breeding programmes.

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          Copy number variation of multiple genes at Rhg1 mediates nematode resistance in soybean.

          The rhg1-b allele of soybean is widely used for resistance against soybean cyst nematode (SCN), the most economically damaging pathogen of soybeans in the United States. Gene silencing showed that genes in a 31-kilobase segment at rhg1-b, encoding an amino acid transporter, an α-SNAP protein, and a WI12 (wound-inducible domain) protein, each contribute to resistance. There is one copy of the 31-kilobase segment per haploid genome in susceptible varieties, but 10 tandem copies are present in an rhg1-b haplotype. Overexpression of the individual genes in roots was ineffective, but overexpression of the genes together conferred enhanced SCN resistance. Hence, SCN resistance mediated by the soybean quantitative trait locus Rhg1 is conferred by copy number variation that increases the expression of a set of dissimilar genes in a repeated multigene segment.
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            Selection of reliable reference genes for gene expression studies in peach using real-time PCR

            Background RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach. Results In this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results. Conclusion Our study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.
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              High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.

              Isolation of unknown DNA sequences flanked by known sequences is an important task in molecular biology research. Thermal asymmetric interlaced PCR (TAIL-PCR) is an effective method for this purpose. However the success rate of the original TAIL-PCR needs to be increased, and it is more desirable to obtain target products with larger sizes. Here we present a substantially improved TAIL-PCR procedure with special primer design and optimized thermal conditions. This high-efficiency TAIL-PCR (hiTAIL-PCR) combines the advantages of the TAIL-cycling and suppression-PCR, thus it can block the amplification of nontarget products and suppress small target ones, but allow efficient amplification of large target sequences. Using this method, we isolated genomic flanking sequences of T-DNA insertions from transgenic rice lines. In our tests, the success rates of the reactions were higher than 90%, and in most cases the obtained major products had sizes of 1-3 kb.
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                Author and article information

                Journal
                J Exp Bot
                J. Exp. Bot
                jexbot
                exbotj
                Journal of Experimental Botany
                Oxford University Press (UK )
                0022-0957
                1460-2431
                April 2016
                5 February 2016
                5 February 2016
                : 67
                : 6
                : 1993-2005
                Affiliations
                1Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Sino-African Joint Research Center, Wuhan Botanical Garden of the Chinese Academy of Sciences , Wuhan 430074, China
                2Graduate University of Chinese Academy of Sciences , 19A Yuquanlu, Beijing 100049, China
                3Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University , Bangkok 10330, Thailand
                4College of Horticulture Science and Engineering, Shandong Agricultural University , Tai-An, Shandong 271018, China
                Author notes
                * To whom correspondence should be addressed. E-mail: yphan@ 123456wbgcas.cn

                Editor: Gerhard Leubner, Royal Holloway, University of London

                Article
                10.1093/jxb/erw021
                4783375
                26850878
                5b708063-4252-48ff-bdaa-5e2f20b62770
                © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Page count
                Pages: 13
                Categories
                Research Paper

                Plant science & Botany
                copy number variation,flesh texture,melting flesh,peach,polygalacturonase,stone adhesion.

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