Physical localization of highly nutritional protein coding AmA1 gene in amaranths through molecular cytogenetic tools
TL;DR: A rapid and reliable method for physical localization of single gene on plant chromosome was described and both the methods for in situ localization i.e. PRINS and C-PRINS were compared.
Abstract: — A highly nutritional protein coding gene AmA1 of Amaranthus hypochondriacus was searched in different species of Amaranthus by using various molecular biological techniques. PCR, Southern hybridization, primed in situ labeling (PRINS) and Cycling-primed in situ labeling (C-PRINS) confirmed the presence of AmA1 coding region only in Amaranthus hypochondriacus. The AmA1 gene was successfully localized on interphase nuclei and also on the metaphase chromosomes. The single copy, unique AmA1 gene was found to be present in the subtelomeric region of two homologous chromosomes in the group of 2-2.2 μm in length. In the present study, a rapid and reliable method for physical localization of single gene on plant chromosome was described and both the methods for in situ localization i.e. PRINS and C-PRINS were compared.
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TL;DR: A direct cycling primed in situ (C-PRINS) labeling technique for chromosome mapping of cultivated strawberry was developed and it was found that a maceration time of 25 min was appropriate for obtaining many somatic cells with 56 chromosomes and that 45% acetic acid was effective for reducing the amount of damage to the chromosomes and obtaining clear chromosome images.
Abstract: Few studies have aimed to develop physical chromosome mapping methods for cultivated strawberry (Fragaria×ananassa). Recently, linkage maps have been developed in this species using many single sequence repeat (SSR) markers. However, the accuracy of these needs to be evaluated because cultivated strawberries are allo-octoploids. This could be achieved by fluorescently labeling chromosomes that contain SSR markers on the same linkage group and observing whether other fluorescent signals can also be detected on the same chromosomes. The aim of this study was to develop a direct cycling primed in situ (C-PRINS) labeling technique for chromosome mapping of cultivated strawberry. To achieve this, we investigated the effect of the 1) maceration time for softening the root tips, 2) concentration of acetic acid for spreading chromosomes on the glass slide, 3) incubation time after slide preparation and before PRINS labeling, 4) number of PCR cycles for obtaining fluorescent signals, and 5) temperature accuracy for PCR. All of these factors were considered important for developing a chromosome labeling method. We found that a maceration time of 25 min was appropriate for obtaining many somatic cells with 56 chromosomes and that 45% acetic acid was effective for reducing the amount of damage to the chromosomes and obtaining clear chromosome images. A 72-h incubation time was appropriate for detecting chromosomes that had fluorescent signals, and the number of signals on the chromosomes increased with an increase in the number of PCR cycles. Finally, an aluminum tape treatment was effective for maintaining the PCR temperatures.
2 citations
Cites methods from "Physical localization of highly nut..."
...In particular, some studies have detected the localization of single copy sequence in situ using C-PRINS (Zhu et al. 1995, Mukai and Appels 1996, Kubaláková and Doležel 1998, Kaczmarek et al. 2007, Tui and Roy 2008, Talia et al. 2011)....
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TL;DR: The cleavage amplified polymorphic sequence (CAPS) marker was confirmed to have a chromosome specificity by chromosome observation using a primed in situ (PRINS) technique.
Abstract: The cleavage amplified polymorphic sequence (CAPS) markers were developed to identify strawberry cultivars from unknown samples. These markers seemed to have a chromosome specificity. However, no reports confirmed it. The present study was conducted to verify the location of a CAPS marker on the chromosomes using a primed in situ (PRINS) technique. A CAPS marker was hybridized with the ‘Sachinoka’ chromosomes using the PRINS technique. The samples were observed under a fluorescence microscope. When the chromosomes were hybridized with the single marker, fluorescent signals were found on two chromosomes. From the results, the CAPS marker was confirmed to have a chromosome specificity by chromosome observation.
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01 Apr 2017
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15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: The topic of this report is rap,d m,croscale methods for,solat,on of plant D N A without tile use of ul tracentr ,fugatlon wEth CsCI, which is of moderately high molecular weight and serves as a satisfactory substrate for most restrlctum cndonucleases and is statable for genom,c blot analys,s.
Abstract: The topic of this report is rap,d m,croscale methods for ,solat,on of plant D N A without tile use of ul tracentr ,fugatlon wEth CsCI. The D N A produced ,s of moderately high molecular weight and serves as a satisfactory substrate for most restrlctum cndonucleases and is statable for genom,c blot analys,s. In addi t ion to the rapidi ty and convenience of mlmpreps which permit a large number of samples to be processed in just a few hours, the small amount of tissue reqmred (less than 1.0 grams) allows tbr molecular analysis of plants at a very young stage Mm,prep D N A y,elds from leaf tissue of most species tested to date are typ,cally 30-100 big per gram tissue, greater than 50 kb, and remarkably uniform from sample to sample. The first mmlprep procedure we reported fi3r maize D N A isolation (Dellaporta et al , ;'*l,;tze Geneta3 Cr162162 Neu'_~letlrt. 1983) was adapted from a procedure commonly used for }'east D N A preparatmn (Dav,s et al. , 1980) Since th,s report, numerous personal commun,cat ,ons have demonstrated that the mm,prep procedure or a modification thereof, can be apphed to most plant species tested. For example, the method has been successfully used on Ntcottana hlgl~um. N. plumklgmgidtum. N. 3)/t'eJtrt~. L)s~opertcum sp.. Amar,mthm sp . Gl)~me max. Petuma h.~hra&. Several modifications have been apphed by these ,nvestlgators and in our own laboratory m order to extend the appl ,catmn of ram,prep procedures to other plant species. The select,on of a part icular protocol depends to a large degree on the plant spec,es used. However, the procedure reported here was selected to be statable for most situations.
7,263 citations
"Physical localization of highly nut..." refers methods in this paper
...Isolation of genomic DNA - Genomic DNA was isolated from young leaves of both leafy vegetables and grain amaranths using a protocol modified from DELLAPORTA et al. (1983)....
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TL;DR: It is demonstrated that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L.) chromosomes by FISH, demonstrating the utility of FISH in plant genome analysis.
Abstract: Fluorescence in situ hybridization (FISH) is a powerful tool for physical mapping in human and other mammalian species However, application of the FISH technique has been limited in plant species, especially for mapping single- or low-copy DNA sequences, due to inconsistent signal production in plant chromosome preparations Here we demonstrate that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L) chromosomes by FISH Repetitive DNA sequences in BAC clones can be suppressed efficiently by using rice genomic DNA as a competitor in the hybridization mixture BAC clones as small as 40 kb were successfully mapped To demonstrate the application of the FISH technique in physical mapping of plant genomes, both anonymous BAC clones and clones closely linked to a rice bacterial blight-resistance locus, Xa21, were chosen for analysis The physical location of Xa21 and the relationships among the linked clones were established, thus demonstrating the utility of FISH in plant genome analysis
344 citations
"Physical localization of highly nut..." refers background in this paper
...…(FISH) has been extensively used to visualize DNA sequences in interphase nuclei (DONG and JIANG 1998), mitotic and meiotic chromosomes (NENNO et al. 1994; PEDERSEN et al. 1995) as well as on extended DNA fibres (DE JONG et al. 1999) and bacterial artificial chromosomes (JIANG et al. 1995)....
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TL;DR: A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.
Abstract: Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for...
270 citations
"Physical localization of highly nut..." refers methods in this paper
...It has been reported that localization of DNA sequences shorter than 10 kb on plant chromosomes using ISH or FISH is still very difficult (JIANG and GILL 1994)....
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TL;DR: In an attempt to improve the nutritional value of potato, the AmA1 coding sequence was successfully introduced and expressed in tuber-specific and constitutive manner and there was a striking increase in the growth and production of tubers in transgenic populations and also the total protein content with an increase in most essential amino acids.
Abstract: Improvement of nutritive value of crop plants, in particular the amino acid composition, has been a major long-term goal of plant breeding programs. Toward this end, we reported earlier the cloning of the seed albumin gene AmA1 from Amaranthus hypochondriacus. The AmA1 protein is nonallergenic in nature and is rich in all essential amino acids, and the composition corresponds well with the World Health Organization standards for optimal human nutrition. In an attempt to improve the nutritional value of potato, the AmA1 coding sequence was successfully introduced and expressed in tuber-specific and constitutive manner. There was a striking increase in the growth and production of tubers in transgenic populations and also of the total protein content with an increase in most essential amino acids. The expressed protein was localized in the cytoplasm as well as in the vacuole of transgenic tubers. Thus we have been able to use a seed albumin gene with a well-balanced amino acid composition as a donor protein to develop a transgenic crop plant. The results document, in addition to successful nutritional improvement of potato tubers, the feasibility of genetically modifying other crop plants with novel seed protein composition.
229 citations
"Physical localization of highly nut..." refers methods in this paper
...The AmA1 coding region was amplified by PCR using forward primer F51 (5’-CACCATGGCGGGATTACCAGTG3’) and reverse primer R1044 (5’-CAAGGAAGAACCCTCTTGTTTCC-3’) as reported by CHAKRABORTY et al. (2000)....
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...3 plasmid using EcoRI restriction enzyme according to the map provided by CHAKRABORTY et al. (2000), gel was eluted using Qiagen gel elution kit (Qiagen, Valencia, CA, USA) and labeled with [a-32P] dCTP (Perkin Elmer) using ‘‘Ready Prime’’ random labeling system (Amersham Biosciences)....
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