Name: an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants
Uploaded: 2017-11-08T15:00:00
Description: Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

Finansieringen af dette arbejde er fastsat i en del af et tilskud fra NSF plante genomforskning (IOS-1127155).

Bemærk: figur 1 viser de fem trin til array CGH protokol. De er: 1) forberedelse af plantematerialer; 2) isolering af høj kvalitet DNA prøver; 3) mærkningsbestemmelserne og oprensning af DNA-prøver; 4) hybridisering, vask, og scanning af hele genom arrays; og 5) CGH dataanalyse. M. truncatula samlede genom tiling arrays indeholder ialt 971,041 unikke oligo prober rettet mod mere end 50.000 gener eller gen modeller i genomet (Se tabel af materialer). De unikke...

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	<li>Tang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+genome+release+(version+Mt4.0)+for+the+model+legume+Medicago+truncatula.">An improved genome release (version Mt4.0) for the model legume Medicago truncatula.</a> BMC Genomics. 15, 312 (2014).</li><li>Young, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+genome+provides+insight+into+the+evolution+of+rhizobial+symbioses.">The Medicago genome provides insight into the evolution of rhizobial symbioses.</a> Nature. 480 (7378), 520-524 (2011).</li><li>Wang, H., Li, G., Chen, R., da Silva, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+neutron+bombardment+(FNB)+induced+deletion+mutagenesis+for+forward+and+reverse+genetic+studies+in+plants.">Fast neutron bombardment (FNB) induced deletion mutagenesis for forward and reverse genetic studies in plants.</a> Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues. , 629-639 (2006).</li><li>Rogers, C., Wen, J., Chen, R., Oldroyd, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion-based+reverse+genetics+in+Medicago+truncatula.">Deletion-based reverse genetics in Medicago truncatula.</a> Plant Physiol. 151 (3), 1077-1086 (2009).</li><li>Alonso, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+components+of+the+ethylene-response+pathway+identified+in+a+screen+for+weak+ethylene-insensitive+mutants+in+Arabidopsis.">Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis.</a> Proc Natl Acad Sci U S A. 100 (5), 2992-2997 (2003).</li><li>Silverstone, A. L., Ciampaglio, C. N., Sun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+RGA+gene+encodes+a+transcriptional+regulator+repressing+the+gibberellin+signal+transduction+pathway.">The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway.</a> Plant Cell. 10 (2), 155-169 (1998).</li><li>Li, X., Lassner, M., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleteagene:+a+fast+neutron+deletion+mutagenesis-based+gene+knockout+system+for+plants.">Deleteagene: a fast neutron deletion mutagenesis-based gene knockout system for plants.</a> Comp Funct Genomics. 3 (2), 158-160 (2002).</li><li>Bolon, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+genomic+analyses+of+a+fast+neutron+mutant+population+resource+in+soybean.">Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean.</a> Plant Physiol. 156 (1), 240-253 (2011).</li><li>Men, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Neutron+Mutagenesis+of+Soybean+(Glycine+soja+L.)+Produces+a+Supernodulating+Mutant+Containing+a+Large+Deletion+in+Linkage+Group+H.">Fast Neutron Mutagenesis of Soybean (Glycine soja L.) Produces a Supernodulating Mutant Containing a Large Deletion in Linkage Group H.</a> Genome Letters. 1 (3), 147-155 (2002).</li><li>Hoffmann, D., Jiang, Q., Men, A., Kinkema, M., Gresshoff, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodulation+deficiency+caused+by+fast+neutron+mutagenesis+of+the+model+legume+Lotus+japonicus.">Nodulation deficiency caused by fast neutron mutagenesis of the model legume Lotus japonicus.</a> J Plant Physiol. 164 (4), 460-469 (2007).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+neutron+deletion+mutagenesis-based+reverse+genetics+system+for+plants.">A fast neutron deletion mutagenesis-based reverse genetics system for plants.</a> Plant J. 27 (3), 235-242 (2001).</li><li>Bourcy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicago+truncatula+DNF2+is+a+PI-PLC-XD-containing+protein+required+for+bacteroid+persistence+and+prevention+of+nodule+early+senescence+and+defense-like+reactions.">Medicago truncatula DNF2 is a PI-PLC-XD-containing protein required for bacteroid persistence and prevention of nodule early senescence and defense-like reactions.</a> New phytol. 197 (4), 1250-1261 (2013).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+dissected+leaf+morphology+by+a+Cys(2)His(2)+zinc+finger+transcription+factor+in+the+model+legume+Medicago+truncatula.">Control of dissected leaf morphology by a Cys(2)His(2) zinc finger transcription factor in the model legume Medicago truncatula.</a> Proc Natl Acad Sci U S A. 107 (23), 10754-10759 (2010).</li><li>Ge, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+seed+size+and+quality+by+manipulating+BIG+SEEDS1+in+legume+species.">Increasing seed size and quality by manipulating BIG SEEDS1 in legume species.</a> Proc Natl Acad Sci U S A. 113 (44), 12414-12419 (2016).</li><li>Kalo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodulation+signaling+in+legumes+requires+NSP2,+a+member+of+the+GRAS+family+of+transcriptional+regulators.">Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators.</a> Science. 308 (5729), 1786-1789 (2005).</li><li>Oldroyd, G. E., Long, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+nodulation-signaling+pathway+2,+a+gene+of+Medicago+truncatula+involved+in+Nod+actor+signaling.">Identification and characterization of nodulation-signaling pathway 2, a gene of Medicago truncatula involved in Nod actor signaling.</a> Plant Physiol. 131 (3), 1027-1032 (2003).</li><li>Peng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+compound+leaf+development+in+Medicago+truncatula+by+fused+compound+leaf1,+a+class+M+KNOX+gene.">Regulation of compound leaf development in Medicago truncatula by fused compound leaf1, a class M KNOX gene.</a> Plant Cell. 23 (11), 3929-3943 (2011).</li><li>Tsujimoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+TOBAMOVIRUS+MULTIPLICATION+(TOM)+2+locus+encodes+a+transmembrane+protein+that+interacts+with+TOM1.">Arabidopsis TOBAMOVIRUS MULTIPLICATION (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1.</a> EMBO J. 22 (2), 335-343 (2003).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nodule-specific+protein+secretory+pathway+required+for+nitrogen-fixing+symbiosis.">A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis.</a> Science. 327 (5969), 1126-1129 (2010).</li><li>Bejjani, B. A., Shaffer, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+array-based+comparative+genomic+hybridization+to+clinical+diagnostics.">Application of array-based comparative genomic hybridization to clinical diagnostics.</a> J Mol Diagn. 8 (5), 528-533 (2006).</li><li>Emerson, J. J., Cardoso-Moreira, M., Borevitz, J. O., Long, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+selection+shapes+genome-wide+patterns+of+copy-number+polymorphism+in+Drosophila+melanogaster.">Natural selection shapes genome-wide patterns of copy-number polymorphism in Drosophila melanogaster.</a> Science. 320 (5883), 1629-1631 (2008).</li><li>Gong, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+rapid+cloning+of+an+ion+accumulation+deletion+mutant+in+Arabidopsis+thaliana.">Microarray-based rapid cloning of an ion accumulation deletion mutant in Arabidopsis thaliana.</a> Proc Natl Acad Sci U S A. 101 (43), 15404-15409 (2004).</li><li>Guryev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+functional+impact+of+DNA+copy+number+variation+in+the+rat.">Distribution and functional impact of DNA copy number variation in the rat.</a> Nat Genet. 40 (5), 538-545 (2008).</li><li>Haun, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+composition+and+origins+of+genomic+variation+among+individuals+of+the+soybean+reference+cultivar+Williams+82.">The composition and origins of genomic variation among individuals of the soybean reference cultivar Williams 82.</a> Plant Physiol. 155 (2), 645-655 (2011).</li><li>Infante, J. J., Dombek, K. M., Rebordinos, L., Cantoral, J. M., Young, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+amplifications+caused+by+chromosomal+rearrangements+play+a+major+role+in+the+adaptive+evolution+of+natural+yeast.">Genome-wide amplifications caused by chromosomal rearrangements play a major role in the adaptive evolution of natural yeast.</a> Genetics. 165 (4), 1745-1759 (2003).</li><li>Jones, M. R., Maydan, J. S., Flibotte, S., Moerman, D. G., Baillie, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide+Array+Comparative+Genomic+Hybridization+(oaCGH)+based+characterization+of+genetic+deficiencies+as+an+aid+to+gene+mapping+in+Caenorhabditis+elegans.">Oligonucleotide Array Comparative Genomic Hybridization (oaCGH) based characterization of genetic deficiencies as an aid to gene mapping in Caenorhabditis elegans.</a> BMC Genomics. 8, 402 (2007).</li><li>Lakshmi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+genomic+representational+oligonucleotide+microarray+analysis:+detection+of+copy+number+variations+in+normal+and+tumor+specimens.">Mouse genomic representational oligonucleotide microarray analysis: detection of copy number variations in normal and tumor specimens.</a> Proc Natl Acad Sci U S A. 103 (30), 11234-11239 (2006).</li><li>Mitra, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Ca2+/calmodulin-dependent+protein+kinase+required+for+symbiotic+nodule+development:+Gene+identification+by+transcript-based+cloning.">A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning.</a> Proc Natl Acad Sci U S A. 101 (13), 4701-4705 (2004).</li><li>Rios, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemizygous+deletions+in+citrus+using+array-comparative+genomic+hybridization+and+microsynteny+comparisons+with+the+poplar+genome.">Characterization of hemizygous deletions in citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome.</a> BMC Genomics. 9, 381 (2008).</li><li>Skvortsov, D., Abdueva, D., Stitzer, M. E., Finkel, S. E., Tavare, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+expression+arrays+for+copy+number+detection:+an+example+from+E.+coli.">Using expression arrays for copy number detection: an example from E. coli.</a> BMC Bioinformatics. 8, 203 (2007).</li><li>Werner, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+locus+mapping+and+DNA+array+hybridization+identify+an+FLM+deletion+as+a+cause+for+natural+flowering-time+variation.">Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation.</a> Proc Natl Acad Sci U S A. 102 (7), 2460-2465 (2005).</li><li>Xi, J., Chen, Y., Nakashima, J., Wang, S. M., Chen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicago+truncatula+esn1+defines+a+genetic+locus+involved+in+nodule+senescence+and+symbiotic+nitrogen+fixation.">Medicago truncatula esn1 defines a genetic locus involved in nodule senescence and symbiotic nitrogen fixation.</a> Mol Plant Microbe Interact. 26 (8), 893-902 (2013).</li><li>Schnabel, E., Journet, E. P., de Carvalho-Niebel, F., Duc, G., Frugoli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+truncatula+SUNN+gene+encodes+a+CLV1-like+leucine-rich+repeat+receptor+kinase+that+regulates+nodule+number+and+root+length.">The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase that regulates nodule number and root length.</a> Plant Mol Biol. 58 (6), 809-822 (2005).</li><li>Horvath, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+nodule-specific+cysteine+rich+peptide,+NCR169,+abolishes+symbiotic+nitrogen+fixation+in+the+Medicago+truncatula+dnf7+mutant.">Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.</a> Proc Natl Acad Sci U S A. 112 (49), 15232-15237 (2015).</li><li>Kim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+antimicrobial+peptide+essential+for+bacterial+survival+in+the+nitrogen-fixing+symbiosis.">An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis.</a> Proc Natl Acad Sci U S A. 112 (49), 15238-15243 (2015).</li><li>. Medicago truncatula Mutant Database Available from: <a target="_blank" href="https://medicago-mutant.noble.org/mutant/FNB.php">https://medicago-mutant.noble.org/mutant/FNB.php</a> (2017)</li><li>Burton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+with+the+next+generation+of+CGH+microarray.">SNP genotyping with the next generation of CGH microarray.</a> MLO Med Lab Obs. 45 (7), (2013).</li></ol>

Vi har udviklet en array-baserede CGH platform til påvisning og karakterisering af hurtig neutron bombardement (FNB)-induceret mutanter i M. truncatula cv. Jemalong A17. For at demonstrere brugen af array CGH metode til at opdage genmutationer, udførte vi aCGH analyse af mutant FN6191, som udstillede en hyper-nodulation fænotype i modsætning til vildtype planter, når podet med S. meliloti Sm1021. Segmenteringsanalyse, et segment var anses for væsentlig, hvis log2 forhold betyde af sonde...

Bælgplanter (ærteblomstfamilien) er den tredjestørste familie af dækfrøede planter, med mange økonomisk vigtige arter såsom sojabønner (Glycine max) og lucerne (Medicago sativa). Bælgplanter planter kan interagere med kvælstofbindende jord bakterier, generelt kaldet Rhizobia at udvikle rodknolde, hvor de atmosfæriske dinitrogenoxider er reduceret til ammoniak til brug af anlægget vært. Som sådan, dyrkning af bælgplanter afgrøder kræver lidt input af kvælstofgødning og dermed bidrager til bæredygtigt landbrug. Bælgplanter afgrøder producere blade og frø med højt proteinindhold, der tjener som udmærkede foder og korn afgrøder. Dyrkede bælgplanter arter har dog generelt kompleks genom strukturer, at funktionelle studier af gener, der spiller vigtige roller i bælgplanter-specifikke processer besværlige. Medicago truncatula er blevet bredt vedtaget som en model art for bælgplanter undersøgelser, primært fordi (1) det har en diploide genom med en relativt lille haploide genom størrelse (~ 550 Mbps); (2) planter kan blive stabilt omdannet til gen funktionelle studier; og (3) når det er nært beslægtet med Lucerne (M. sativa), Dronningen af foderafgrøder og mange andre økonomisk vigtige afgrøder for Translationel undersøgelser. For nylig, genomet sekvens af M. truncatula cv Jemalong A17 har været frigivet1,2. Anmærkning af genom viser, at der er mere end 50.000 forudsagte gener eller gen modeller i genomet. For at bestemme funktionen af de fleste af gener i M. truncatula er genom en udfordrende opgave. For at lette funktionelle studier af gener, der er oprettet en omfattende samling af mutanter i rækken af mere end 150.000 M1 linjer ved hjælp af hurtig neutron bombardement (FNB) mutagenese i M. truncatula cv Jemalong A173 ,4. Hurtig neutron, en høj energi ionisering mutagen, har været brugt til at generere mutanter i mange plantearter, herunder Arabidopsis5,6, ris (Oryza sativa)7, tomat (Solanum lycopersicum), sojabønner (Glycine soja; G. max)8,9, byg (Hordeum vulgare) og Lotus japonicus10. En stor del af mutationer afledt af FNB mutagenese skyldes DNA sletninger der spænder i størrelse fra et par basepar til mega basepar9,11. Mange fænotype-associerede gener er blevet identificeret og karakteriseret4,12,13,14,15,16, 17 , 18 , 19. tidligere, Molekylær kloning af de underliggende gener fra FNB mutanter har påberåbt sig en kort-baseret tilgang, som er tidskrævende og begrænser antallet af mutanter at være kendetegnet på molekylært niveau. For nylig, flere gratis metoder herunder udskrift-baserede metoder, genom flisebelægning array-baserede komparativ genomisk hybridisering (CGH) for DNA copy number variation påvisning og hele genome sequencing, har været ansat til at lette de karakterisering af sletning mutanter i forskellige organismer, herunder dyr og planter20,21,22,23,24,25, 26,27,28,29,30,31.
For at lette karakterisering af FNB mutanter i M. truncatula, en helhed-genom array-baserede komparativ genomisk hybridisering (CGH) er platform udviklet og valideret. Som rapporteret i animalsk systemer, array-baserede CGH platform giver mulighed for påvisning af kopi nummer variationer (CNVs) på det samlede genom niveau i M. truncatula FNB mutanter. Derudover læsioner kan bekræftes ved PCR og sletning grænser kan identificeres af sekventering. Samlet set er array CGH platform et effektivt værktøj til at identificere læsioner i M. truncatula FNB mutanter. Her, er array CGH procedure og PCR karakterisering af sletning grænser i en M. truncatula FNB mutant illustreret.
Følgende protokol giver eksperimenterende trin og oplysninger om reagenser, udstyr og værktøjer til analyse for forskere, der er interesseret i at foretage samlede genom array-baserede komparativ genomisk hybridisering (CGH) analyse af kopi nummer variationer i planter. Som et eksempel, blev Medicago truncatula FN6191 mutant brugt til at identificere sletning regioner og kandidat gener forbundet med mutant fænotyper. M. truncatula FN6191 mutant, oprindeligt isoleret fra en hurtig neutron bombardement-induceret sletning mutant samling32 (Se Tabel af materialer), udstillet en hyper-nodulation fænotype efter podning med jorden bakterien, Sihorhizobium meliloti Sm1021, i modsætning til vildtype planter.

<table>
	<tbody>
		<tr>
			<td>Medicago truncatula genome array, 1 x 1 M</td>
			<td>Agilent</td>
			<td>G4123A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula FN6191 (mutant)</td>
			<td>In house</td>
			<td>FN6191</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula Jemalong A17 (reference)</td>
			<td>In house</td>
			<td>A17</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>1000D</td>
			<td></td>
		</tr>
		<tr>
			<td>SureTag DNA Labeling Kit</td>
			<td>Agilent</td>
			<td>5190-3400</td>
			<td></td>
		</tr>
		<tr>
			<td>Random primer</td>
			<td>Agilent</td>
			<td>5190-3399</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>MJ research</td>
			<td>PTC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Labnet international Inc</td>
			<td>Spectrafuge 24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and Drying Solution</td>
			<td>Agilent</td>
			<td>5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Hybridization Kit</td>
			<td>Agilent</td>
			<td>5188-5380</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber gasket slides</td>
			<td>Agilent</td>
			<td>G2505</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Agilent</td>
			<td>5190-3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Wash Buffer 1 and 2</td>
			<td>Agilent</td>
			<td>5188-5221</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber, stainless</td>
			<td>Agilent</td>
			<td>G2534A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Agilent</td>
			<td>G2545A</td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Columns</td>
			<td>Agilent</td>
			<td>5190-3391</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanner</td>
			<td>Roche</td>
			<td>MS200</td>
			<td></td>
		</tr>
		<tr>
			<td>NimbleScan 2.6</td>
			<td>Roche Nimblegen</td>
			<td>5225035001</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal Map 1.9</td>
			<td>Roche Nimblegen</td>
			<td>Signalmap1.9</td>
			<td></td>
		</tr>
	</tbody>
</table>

an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants

Mutanter er uvurderlige genetiske ressourcer for gen funktion undersøgelser. For at generere mutant samlinger, kan tre typer af mutagener udnyttes, herunder biologiske såsom T-DNA eller transposon, kemiske såsom ethyl methanesulfonate (EMS), eller fysisk såsom ioniserende stråling stråling. Typen af mutation observeret varierer afhængigt af den mutagen anvendes. For ioniserende stråling stråling induceret mutanter omfatter mutationer sletning, kopiering eller omlægning. Mens T-DNA eller transposon-baserede mutagenese er begrænset til arter, der er modtagelige for forandring, kan kemiske eller fysiske mutagenese anvendes til en bred vifte af arter. Men karakterisering af mutationer afledt af kemiske eller fysiske mutagenese traditionelt bygger på en kort-baseret kloning tilgang, der er arbejdskraft intensiv og tidskrævende. Her viser vi, at en high-density genom array-baserede komparativ genomisk hybridisering (aCGH) platform kan anvendes til effektivt at afsløre og karakterisere kopi nummer variationer (CNVs) i mutanter afledt af hurtig neutron bombardement (FNB) mutagenese i Medicago truncatula, en bælgplante arter. Hele genomet Sekvensanalyse viser, at der er mere end 50.000 gener eller gen modeller i M. truncatula. På nuværende, FNB-induceret mutanter i M. truncatula er afledt af mere end 150.000 M1 linjer, der repræsenterer uvurderlige genetiske ressourcer for funktionelle studier af gener i genomet. ACGH platformen beskrevet her er et effektivt redskab til kendetegner FNB-induceret mutanter i M. truncatula.

Figur 2 viser fordeling af normaliserede log2 nøgletal mutant versus WT signaler på tværs af den samlede genom. Analyse af CGH data viste en omtrentlig 22 kb sletning på kromosom 4, der omfatter hele SUNN gen33 og flere andre kommenteret gener i FN6191 mutant (figur 2, figur 3). Kandidat slettede regionen var omfattet af 73 træk sonder på matrixen med...

Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Denne protokol giver eksperimenterende trin og oplysninger om reagenser, udstyr og værktøjer til analyse for forskere, der er interesseret i gennemføre samlede genom array-baserede komparativ genomisk hybridisering (CGH) analyse af kopi nummer variationer i planter.

En Array-baserede komparativ genomisk hybridisering Platform for effektiv påvisning af kopi antal variationer i hurtig Neutron-induceret Medicago truncatula mutanter

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

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An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

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