Bestemmelse af selv- og inter-(in)kompatibilitetsforhold i abrikos, der kombinerer håndbestøvning, mikroskopi og genetiske analyser
Summary
Vi præsenterer en metode til at fastsætte bestøvningskravene for abrikos(Prunus armenienca L.) sorter, der kombinerer bestemmelse af selv-(in)kompatibilitet ved fluorescensmikroskopi med identifikation af S-genotype ved PCR-analyse.
Abstract
Selv-uforenelighed i Rosaceae bestemmes af en Gametophytic Self-Incompatibility System (GSI), der hovedsagelig kontrolleres af den multiallelic locus S. I abrikos er det stadig vigtigere at bestemme selv- og inter-(in)kompatibilitetsforhold, da frigivelsen af et betydeligt antal nye sorter har resulteret i en stigning i sorter med ukendte bestøvningskrav. Her beskriver vi en metode, der kombinerer bestemmelse af selv-(in)kompatibilitet ved håndbestøvninger og mikroskopi med identifikation af S-genotype ved PCR-analyse. For selv-(in) kompatibilitet bestemmelse, blomster på ballon fase fra hver sort blev indsamlet i marken, hånd-bestøvet i laboratoriet, fast, og farves med anilin blå til observation af pollen rør adfærd under fluorescens mikroskopi. Med henblik på etablering af uforenelighedsrelationer mellem sorter blev DNA fra hver sort Sudvundet af unge blade, og S-alleler blev identificeret ved PCR. Denne fremgangsmåde gør det muligt at etablere uforenelighed grupper og belyse uforenelighed relationer mellem sorter, som giver en værdifuld information til at vælge egnede bestøvere i udformningen af nye plantager og til at vælge passende forældre i avlsprogrammer.
Introduction
Selv-uforenelighed er en strategi for blomstrende planter for at forhindre selvbestøvning og fremme outcrossing1. I Rosaceae bestemmes denne mekanisme af et Gametophytic Self-Incompatibility System (GSI), der hovedsageligt styres af den multiallelic locus S2. I den stil, RNase genet koder S-stylar determinant, en RNase3, mens en F-box protein, som bestemmer S-pollen determinant, er kodificeret af SFB genet4. Samspillet mellem sig selvi kompatibilitet finder sted gennem hæmning af pollenrørvækst i stil med at forhindre befrugtning af ovule5,6.
I abrikos, en sort fornyelse har fundet sted på verdensplan i de sidste to årtier7,8. Denne indførelse af et stort antal nye sorter, fra forskellige offentlige og private avlsprogrammer, har resulteret i stigningen af abrikos sorter med ukendte bestøvning krav8.
Forskellige metoder er blevet anvendt til at bestemme bestøvning krav i abrikos. På marken kan selv-in-kompatibilitet etableres ved kontrollerede bestøvninger i burtræer eller i kastrerede blomster og derefter registrere procentdelen af frugtsæt9,10,11og12. Desuden er kontrollerede bestøvninger blevet udført i laboratoriet ved semi-in vivo kultur af blomster og analyse af pollen rør adfærd under fluorescens mikroskopi8,13,14,15,16,17. For nylig, molekylære teknikker, såsom PCR analyse og sekventering, har gjort det muligt karakterisering af uforenelighed relationer baseret på studiet af RNase og SFB gener18,19. I abrikos er der rapporteret 33 S-alleler (S1 til S20, S22 til S30, S52, S53, Sv, Sx), herunder en allel relateret til selvkompatibilitet (Sc)12,18,20,21,22,23,24. Indtil nu er 26 inkompatibilitetsgrupper blevet ukket ned i denne art i henhold til S-genotype S8,9,,17,25,26,27. Kultiønder med Sde samme S-alleler er indbyrdes uforenelige, mens sorter med mindst én anden S-allelog derfor fordelt i forskellige uforenelige grupper er indbyrdes kompatible.
For at definere kravene til bestøvning af abrikoskultstoffer beskriver vi en metode, der kombinerer bestemmelse af selv-(in)kompatibilitet ved fluorescensmikroskopi med identifikation af S-genotype ved PCR-analyse i abrikoskulterer. Denne fremgangsmåde gør det muligt at etablere uforenelighedsgrupper og belyse uforenelighedsrelationer mellem sorter.
Protocol
Representative Results
Discussion
Traditionelt var de fleste kommercielle abrikoseuropæiske sorter selvkompatible36. Ikke desto mindre har brugen af nordamerikanske selv uforenelige sorter som forældre i avlsprogrammer i de seneste årtier resulteret i frigivelsen af et stigende antal nye selv uforenelige sorter med ukendte bestøvningskrav7,8,37. Det bliver således stadig vigtigere at bestemme selv- og indbyrdes kompatibilitetsforhold…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev finansieret af Ministerio de Ciencia, Innovación y Universidades-European Regional Development Fund, Den Europæiske Union (AGL2016-77267-R og AGL2015-74071-JIN); Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RFP2015-00015-00, RTA2017-00003-00); Den Europæiske Socialfond for Gobierno de Aragón, Den Europæiske Union (Grupo Consolidado A12_17R), Fundación Biodiversidad og Agroseguro S.A.
Materials
| Agarose D1 Low EEO | Conda | 8010.22 | |
| BIOTAQ DNA Polymerase kit | Bioline | BIO-21060 | |
| Bright field microscope | Leica Microsystems | DM2500 | |
| CEQ System Software | Beckman Coulter | ||
| DNeasy Plant Mini Kit | QIAGEN | 69106 | |
| dNTP Set, 4 x 25 µmol | Bioline | BIO-39025 | |
| GenomeLab DNA Size Standard Kit – 400 | Beckman Coulter | 608098 | |
| GenomeLab GeXP Genetic Analysis System | Beckman Coulter | ||
| GenomeLab Separation Buffer | Beckman Coulter | 608012 | |
| GenomeLab Separation Gel LPA-1 | Beckman Coulter | 391438 | |
| HyperLadder 100bp | Bioline | BIO-33029 | |
| HyperLadder 1kb | Bioline | BIO-33025 | |
| Image Analysis System | Leica Microsystems | ||
| Molecular Imager VersaDoc MP 4000 system | Bio-Rad | 170-8640 | |
| NanoDrop One Spectrophotometer | Thermo Fisher Scientific | 13-400-518 | |
| pH-Meter BASIC 20 | Crison | ||
| Phusion High-Fidelity PCR Kit | Thermo Fisher Scientific | F553S | |
| Power Pack P 25 T | Biometra | ||
| Primer Forward | Isogen Life Science | ||
| Primer Reverse | Isogen Life Science | ||
| Quantity One Software | Bio-Rad | ||
| Stereoscopic microscope | Leica Microsystems | MZ-16 | |
| Sub-Cell GT | Bio-Rad | ||
| SYBR Safe DNA Gel Stain | Thermo Fisher Scientific | S33102 | |
| T100 Thermal Cycler | Bio-Rad | 1861096 | |
| Taq DNA Polymerase | QIAGEN | 201203 | |
| Vertical Stand Autoclave | JP Selecta |
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