Development And Application Of Oligonucleotide Probes In Maize,Barley And Wheat For Fluoresence In Situ Hybridization

Fluorescence in situ hybridization(FISH)is a critical technique of modern molecular cytogenetics which plays important roles in chromosome recognition,structure and function research,gene localization and expression analysis.Single stranded oligonucleotide(SSON)probes developed from the known repetitive and genomic sequences are simple,economical and efficient probes,which are gradually replacing the conventional plasmid clones and widely used in chromosome research.In this study,SSON probes of maize,barley and wheat have been developed or identified and used to form different SSON multiplexes for chromosome identification of these species after one round FISH or ND-FISH(Non-denaturing FISH),the major results are as following.Firstly,based on the known repeat sequences and genomic sequence of maize,10 SSON probes(ACT)5,(ACT)10,(ACT)19,Knob-1,Knob-2,Knob-3,CentC69-1,MR68-3,K10-72-1 and TR1-357-2 were successfully developed or indentified,which produced clear hybridization signals and could replace the roles of their conventional plasmid clones.At same conditions(ACT)10 and(ACT)19 produced clearer and more stable signals than(ACT)5.ND-FISH found that all these probes could produce expected signals in maize.Sequential ND-FISH and multi-color FISH determined that SSON probes MR68-3,K10-72-1 and TR1-357-2 were all located on the satellites of chromosome 6 and overlapped with signals of the 45S rDNA plasmid clones.Three Knob derived SSON probes(Knob-1,Knob-2 and Knob-3)all generated the same signal and can be used to distinguish different maize chromosomes.Based on the signal characteristics of single SSON,four SSON probes(ACT)10,Knob-2,CentC69-1 and MR68-3 were combined into a maize SSON multiplex probe Maize SSON#1,which could clearly distinguish all chromosomes of inbred line B73 and allowed to construct a high resolution standard karyotype of maize just after one round ND-FISH.This multiplex and reference karyotype were then used to analyze 16 maize inbred lines and hybrids,which allowed to develop high resolution karyotyes of all accessions and revealed rich signal patterns.All inbred lines,hybrids and different genotypes could be clearly differentiated,particularly for chromosome 6,which present most polymorphism and allowed to distinguish all inbred lines and different hybrids.Further analysis found that SSON-FISH signals of such probes were consistent with their presence in reference genomic sequences.Secondly,to identify effective SSON probes and construct high resolution barley karyotypes,52 probes previously developed in our lab were identified in barley.30 SSON probes could produce clear and stable signals on barley chromosomes,including 21 SSR-SSONs and 9 tandem repeats derived SSONs.According to the signal characteristics of single probe,9 probes including(GAA)10,pAs1-1,pAsl-4,AFA-3,AFA-4,Dig-45S rDNA,(GA)15,Bio-(TTA)7 and FAM-(TTTAGGG)8-FAM were used to form a multiplex probe Barely SSON#1.Through FISH analysis,all the chromosomes can be clearly distinguished and the high-resolution karyotype of barley 08-49 wild type was constructed for reference.Using this multiplex 22 barley varieties and 11 mutants were analyzed,and obvious chromosome polymorphisms were present.Among them,chromosome 4H showed the most polymorphic types(6),followed by 1H(5),2H(5),3H(4),5H(4),7H(4)and 6H(2),indicating rich variations occurred during barley cultivar evolution.However,no obvious chromosomal variation was found between the wild type and its mutants of barley 08-49,indicating invisible or gene mutation might have occurred in these different mutants.Finally,by using SSON multiplex probe Wheat SSON#4(containing TAMRA-modified pAsl-1,pAsl-3,pAsl-4,pAs1-6,AFA-3 and AFA-4 and FAM-modified pSc119.2-1 and(GAA)10)previously developed by Du et al.(2017),14 types of translocations or deletions were identified from 373 Chinese cultivars founders.In this study,4 reciprocal translocation and deletion types including RT 4AS·4AL-1DS/1DL·1DS-4AL(17Z056,Youmanghong18),De11B+T 1BS-3DS· 3DL(17Z172,Shannongfu63),RT 4AS·4AL-1BL/IBS· 1BL-4AL(17Z283,Xiangnong2170-7-27)and T 1RS/1BL+RT 2AL·2AS-5BL/5BS·5BL-2AS(17Z340,Xiangnong 153-27)were selected and further validated by sequential FISH and different genomic in situ hybridization(GISH),which not only accurately confirmed the chromosome identities of these lines but also reveal the clear breakpoints in corresponding chromosomes,indicating the potential of such SSON probes in identifying chromosomes of wheat.