Development Of Oligonucleotide Probes Using Wheat A-genome Sequencing Reads And Application In Identifying Wheat-thinopyrum Bessarabicum Alien Chromosome Introgression Lines

Oligonucleotide(as oligo hereafter)as probe in FISH is a new but powerful tool for chromosome identification.Oligo has many advantages such as shorter sequence,high invading ability,easy synthesis and cost efficient,thus it has been used in many species replacing the traditional plasmid clones and PCR amplification.Meanwhile,the single strand oligos as probes do not need probe and chromosome denaturation,thus the FISH procedure can be greatly simplified and it becomes the new generation of cytological markers for chromosome identification and engineering.Several oligos multiplex have been successfully developed by Cytogenetics Institute,Nanjing Agricultural University and used in chromosome identification of wheat,Thinopyrum bessarabicum and rye.The current oligo multiplex could distinguish all wheat chromosomes after one round FISH,however,some A genome chromosomes are still difficult to be distinguished due to the lack of specific markers.In this study,4.6 Gb A genome reads were obtained after genome sequencing of einkorn(Triticum monococcum L.2n=14 AA),and 100 repeat sequences were identified for developing A genome-specific oligos.FISH analysis showed that 16 oligos after random primers or tailing reaction labeling generated hybridization signals,but only 2 oligos produced stable and clear signals after end modification using FAM or TAMRA,where oligo-DPYL36916 produced signals on 16 wheat chromosomes including 1A and 7A with specific signals,while oligo-DPYL35874 produced overlapped signals with that of oligo-pAsl,which could replaced the roles of oligo-pAsl to identifyD-genome chromosomes.This provides new markers for chromosome identification in wheat and new information for developing A genome-specific oligos in future.Using the multiplex#4 and oligo-DPYL36916,wheat landrace Chinese Sprng(CS),CS-Thinopyrum bessarabicum amphiploid and six wheat-Th.bessarabicum chromosome introgression lines including DS1J(1B),T2JS-2BS-2BL,DA3JS-4JL,DS5J(5A),DS6J(6A)and T6JS·2JL were clearly identified and oligopainting karyotypes of CS andTh.bessarabicum were developed which allowed to distinguish five homoeologous chromosomes of Th.bessarabicum except 3J and 7J.Using this oligo multiplex,one isoarm chromosome i5JL-5JL and one large segmental translocation line T1BL-1JL·1JS were identified.Additionally,total 20 chromosome aberrations involving 1J were prelimimarily developed after molecular and cytological analysis,which provides genetic matrials for further transferring and application of beneficial genes of 1J.