Non-denaturing FISH (ND-FISH) technology has been widely used to study the chromosomes of Triticeae species because of its convenience. The oligo probes for ND-FISH analysis of wheat (Triticum aestivum L.) chromosomes are still limited. In this study, the whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of Chinese Spring (T. aestivum L.) were used to find new tandem repeats. One hundred and twenty oligo probes were designed according to the new tandem repeats and used for ND-FISH analysis of chromosomes of wheat Chinese Spring. Twenty nine of the 120 oligo probes produce clear or strong signals on wheat chromosomes. Two of the 29 oligo probes can be used to conveniently distinguish wheat A-, B-, and D-genome chromosomes. Sixteen of the 29 oligo probes only produce clear or strong signals on the subtelomeric regions of 1AS, 5AS, 7AL, 4BS, 5BS, and 3DS arms, on the telomeric regions of 1AL, 5AL, 2BS, 3BL, 6DS, and 7DL arms, on the intercalary regions of 4AL and 2DL arms, and on the pericentromeric regions of 3DL and 6DS arms. Eleven of the 29 oligo probes generate distinct signal bands on several chromosomes and they are different from those previously reported. In addition, the short and long arms of 6D chromosome have been confirmed. The new oligo probes developed in this study are useful and convenient for distinguishing wheat chromosomes or specific segments of wheat chromosomes.

Oligonucleotide probes that can be used for non-denaturing fluorescence in situ hybridization (ND-FISH) analysis are convenient tools for identifying chromosomes of wheat (Triticum aestivum L.) and its relatives. New oligonucleotide probes, Oligo-HvT01, Oligo-pTa71-1, Oligo-s120.1, Oligo-s120.2, Oligo-s120.3, Oligo-275.1, Oligo-275.2, Oligo-k566 and Oligo-713, were designed based on the repetitive sequences HVT01, pTa71, pTa-s120, pTa-275, pTa-k566 and pTa-713. All these probes can be used for ND-FISH analysis and some of them can be used to detect polymorphisms of wheat chromosomes. Probes Oligo-HvT01, Oligo-pTa71-1, Oligo-s120.3, Oligo-275.1, Oligo-k566 and Oligo-713 can, respectively, replace the roles of their original sequences to identify chromosomes of some barley (Hordeum vulgare ssp. vulgare) and the common wheat variety Chinese Spring. Oligo-s120.1, Oligo-s120.2 and Oligo-275.2 produced different hybridization patterns from the ones generated by their original sequences. In addition, Oligo-s120.1, Oligo-s120.2 and Oligo-s120.3, which were derived from pTa-s120, revealed different signal patterns. Likewise, Oligo-275.1 and Oligo-275.2, which were derived from pTa-275, also displayed different hybridization patterns. These results imply that differently arranged or altered structural statuses of tandem repeats might exist on different chromosome regions. These new oligonucleotide probes provide extra convenience for identifying some wheat and barley chromosomes, and they can display polymorphisms of wheat chromosomes.

Oligonucleotide probes and the non-denaturing fluorescence in situ hybridization (ND-FISH) technique are widely used to analyze plant chromosomes because they are convenient tools. New oligonucleotide probes, Oligo-Ku, Oligo-3B117.1, Oligo-3B117.2, Oligo-3B117.2.1, Oligo-3B117.3, Oligo-3B117.4, Oligo-3B117.5, Oligo-3B117.6, Oligo-pTa71A-1, Oligo-pTa71A-2, Oligo-pTa71B-1, Oligo-pTa71B-2, Oligo-pTa71C-1, Oligo-pTa71C-2, Oligo-pTa71C-3 and Oligo-pTa71D were designed based on the repetitive sequences KU.D15.15, pSc119.2-like sequence 3B117 and pTa71. Oligonucleotide probe (GT)7 was also used. Oligo-Ku and (GT)7 can be together used to identify Dasypyrum villosum from wheat chromosomes and to distinguish individual D. villosum chromosomes. The oligonucleotide probes that were derived from the same repeat sequence displayed different signal intensity and hybridization sites on the same chromosomes. Both the length and the nucleotide composition of oligonucleotide probes determined their signal intensity. For example, Oligo-3B117.2 (25 bp) and Oligo-pTa71A-2 (46 bp) produced the strongest signals on chromosomes of wheat (Triticum aestivum L.), rye (Secale cereale L.), barley (Hordeum vulgare ssp. vulgare) or D. villosum, the signal of Oligo-3B117.4 (18 bp) on the short arm of 7B chromosome was weaker than that of Oligo-3B117.2.1 (15 bp) and Oligo-3B117.3 (16 bp), and Oligo-pTa71A-1 (38 bp) produced the same strong signals as Oligo-pTa71A-2 did on 1B and 6B chromosomes, but its signals on 1R and 1V chromosomes were weaker than the ones of Oligo-pTa71A-2. Oligonucleotide probes and ND-FISH analysis can reflect the distribution and structural statues of different segments of tandem repeats on chromosomes. The possible reasons why different segments derived from the same repeat sequence produced different signal patterns are discussed.

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Thinopyrum has been widely used to improve wheat (Triticum aestivum L.) cultivars. Non-denaturing fluorescence in situ hybridization (ND-FISH) technology using oligonucleotides (oligo) as probes provides a convenient and efficient way to identify alien chromosomes in wheat backgrounds. However, suitable ND-FISH-positive oligo probes for distinguishing Thinopyrum chromosomes from wheat are lacking. Two oligo probes, Oligo-B11 and Oligo-pThp3.93, were designed according to the published Thinopyrum ponticum (Th. ponticum)-specific repetitive sequences. Both Oligo-B11 and Oligo-pThp3.93 can be used for ND-FISH analysis and can replace conventional GISH and FISH to discriminate some chromosomes of Th. elongatum, Th. intermedium, and Th. ponticum in wheat backgrounds. The two oligo probes provide a convenient way for the utilization of Thinopyrum germplasms in future wheat breeding programs.