Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis

Abstract

Hybrids derived from wheat (Triticum aestivum L.) × rye (Secale cereale L.) have been widely studied because of their important roles in wheat cultivar improvement. Repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 are usually used as probes in fluorescence in situ hybridization (FISH) analysis of wheat, rye, and hybrids derived from wheat × rye. Usually, some of these repetitive sequences for FISH analysis were needed to be amplified from a bacterial plasmid, extracted from bacterial cells, and labeled by nick translation. Therefore, the conventional procedure of probe preparation using these repetitive sequences is time-consuming and labor-intensive. In this study, some appropriate oligonucleotide probes have been developed which can replace the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 in FISH analysis of wheat, rye, and hybrids derived from wheat × rye. These oligonucleotides can be synthesized easily and cheaply. Therefore, FISH analysis of wheat and hybrids derived from wheat × rye using these oligonucleotide probes becomes easier and more economical.

The Chinese bread wheat cultivar Xiaoyanmai 7 harbours genes encoding a pair of novel high-molecular-weight glutenin subunits inherited from cereal rye

“…The chromosome preparations were mounted in propidium iodide dissolved in Vectashield antifade solution (Vector Laboratories, Burlingame, CA, USA). The FISH method was based on the protocol described by Tang et al (2014). Oligo-pSc119.2-1 repeats were end-labelled with 6-carboxyfluorescein (6-FAM), whereas oligo-pTa535-1 and (GAA)7 were labelled with 6-carboxytetramethylrhodamine.…”

“…6a). Probing with pTa535, a sequence which discriminates D genome chromosomes from those of A and B, and with pSc119.2, which preferentially hybridises to the B and R genome chromosomes (Tang et al 2014), showed that the Xiaoyanmai 7 genome lacks six of the seven D genome chromosomes (the exception was chromosome 2D), and that these have been replaced by six pairs of homoeologous rye chromosomes (Fig. 6a).…”

Characterization of a New Wheat-Aegilops biuncialis1Mb(1B) Substitution Line with Good Quality-associated HMW Glutenin Subunit

“…Cereal Research Communications 44, 2016 TATTACTCA CCGCTTTGGGGTCCCATAGCTAT-3') and oligo-pTa535-1 (Tamra-5'-AAAAACTTGACG CACGTCACGTACAAATTGGACAAACTCTTTCGGAGTAT-CAGGGTTTC-3') were prepared and fluorescence in situ hybridization (FISH) were done as described previously (Tang et al 2014). Slides were examined on an Olympus BX-51 fluorescence microscope.…”

“…Oligonucleotide probes end-labelled with 6-carboxyfluorescein (6-FAM) or 6-carboxytetramethylrhodamine (Tamra) are usually used in multi-colour FISH analysis to distinguish wheat A-, B-, and D-genome chromosomes and wild relative species of wheat (Tang et al 2014). Probes oligo-pSc119.2-1 and oligo-pTa535-1 were used to identify chromosomes in line 12-5-1.…”

“…1, twenty-one chromosome pairs were detected. Except for two 1B chromosomes, the other wheat chromosomes were distinguished according to the standard FISH signal patterns of wheat (Tang et al 2014). In addition, Oligo-pSc119.2-1 (green) generated strong signals on two distal arms of a pair chromosome, which should be chromosome 1M b compared with the 1U b chromosomes described previously .…”