qPCR as a Selective Tool for Cytogenetics

Abstract

qPCR is widely used in quantitative studies of plant genomes and transcriptomes. In this article, this method is considered as an auxiliary step in the preparation and selection of markers for FISH analysis. Several cases from the authors' research on populations of the same species were reviewed, and a comparison of the closely related species, as well as the adaptation of the markers, based on satellite tandem repeats (TRs) using quantitative qPCR data was conducted. In the selected cases, TRs with contrast abundance were identified in the cases of the Dasypyrum, Thinopyrum and Aegilops species, and the transfer of TRs between the wheat and related species was demonstrated. TRs with intraspecific copy number variation were revealed in Thinopyrum ponticum and wheat-wheatgrass partial amphidiploids, and the TR showing predominant hybridization to the sea buckthorn Y chromosome was identified. Additionally, problems such as the absence of a reference gene for qPCR, and low-efficiency and self-complementary primers, were illustrated. In the cases considered here, the qPCR results clearly show high correlation with the subsequent results of the FISH analysis, which confirms the value of this method for cytogenetic studies.

Keywords: DNA repeats; copy number; fluorescent in situ hybridization; sea buckthorn; tandem satellite repeats; wheat; wheatgrass; whole genome sequencing.

Figure 1

Methods of qPCR applied in different cases, including the development of FISH markers.

Figure 2

Localization of pHv-961 tandem repeat on the metaphase chromosomes of (A) Dasypyrum villosum PI 21717 and (B) Dasypyrum breviaristatum PI 516547 using fluorescence in situ hybridization. Red signals indicate the chromosomal localization of the pHv-961 tandem repeat. The bar indicates 10 µm. (C) Histogram of the copy number of the tandem repeat pHv-961 in Dasypyrum villosum PI 21717 and Dasypyrum breviaristatum PI 516547.

Figure 3

Localization of the 19-202 tandem repeat on the metaphase chromosomes of (A) Thinopyrum ponticum PI 636523; (B) Thinopyrum intermedium PI 401200; (C) Dasypyrum breviaristatum PI 516547; and (D) Thinopyrum sartorii PI 531745, using fluorescence in situ hybridization. Green signals indicate the chromosomal localization of 19-202 tandem repeat. The bar indicates 10 µm. (E) Histogram of the copy number of tandem repeat 19-202 in Thinopyrum sartorii PI 531745, Thinopyrum intermedium PI 401200, Dasypyrum breviaristatum PI 516547, and Thinopyrum ponticum PI 636523.

Figure 4

Localization of CL244 tandem repeat on metaphase chromosomes of (A)—Aegilops crassa AE 742; (B)—Aegilops tauschii K-112; (C)—Thinopyrum bessarabicum PI 201890, using fluorescence in situ hybridization. Green signals show chromosomal localization of CL244 tandem repeat, bar indicates 10 µm.

Figure 5

Localization of the P720 tandem repeat on the metaphase chromosomes of triticale cv. Solovey Kharkovskiy using fluorescence in situ hybridization. Red signals indicate the chromosomal localization of the P720 tandem repeat [25]. The bar indicates 10 µm.

Figure 6

Localization of the 17-202 tandem repeat on the metaphase chromosomes of (A) wheat-wheatgrass hybrid 548 using fluorescence in situ hybridization and (B) genomic in situ hybridization (GISH) on the same chromosome spread hybridized to total genomic DNA of P. spicata (green) and D. villosum (red) as probes. Green signals indicate the chromosomal localization of the 17-202 tandem repeat. The bar indicates 10 µm.

Figure 7

Localization of the 17-62 tandem repeat on the metaphase chromosomes of the wheat-wheatgrass hybrids (A) ZP26; (B) 166; (C) 4044; and (D) 548, using fluorescence in situ hybridization. Red signals indicate the chromosomal localization of the 17-202 tandem repeat. The bar indicates 10 µm.

Figure 8

Localization of the 19-202 tandem repeat on the metaphase chromosomes of (A) Thinopyrum ponticum PI 693508 and (B) Thinopyrum ponticum 1158A/19 using fluorescence in situ hybridization. Green signals indicate the chromosomal localization of the 19-202 tandem repeat. The bar indicates 10 µm.

Figure 9

Localization of the HRTR12 tandem repeat on the metaphase chromosomes of Hippophae rhamnoides using fluorescence in situ hybridization. Red signal indicates the chromosomal localization of the HRTR12 tandem repeat. The letter “Y” corresponds to Y chromosome. The bar indicates 5 µm.

Figure 10

Localization of the CL131 tandem repeat on the Aegilops crassa AE 742 metaphase chromosomes using fluorescence in situ hybridization. Green signals indicate the chromosomal localization of the CL131 tandem repeat. The bar indicates 10 µm.