Karyotyping Dasypyrum breviaristatum chromosomes with multiple oligonucleotide probes reveals the genomic divergence in Dasypyrum

Abstract

The perennial species <i>Dasypyrum breviaristatum</i> (genome V<sup>b</sup>) contains many potentially valuable genes for the improvement of common wheat. Construction of a detailed karyotype of <i>D. breviaristatum</i> chromosomes will be useful for the detection of <i>Dasypyrum</i> chromatin in wheat background. We established the standard karyotype of 1V<sup>b</sup>-7V<sup>b</sup> chromosomes through non-denaturing fluorescence <i>in situ</i> hybridization (ND-FISH) technique using 28 oligonucleotide probes from the wheat-<i>D. breviaristatum</i> partial amphiploid TDH-2 (AABBV<sup>b</sup>V<sup>b</sup>) and newly identified wheat-<i>D. breviaristatum</i> disomic translocation and addition lines D2138 (6V<sup>b</sup>S.2V<sup>b</sup>L), D2547 (4V<sup>b</sup>) and D2532 (3V<sup>b</sup>S.6V<sup>b</sup>L) by comparative molecular marker analysis. The ND-FISH with multiple oligo probes were conducted on the durum wheat-<i>D. villosum</i> amphiploid TDV-1 and large karyotype differences between <i>D. breviaristatum</i> and <i>D. villosum</i> was revealed. These ND-FISH probes will be valuable for screening the wheat-<i>Dasypyrum</i> derivative lines for chromosome identification, and newly developed wheat-<i>D. breviaristatum</i> addition lines may broaden the gene pool of wheat breeding. The differences between <i>D. villosum</i> and <i>D. breviaristatum</i> chromosomes revealed by ND-FISH will help us understand evolutionary divergence of repetitive sequences within the genus <i>Dasypyrum</i>.

Characterization of a Wheat-Dasypyrum breviaristatum Chromosome Addition and Its Derived Progenies Carrying Novel Dasypyrum-Specific Gliadin Genes

“…The molecular and cytogenetic evidence have suggested the distinct chromosomal divergence between D. villosum and D. breviaristatum species [3,4,36,37]. Recently, Yu et al [25] constructed a standard karyotype of 1V b -7V b chromosomes through ND-FISH identification of the wheat-D. breviaristatum partial amphiploid TDH-2. In the present study, we firstly produced the standard FISH karyotype of four copies of 1V b -7V b chromosomes of wild 4× D. breviaristatum based on the sequential ND-FISH and Oligo-FISH painting technique (Figure 1).…”

“…The wheat-D. breviaristatum partial amphiploid TDH-2 (genome AABBV b V b ) was as described by Yang et al [14]. Lines TDV-1 (AABBVV), D11-5 (2V b substitution, Li et al [16]), D2176 (1V b addition, Wang et al [20]), D2150 (5V b addition, Zhang et al [18]), D2139 (7V b addition, Li et al [17]) and D2532 (3V b S.2V b L, Yu et al [25]) were maintained in our lab at the University of Electronic Science and Technology of China. Line Pm97034 was obtained from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and line 92R137 was provided by the Cytogenetics Institute, Nanjing Agricultural University.…”

“…The root tips chromosome preparation protocol followed the procedure of Han et al [27]. The synthetic oligonucleotides Oligo-B11, Oligo-pDb12H, Oligo-k288, Oligo-D, Oligo-pSc119.2 and Oligo-pTa535 [22,23,25] were either 5 end-labelled with 6-carboxyfluorescein (6-FAM) for green or 6-carboxytetramethylrhodamine (TAMRA) for red signals. The non-denaturing FISH (ND-FISH) by the synthesized probes was described by Yu et al [25].…”

“…The synthetic oligonucleotides Oligo-B11, Oligo-pDb12H, Oligo-k288, Oligo-D, Oligo-pSc119.2 and Oligo-pTa535 [22,23,25] were either 5 end-labelled with 6-carboxyfluorescein (6-FAM) for green or 6-carboxytetramethylrhodamine (TAMRA) for red signals. The non-denaturing FISH (ND-FISH) by the synthesized probes was described by Yu et al [25]. After the oligobased FISH, the sequential FISH with bulk painting with oligos was conducted following the description by Li and Yang [28].…”

“…Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) are the most efficient techniques to detect alien chromosomes or segments in common wheat background [21]. A low-cost and high throughput non-denaturing FISH (ND-FISH) technology is powerful for identifying a large number of plants from wheat-alien hybridization [22], and the ND-FISH probes suitable for chromosome recognition of different wild Triticeae species were also developed [23][24][25]. Moreover, the Oligo-FISH painting systems using the bulked pools of 40-50 bp lengths of single-copy sequences have successfully enabled the assignment of chromosomes to specific linkage groups in Triticeae species [26], and hence are potentially powerful for the detection of Dasypyrum chromatin in diversified wheat backgrounds.…”