Research Article |
Corresponding author: Elena V. Evtushenko ( evt@mcb.nsc.ru ) Academic editor: Julio R. Daviña
© 2019 Elena V. Evtushenko, Yulia A. Lipikhina, Petr I. Stepochkin, Alexander V. Vershinin.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Evtushenko EV, Lipikhina YA, Stepochkin PI, Vershinin AV (2019) Cytogenetic and molecular characteristics of rye genome in octoploid triticale (× Triticosecale Wittmack). Comparative Cytogenetics 13(4): 423-434. https://doi.org/10.3897/CompCytogen.v13i4.39576
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Alloploidization resulting from remote (interspecific or intergeneric) hybridization is one of the main factors in plant evolution, leading to the formation of new species. Triticale (× Triticosecale Wittmack, 1889) is the first artificial species created by crossing wheat (Triticum spp.) and rye (Secale cereale Linnaeus, 1753) and has a great potential as a grain and forage crop. Remote hybridization is a stress factor that causes a rapid reorganization of the parental genomes in hybrid progeny (“genomic shock”) and is accompanied by abnormalities in the chromosome set of hybrids. The formation of the hybrid genome and its subsequent stabilization are directly related to the normalization of meiosis and the correct chromosome segregation. The aim of this work was to cytogenetically characterize triticale (× Triticosecale rimpaui Wittmack, 1899, AABBDDRR) obtained by crossing Triticum aestivum Linnaeus, 1753. Triple Dirk D × Secale cereale L. Korotkostebel’naya 69 in F3–F6 generations of hybrids, and to trace the process of genetic stabilization of hybrid genomes. Also, a comparative analysis of the nucleotide sequences of the centromeric histone CENH3 genes was performed in wheat-rye allopolyploids of various ploidy as well as their parental forms. In the hybrid genomes of octoploid triticale an increased expression of the rye CENH3 variants was detected. The octoploid triticale plants contain complete chromosome sets of the parental subgenomes maintaining the chromosome balance and meiotic stability. For three generations the percentage of aneuploids in the progeny of such plants has been gradually decreasing, and they maintain a complete set of the paternal rye chromosomes. However, the emergence of hexaploid and new aneuploid plants in F5 and F6 generations indicates that stabilization of the hybrid genome is not complete yet. This conclusion was confirmed by the analysis of morphological features in hybrid plants: the progeny of one plant having the whole chromosome sets of parental subgenomes showed significant morphological variations in awn length and spike density. Thus, we expect that the results of our karyotyping of octoploid triticales obtained by crossing hexaploid wheat to diploid rye supplemented by comparative analysis of CENH3 sequences will be applicable to targeted breeding of stable octo- and hexaploid hybrids.
Aneuploidy, centromeric histone H3 (CENH3), fluorescence in situ hybridization (FISH), remote hybridization, triticale
Triticale, derived from crossing wheat (Triticum spp.) and rye (Secale cereale Linnaeus, 1758) was the first synthetic allopolyploid cereal. It incorporates favorable alleles from both progenitor species (wheat and rye), enabling adaptation to environments that are less favorable for wheat yet providing better biomass yield and forage quality (
The formation of a hybrid genome and its subsequent stabilization are directly related to the normalization of the meiosis process and the correct chromosome segregation (
The variations in the amount and distribution of heterochromatin have facilitated the identification of rye chromosomes in different triticales (
The aim of this work was to cytogenetically characterize triticale, obtained by crossing Triticum aestivum L. line Triple Dirk D × S. cereale L., cultivar Korotkostebel’naya 69, by FISH analysis of their rye chromosomes. The parental forms have a number of specific characteristics. The wheat near-isogenic line Triple Dirk D has only one dominant spring allele Vrn-A1 (
Octoploid triticales (genome constitution AABBDDRR) were created by crossing the near isogenic line of common wheat (Triticum aestivum L.) Triple Dirk D (genome AABBDD) with diploid rye (Secale cereale L.) cultivar Korotkostebel’naya 69 (genome RR) (
For chromosome counts in triticale somatic cells, root-tips of seedlings were treated with saturated α-bromonaphthalene solution and visualized through Feulgen staining (
Total RNA was isolated from leaves of individual young seedlings with TRI Reagent RT (MRC Inc., United States) and treated with RQRNaseFree DNase (Promega Corporation, Madison, WI, USA) according to manufacturers’ recommendations. RNA was reverse-transcribed to cDNA with a RevertAid H Minus First Strand cDNA Synthesis Kit (Thermoscientific). Amplification primers specific to the N-terminal tail of the αCENH3 gene from rye, wheat (Genbank accession nos. MG384772.1, JF969285.1) and triticale cDNA had been designed in (
Karyotype analysis of 30 hybrid plants in the F3 generation showed that the number of chromosomes in their somatic cells varies from 53 to 56 (Table
Identification of rye chromosomes by FISH using the pSc200 (green) and pTa71 (red) probes on metaphase chromosomes of the paternal parent and allopolyploid triticale hybrids a rye Korotkostebel’naya 69 b the line TDK96F3 (56 chromosomes) c the line TDK94F3 (55 chromosomes) d TDK96.3.F4 (49 chromosomes) e TDK96.3.F6 (42 chromosomes) f TDK96.3.F6 (41 chromosome). The arrows indicate rye chromosomes.
The chromosome numbers in the karyotypes of somatic cells of triticale lines.
Triticale lines | Generation | Percentages of plants with | Number of chromosomes | |||
---|---|---|---|---|---|---|
56 chromosomes | 42 chromosomes | Mean | Min-max | Rye | ||
TDK 94 | F3 | 0 | 0 | 54.3 | 53–55 | 14 |
TDK 94 | F4 | 16.7 | 0 | 54.5 | 53–56 | 14 |
TDK 96 | F3 | 100 | 0 | 56.0 | 56 | 14 |
TDK 96 | F4 | 46.2 | 0 | 54.5 | 49–56 | 14 |
TDK 96.1 | F5 | 62.5 | 0 | 55.3 | 55–56 | 14 |
TDK 96.2 | F5 | 80 | 0 | 55.7 | 55–56 | 14 |
TDK 96.3 | F5 | 0 | 0 | 44.4 | 43–47 | 14 |
TDK 96.3 | F6 | 0 | 37.5 | 42.1 | 41–45 | 14 |
The hybrid plant TDK96F3 containing a set of 56 chromosomes was also reproduced by self-pollination and its progeny (designated as TDK96F4 in Table
This conclusion is confirmed by the analysis of some morphological features in hybrid plants. Figure
Intergeneric hybridization in plants is often accompanied by elimination of some chromosomes or whole genomes. One of the putative ways to the emergence of aneuploid plants is disturbances in the functioning of centromeres of one of the parents owing to the differences in the molecular structure of centromeric histones (
The positions of species-specific non-synonymous SNPs across NTT domain of CENH3 of wheat, rye and octoploid triticale.
Plants | The percentages of substitutions at positions across NTT domain | |||||||
---|---|---|---|---|---|---|---|---|
28 | 32 | 73 | 82 | 84 | 99 | 122 | 145 | |
T. aestivum Triple Dirk D (AABBDD), 2n=42 | 11.1 | 55.6 | 55.6 | 27.8 | ||||
S. cereale Korotkostebel’naya 69 (RR), 2n=14 | 8.7 | 7.4 | 7.4 | 30.4 | 21.7 | |||
Octoploid triticale F3 | ||||||||
Plant 1, (2n=56), TDK 96 | 5.6 | 16.7 | 5.6 | 5.6 | 11.1 | 5.6 | 5.6 | |
Plant 2 (2n=52), TDK 92.4 | 16.7 | – | 16.7 | 33.3 | ||||
Plant 3 (2n=54), TDK 94.2 | 30 | 10 | – | 10 | ||||
Octoploid triticale F4 (derived from F3, plant 1) | ||||||||
Plant 1 (2n=56), TDK 96.1 | 5 | 35 | 10 | 10 | 10 | 15 | 5 | |
Plant 2 (2n=56), TDK 96.2 | 10 | 30 | 10 | 10 | 10 | |||
Plant 3 (2n=49), TDK 96.3 | 6.7 | 33.3 | 6.7 | 6.7 | 6.7 | 6.7 | ||
Octoploid triticale F5 (derived from F4, plant 1) | ||||||||
Plant 1 (2n=56), TDK 96.1.1 | 7.1 | 50 | 7.1 | |||||
Octoploid triticale F5 (derived from F4, plant 3) | ||||||||
Plant 2 (2n=43), TDK 96.3.1 | 57.1 | 7.1 | 14.3 | 7.1 |
Various chromosomal rearrangements in allopolyploid hybrids are among the most frequently described effects of remote hybridization. Significant genomic changes at remote hybridization cause instability of the hybrid genome and chromosome number imbalance. An imbalance of the hybrid genome results in a high percentage of aneuploid plants immediately after the crossing (
Deletions and translocations of individual chromosomal regions and chromosome arms are also among the most common chromosomal alterations and have been found in the cytogenetic analysis of wheat-rye substitution and addition lines (
Data on the effect of “genomic shock” on the chromosomes of the rye subgenome in hybrid plants are contradictory. Early studies indicated that both rye and wheat chromosomes contributed to aneuploidy (
Differences in the CENH3 structure between the parental forms allow us to judge the regulation of the expression level of the parental protein forms in a new genomic environment that arises in a hybrid cell in case of remote hybridization. The first study of the possible relationship between differences in the CENH3 structure in parental forms and the processes of parental genome chromosome segregation during the division of hybrid cells was carried out on hybrids obtained by crossing cultured barley H. vulgare L. and its closest wild relative H. bulbosum Linnaeus, 1756. The CENH3 molecules were not included in the centromeres of H. bulbosum chromosomes, which were herewith inactivated and eliminated from hybrid embryos. Perhaps this was due to significant differences in protein structure between the barley species, especially in the structure of the N-terminal tail (
Our results on CENH3 sequences analysis in hybrid plants (Table
The work was supported by the Russian fundamental scientific research program (project 0310-2019-0003); the experiments on plant crossing were funded by the Russian Foundation for Basic Research (project № 18-54-00013), cytogenetic analysis and determination of gene expression were funded by the Russian Foundation for Basic Research (project № 17-04-00748), the experiments on identifying differences in the DNA structure of parental forms were funded by the Russian Science Foundation (project № 19-14-00051).