Review Article |
Corresponding author: Michał T. Kwiatek ( michal.kwiatek@up.poznan.pl ) Academic editor: Elena Mikhailova
© 2019 Michał T. Kwiatek, Danuta Kurasiak-Popowska, Sylwia Mikołajczyk, Janetta Niemann, Agnieszka Tomkowiak, Dorota Weigt, Jerzy Nawracała.
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:
Kwiatek MT, Kurasiak-Popowska D, Mikołajczyk S, Niemann J, Tomkowiak A, Weigt D, Nawracała J (2019) Cytological markers used for identification and transfer of Aegilops spp. chromatin carrying valuable genes into cultivated forms of Triticum. Comparative Cytogenetics 13(1): 41-59. https://doi.org/10.3897/CompCytogen.v13i1.30673
|
There are many reports describing chromosome structure, organization and evolution within goatgrasses (Aegilops spp.). Chromosome banding and fluorescence in situ hybridization techniques are main methods used to identify Aegilops Linnaeus, 1753 chromosomes. These data have essential value considering the close genetic and genomic relationship of goatgrasses with wheat (Triticum aestivum Linnaeus, 1753) and triticale (× Triticosecale Wittmack, 1899). A key question is whether those protocols are useful and effective for tracking Aegilops chromosomes or chromosome segments in genetic background of cultivated cereals. This article is a review of scientific reports describing chromosome identification methods, which were applied for development of prebreeding plant material and for transfer of desirable traits into Triticum Linnaeus, 1753 cultivated species. Moreover, this paper is a resume of the most efficient cytomolecular markers, which can be used to follow the introgression of Aegilops chromatin during the breeding process.
Aegilops , chromosome, banding, fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH), prebreeding, triticale, wheat
There are twenty three species of goatgrasses (Aegilops spp.) (
The ability to distinguish alien chromosomes, which were introduced into a genetic background of an acceptor plant, is the initial step in characterization of introgression lines. The first chromosome identification studies in wheat were done by
The first molecular probes used for FISH purposes on Aegilops-Triticum chromosomes contained conserved high-copy sequences, such as telomere sequences or 5S and 45S ribosomal RNA genes (
The structure and organization of chromosomes of species belonging to the genera Aegilops and Triticum are collinear, as chromosomes within each homoeologous group are related by descent from a chromosome of the ancestor of the Triticum-Aegilops complex (
Since the 1970s C-banding and N-banding techniques were used to distinguish the chromosomes of Aegilops-Triticum complex (
Polymorphisms in C-banding patterns were also utilised to distinguish Aegilops chromosomes in wheat genetic background. Ae. speltoides turned out to be one of the largest sources of valuable genes and was used to develop Aegilops-Triticum introgression lines.
One of the most notable applications of the C-banding technique was the identification of radiation-induced translocation lines resistant to leaf rust (Lr9) and assignment of Lr9 loci to 6UL chromosome of Ae. umbellulata. The following chromosome translocations were identified by means of C-banding analysis: 6BL.6BS-6UL, T4BL.4BS-6UL, 2DS.2DL-6UL, T6BS.6BL-6UL and 7BL.7BS-6UL (
The N-banding method was less often used to investigate Aegilops-Triticum introgression lines.
A combination of molecular techniques and classical cytology became a breakthrough tool for science and crop breeding, especially for the development and characterization of Aegilops-Triticum introgression lines. First reports of adaptation of fluorescence in situ hybridization protocol for analyses of wheat chromosomes were published by
Tandem repeats used as effective FISH markers for identification of Aegilops chromatin introgression.
Tandem repeats | Clones/sequences | References |
---|---|---|
Satellite DNA sequences | pAs1, pSc119.2, pTa-71, pTa-86, pTa-465, pTa-535, pTa-566, pTa-713, pTa-794 |
|
Microsatellite DNA sequences (simple sequence repeats - SSR) | AAC, ACG, GAA |
|
To date the most popular probe used for identification of Aegilops-Triticum chromosomes is a D-genome specific repetitive DNA sequence called pAs1, derived from of Aegilops squarrosa Linnaeus, 1753 (syn. Ae. tauschii Cosson, 1849; 2n = 14, genome DD) (
Karyograms of Aegilops kotschyi 2n=4x=28 chromosomes; UUSS) showing U- and S-genome chromosomes after two rounds of FISH with: a pTa-86 (green; Atto-488 fluorochrome; Jena Bioscience), pTa-535 (red; Atto-550 fluorochrome; Jena Bioscience), pTa-374 (25S rDNA; yellow; Atto-647 fluorochrome; Jena Bioscience) and b pTa-713 (green; Atto-488 fluorochrome; Jena Bioscience), pTa-k566 (red; Atto-550 fluorochrome; Jena Bioscience) and pTa-465 (yellow; Atto-647 fluorochrome; Jena Bioscience) probes (Kwiatek, unpublished)
Apart from the use of long repetitive sequences, one of the most effective ways to saturate chromosome regions with markers as much as possible is to apply microsatellite sequences as cytomolecular probes. Such trinucleotide sequences (i.e. AAC, GAA, ACG) were used to distinguish between chromosomes of wheat (
A combination of C-banding and GISH methods was also used for development of wheat introgression lines with resistance genes against one of the most virulent races of stem rust (Puccinia graminis var. tritici Persoon, 1794), namely Ug99.
In order to screen large populations of Aegilops-Triticum introgression forms, the methods for cytomolecular marker analysis should be easy to handle and cost-efficient. FISH protocols require fluorescent DNA probes, heat treatment and are labour and time consuming. There are reports describing modifications and changes to the protocols used to conduct repetitive sequence preparation for FISH. One of such techniques, primed in situ labeling (PRINS), combines polymerase chain reaction (PCR) with FISH to visualize sequences on chromosomes (
Another way to saturate the chromosome arms with markers is the use of cDNA probes.
Cytogenetic methods seem to be essential to verify genomic constitution in interspecific hybrids. The main problems are: limited sensitivity and spatial resolution, laborious and expensive protocols, which seriously limit the application of cytogenetic markers for large scale selection of Aegilops-Triticum introgressions. High-resolution and high-throughput methods are being progressively developed for identification of micro-introgressions, chromosome breakpoints and spatial localization of alien chromatin in donor nuclei. These require the use of new DNA markers, sequencing and new combinations of cytomolecular techniques. For example, three dimension FISH (3D-FISH) was applied to track the spatial organization of rye chromatin in wheat host genome (
The authors would like to acknowledge and thank Dr. Harrold Bockelman at the USDA/ARS Small Grains Laboratory, Aberdeen (ID, USA) for providing the seeds of Aegilops kotschyi (PI 226615). This publication is being co-financed by the framework of Ministry of Science and Higher Education program as “Regional Initiative Excellence” in years 2019–2022, project no. 005/RID/2018/19. In addition, we would like to thank all of the reviewers and manuscript editor for their careful review of the manuscript and for their excellent suggestions for improving our initial work.