Research Article |
Corresponding author: Elena A. Salina ( salina@bionet.nsc.ru ) Academic editor: Lorenzo Peruzzi
© 2015 Irina G. Adonina, Nikolay P. Goncharov, Ekaterina D. Badaeva, Ekaterina M. Sergeeva, Nadezhda V. Petrash, Elena A. Salina.
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:
Adonina IG, Goncharov NP, Badaeva ED, Sergeeva EM, Petrash NV, Salina EA (2015) (GAA)n microsatellite as an indicator of the A genome reorganization during wheat evolution and domestication. Comparative Cytogenetics 9(4): 533-547. https://doi.org/10.3897/CompCytogen.v9i4.5120
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Although the wheat A genomes have been intensively studied over past decades, many questions concerning the mechanisms of their divergence and evolution still remain unsolved. In the present study we performed comparative analysis of the A genome chromosomes in diploid (Triticum urartu Tumanian ex Gandilyan, 1972, T. boeoticum Boissier, 1874 and T. monococcum Linnaeus, 1753) and polyploid wheat species representing two evolutionary lineages, Timopheevi (T. timopheevii (Zhukovsky) Zhukovsky, 1934 and T. zhukovskyi Menabde & Ericzjan, 1960) and Emmer (T. dicoccoides (Körnicke ex Ascherson & Graebner) Schweinfurth, 1908, T. durum Desfontaines, 1798, and T. aestivum Linnaeus, 1753) using a new cytogenetic marker – the pTm30 probe cloned from T. monococcum genome and containing (GAA)56 microsatellite sequence. Up to four pTm30 sites located on 1AS, 5AS, 2AS, and 4AL chromosomes have been revealed in the wild diploid species, although most accessions contained one–two (GAA)n sites. The domesticated diploid species T. monococcum differs from the wild diploid species by almost complete lack of polymorphism in the distribution of (GAA)n site. Only one (GAA)n site in the 4AL chromosome has been found in T. monococcum. Among three wild emmer (T. dicoccoides) accessions we detected 4 conserved and 9 polymorphic (GAA)n sites in the A genome. The (GAA)n loci on chromosomes 2AS, 4AL, and 5AL found in of T. dicoccoides were retained in T. durum and T. aestivum. In species of the Timopheevi lineage, the only one, large (GAA)n site has been detected in the short arm of 6At chromosome. (GAA)n site observed in T. monococcum are undetectable in the Ab genome of T. zhukovskyi, this site could be eliminated over the course of amphiploidization, while the species was established. We also demonstrated that changes in the distribution of (GAA)n sequence on the A-genome chromosomes of diploid and polyploid wheats are associated with chromosomal rearrangements/ modifications, involving mainly the NOR (nucleolus organizer region)-bearing chromosomes, that took place during the evolution of wild and domesticated species.
Triticum monococcum , T. boeoticum , T. urartu , T. zhukovskyi , T. dicoccoides , (GAA)n microsatellite, FISH
The genus Triticum Linnaeus, 1753 comprises species at different ploidy levels, from diploid to hexaploid. Common wheat T. aestivum L., 1753 is natural allopolyploid with the genome BBAADD, which emerged about 8–10 thousand years ago (TYA) via the cross of tetraploid Emmer species (BBAA genome) with Aegilops tauschii Cosson, 1850 (DD genome). Another hexaploid wheat, T. zhukovskyi Menabde & Ericzjan, 1960 (genome GGAtAtAbAb) was discovered in 1957, in the Zanduri region of Western Georgia and is regarded as natural allopolyploid of T. timopheevii (Zhukovsky) Zhukovsky, 1934 and T. monococcum L., 1753 growing in the same area (
The diploid wheats are the most ancient members of the genus Triticum. Among them taxonomists recognize three species, namely, cultivated T. monococcum and two wild species, T. boeoticum Boissier, 1874 and T. urartu (Goncharov, 2012). Two different types of the A genome, Au (T. urartu) and Ab (T. boeoticum and T. monococcum L., 1753), have been discriminated among the diploid wheats. According to the current concept, the Au and Ab genomes diverged approximately one million years ago (
Despite morphological similarity, the level of genome divergence between T. urartu and T. boeoticum is very high. First of all it is indicated by the sterility of hybrids between T. urartu and T. boeoticum and/or T. monococcum, although in certain combinations of accessions and crossing direction hybrid fertility was elevated from zero to 4.5% (
Genome rearrangements, such as translocations, inversions, and the emergence of large blocks of repeats via amplification, are of considerable importance for the reproductive isolation of species. Such large-scale rearrangements are detectable by meiotic chromosome pairing analysis, comparative genome mapping, and FISH with repetitive probes. The data obtained so far suggest that the emergence of two evolutionary lineages of polyploid wheats, Emmer and Timopheevi, was accompanied by several species-specific translocations (
One of the approaches for the identification of chromosomal rearrangements is cytogenetic analysis. Despite significant progress in sequencing and mapping of cereal genomes, this method is still most powerful for detection of chromosome aberrations; however, it needs a sufficient pool of cytogenetic markers. The number of cytogenetic markers used for the analysis of A genome chromosome is currently rather few. Single hybridization signals can the obtained with probes pSc119.2, pAs1, pTa71 (45S RNA genes), and pTa794 (5S rRNA genes) (
The goal of this work was to study the rearrangement of the A genome chromosomes of wheats during the evolution based on the distribution of (GAA)n microsatellite on the chromosomes.
The following diploid Triticum species were used in our work (see Table
Accessions of the diploid and polyploid Triticum species used in the work.
Accession/ |
Species | Subspecies/variety (if available) | Centre of genetic resource | Accession number | Geographic origin |
---|---|---|---|---|---|
BO2/IG1 | T. boeoticum | subsp. thaoudar | Kyoto Univ. | KU8120 | Iraq |
BO3/IG2 | T. boeoticum | – | VIR | K-25811 | Armenia |
BO9/IG1 | T. boeoticum | – | ICARDA | IG116198 | Turkey |
BO12/IG2 | T. boeoticum | subsp. boeoticum | VIR | K-18424 | Crimea |
BO14/IG1 | T. boeoticum | – | USDA | PI427328 | Iraq |
BO19/IG2 | T. boeoticum | subsp. boeoticum | VIR | K-33869a | Armenia |
MO1/IG3 | T. monococcum | var. macedonicum | VIR | K-18140 | Azerbaijan |
MO3/IG3 | T. monococcum | var. monococcum | VIR | K-20409 | Spain |
T. monococcum | – | VIR | K-18105 | Nagorno-Karabakh Autonomous Region | |
T. monococcum | – | VIR | K-8555 | Crimea | |
T. monococcum | – | USDA | PI119423 | Turkey | |
T. monococcum | var. hornemannii, population Zanduri | VIR | K-46586 | Georgia | |
UR1/IIG4 | T. urartu | – | USDA | PI538736 | Lebanon |
UR2/IIG4 | T. urartu | var. albinigricans | VIR | K-33869b | Armenia |
UR3/IG3 | T. urartu | – | USDA | PI428276 | Lebanon |
UR4/IG1 | T. urartu | – | ICARDA | IG116196 | Turkey |
UR5/IG2 | T. urartu | var. albinigricans | VIR | K-33871 | Armenia |
UR6/IIG4 | T. urartu | – | ICARDA | IG45298 | Syria |
UR44/IIG4 | T. urartu | – | USDA | PI428182 | Armenia |
T. timopheevii | population Zanduri | VIR | K-38555 | Georgia | |
T. zhukovskyi | population Zanduri | VIR | K-43063 | Georgia | |
T. dicoccoides | ICARDA | IG46273 | Israel | ||
T. dicoccoides | ICARDA | IG46288 | Israel | ||
T. dicoccoides | ICARDA | IG139189 | Jordan | ||
T. durum | VIR | K-1931 | Russia | ||
T. aestivum | ICG | cv. Chinese Spring | China | ||
T. aestivum | var. lutescens | ICG | cv. Saratovskaya 29 | Russia |
Polyploid wheat species belonging to either Timopheevi (T. timopheevii, 2n = 4x = 28, GGAtAt, and T. zhukovskyi, 2n = 6x = 42, GGAtAtAbAb), or Emmer evolutionary lineage (T. dicoccoides, 2n = 4x = 28, BBAA, T. durum, 2n = 4x = 28, BBAA, and T. aestivum, 2n = 6x= 42, BBAADD) were analyzed (Table
The plants of all accessions used in our work were grown at the Joint Access Laboratory for Artificial Plant Cultivation for verification of species authenticity by morphological characters using a guide published by
T. zhukovskyi authenticity was verified by electrophoresis of wheat storage proteins (gliadins) (
The (GAA)n microsatellite sequence was cloned from einkorn wheat genome in order to increase the resolution of FISH analysis.
Total DNA was isolated from 5–7-day-old seedlings according to
Giemsa C-banding was performed according to the protocol by
The work was performed at the Vavilov Institute of General Genetics, Russian Academy of Sciences.
Fluorescence in situ hybridization (FISH) was conducted as earlier described (
The chromosomes were identified using the pSc119.2 (120 bp, rye repeats;
The preparations were embedded into Vectashield mounting medium (Vector Laboratories), containing 0.5 µg/ml DAPI (4’,6-diamidino-2-phenylindole, Sigma) for chromosome staining. The chromosomes were examined with an Axioskop 2 Plus (Zeiss) microscope and recorded with a VC-44 (PCO) CCD camera.
The work was performed at the Joint Access Center for Microscopic Analysis of Biological Objects with the Siberian Branch of the Russian Academy of Sciences.
Totally, four clones differing in the length of the insert were selected and sequenced. The clones obtained from T. urartu were designated pTu and from T. monococcum, pTm. All the clones contain (GAA)n microsatellite sequence, but differ in length: pTm30 has a length of 167 bp [(GAA)56]; pTm17, 62 bp [(GAA)21]; pTu33, 56 bp [(GAA)19]; and pTu38, 36 bp [(GAA)12]. The (GAA)56 microsatellite variant pTm30 generating most distinct signals was selected for further work.
The new probe pTm30 containing (GAA)56 sequence was hybridized to chromosomal preparations of T. urartu, T. boeoticum and T. monococcum; the pTa71 (45S rRNA genes) probe was used for chromosome identification. As was expected, the 45S ribosomal RNA genes in all species were localized to the nucleolar organizer region in the distal parts of the 1AS and 5AS chromosomes.
The accessions of wild species T. boeoticum and T. urartu displayed polymorphism in the distribution of (GAA)n microsatellite on the chromosomes. One to three pTm30 sites per haploid genome can be detected in these two species (Table
FISH with probes pTm30 (green signal) and pTa71 (red signal) on the chromosomes of diploid Triticum species: a T. monococcum MO1 b T. boeoticum BO3 c T. boeoticum BO14 and d T. urartu UR6.
Localization of pTm30 probe on the chromosomes of diploid Triticum species.
Chromosome (arm) |
T.
boeoticum
|
T.
urartu
|
T.
monococcum
|
|||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
BO2 IG1 | BO3 IG2 | BO9 IG1 | BO12 IG2 | BO14 IG1 | BO19 IG2 | UR1 IIG4 | UR2 IIG4 | UR3 IG3 | UR4 IG1 | UR5 IG2 | UR6 IIG4 | UR4 IIG4 | ||
1A(S) | + | + | + | + | ||||||||||
2A(S) | + | + | + | + | + | + | ||||||||
5A(S) | + | + | + | + | + | + | + | + | ||||||
4A(L) | + | + | + | + |
The domesticated species T. monococcum differs from the wild species by an almost complete lack of polymorphism in the distribution of pTm30 probe. The (GAA)n site in five of the six examined accessions is localized to the pericentromeric region of 4AL (Fig.
The examined accessions of T. timopheevii originated from the Zanduri population (Western Georgia), where the species T. zhukovskyi was first identified. Triticum timopheevii carries pSc119.2 signals predominantly on the G genome chromosomes and also on 1AtL and 5AtS; the pTa71 signals are present on 6AtS and 6GS chromosomes (Fig.
FISH with probe pTm30 (green signal) on the chromosomes of T. zhukovskyi: a red signal, pSc119.2 and b red signal, pTa71.
Similar results were obtained for hexaploid T. zhukovskyi (2n = 42, GGAtAtAbAb genome), a natural amphiploid resulting from interspecific hybridization between T. timopheevii (2n = 28, GGAtAt genome) and T. monococcum (2n = 14, AA genome). T. zhukovskyi, like T. timopheevii, carries the pTm30 site in the short arm of the 6At chromosome and pSc119.2 on 1AtL (Fig.
Since karyotyping of the A genome of polyploid wheats by FISH alone is not precise, Giemsa C-banding was also used in order to identify all chromosomes of T. dicoccoides, T. durum, and T. aestivum. In addition, the distribution pattern of probe pSc119.2 was considered, when identifying the chromosomes.
Among three T. dicoccoides accessions we identified the conserved pSc119.2 sites in subtelomeric regions of 1AS and 4AL chromosomes (Fig.
Localization of probe pTm30 on the chromosomes of Emmer wheats and C-banding. Accessions of T. dicoccoides: pTm30 (red) and pSc119.2 (green); accessions of T. aestivum: pTm30 (green) and pSc119.2 (red).
A comparative analysis of the A genome chromosomes of T. dicoccoides by FISH with pTm30 probe revealed both conserved and polymorphic (GAA)n signals (Fig.
The distribution of pTm30 hybridization sites on the A genome chromosomes of two hexaploid wheat cultivars, Chinese Spring and Saratovskaya 29, was similar (Fig.
The absence of (GAA)n microsatellite on the 1A chromosome was the common feature for all T. dicoccoides, T. durum, and T. aestivum accessions. In addition, the conserved (GAA)n sites on T. dicoccoides chromosomes 2AS, 4AL, and 5AL were remained in T. durum and T. aestivum.
The goal of the search for cytogenetic markers for the wheat A genome dates back to the very first application of cytogenetic methods to analysis of chromosome structure and phylogeny of Triticum species. This was due to both a small number of C-bands detectable by Giemsa staining and the difficulties in FISH-based distinguishing between the A genome chromosomes. In particular, two cytogenetic markers, pSc119.2 and pAs1, are able to discriminate all B and D genomes chromosomes of Emmer wheat, but only three A genome chromosomes (
The situation with chromosome identification in einkorn wheat is even more complex. The probes that are frequently used in molecular cytogenetic analysis of polyploid wheats, such as pSc119.2 and pAs1, either do not hybridize to einkorn chromosomes at all, or give few fuzzy signals (
The distribution of (GAA)n microsatellite on chromosomes of the A genome diploid species has not been studied until recently.
As has been demonstrated here, the pTm30 produces up to four major hybridization sites on the A genome chromosomes of diploid wheats (1AS, 2AS, 5AS, and 4AL), while any minor hybridization sites are undetectable. All four major hybridization sites are present in T. urartu only, and the site on 1AL is absent in T. monococcum and T. boeoticum. Interestingly, T. urartu accessions belonging to super-cluster II (urartu) mainly display two (GAA)n sites, on the 1AS and 5AS chromosomes, also carrying the 45S RNA genes. The major (GAA)n site on 1AS and minor site on 5AS have been also detected in the T. urartu accession by
Thus, it has been shown that the (GAA)n microsatellite can be used as marker for the 1AS, 2AS, 4AL, and 5AS chromosomes of einkorn wheat; however, it should be kept in mind that depending on species, the number of hybridization sites varies from zero to three in individual accessions. The (GAA)n site on chromosome 1AS is present only in T. urartu, while T. boeoticum and T. monococcum often carry (GAA)n site on the chromosome 4AL.
The evolution of diploid and polyploid wheat species is known to be accompanied by reorganization of the genomes. At the diploid level, genome divergence occurs via accumulation of DNA mutations, amplifications/deletions of tandem repeats, proliferation of mobile elements, and, in some cases, chromosomal rearrangements (
As any other tandem repeats, microsatellites frequently form large clusters on chromosomes, detectable with FISH. The polymorphism of satellite repeats most typically involve changes in the copy number, resulting in the appearance/elimination of large blocks of repeated sequences.
Study of the distribution of (GAA)n hybridization sites in diploid and polyploid wheats allows us to propose that several factors could have led to redistribution of regions housing this microsatellite. In particular, a decrease in the number of major microsatellite blocks in domesticated T. monococcum may only be a result genetic diversity shortage caused by bottleneck effect during domestication. Another important fact is that all studied accessions of polyploid wheat species T. dicoccoides, T. durum, T. aestivum, and T. timopheevii lack hybridization sites on the short arms of their 1A and 5A chromosomes (Fig.
As in the parental species T. timopheevii, T. zhukovskyi displays only one (GAA)n site in the short arm of chromosome 6At, near the nucleolus organizer region. It is known that the T6AS/1GS translocation took place during T. timopheevii speciation (
The (GAA)n sites of the einkorn wheat that are localized to more conserved chromosome regions, namely, pericentromeric regions of 2AS and 4AL, were inherited by all polyploid Emmer species (Fig.
Thus, amplification and cloning of the long fragment of (GAA)n sequence from T. monococcum genome allowed us to obtain the new DNA probe for analysis of the A-genome chromosomes in diploid and polyploid wheat. An increased sequence length provides for higher probe stability, which enhances resolution of hybridization. Using a new probe we defined differences between Ab and Au variants of the A-genomes, revealed variability of labeling patterns among T. boeoticum and T. urartu accessions, and significant shortage of polymorphism in T. monococcum, probably due to domestication. We suppose that distribution of (GAA)n sites in diploid and polyploid species reflects the chromosome reorganizations, mainly including the nucleolus organize region, that have taken place during the evolution of wild and domesticated species.
The study of diploid species was done in framework of the State Budget Program (Project No VI.53.1.5.), the analysis of polyploid wheats was supported by the Russian Scientific Foundation (Project No. 14-14-00161).