Research Article |
Corresponding author: Quanwen Dou ( douqw@nwipb.cas.cn ) Academic editor: Viktoria Shneyer
© 2016 Quanwen Dou, Ruijuan Liu, Feng Yu.
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:
Dou Q, Liu R, Yu F (2016) Chromosomal organization of repetitive DNAs in Hordeum bogdanii and H. brevisubulatum (Poaceae). Comparative Cytogenetics 10(4): 465-481. https://doi.org/10.3897/CompCytogen.v10i4.9666
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Molecular karyotypes of H. bogdanii Wilensky, 1918 (2n = 14), and H. brevisubulatum ssp. brevisubulatum (2n = 28), were characterized by physical mapping of several repetitive sequences. A total of 18 repeats, including all possible di- or trinucleotide SSR (simple sequence repeat) motifs and satellite DNAs, such as pAs1, 5S rDNA, 45S rDNA, and pSc119.2, were used as probes for fluorescence in situ hybridization on root-tip metaphase chromosomes. Except for the SSR motifs AG, AT and GC, all the repeats we examined produced detectable hybridization signals on chromosomes of both species. A detailed molecular karyotype of the I genome of H. bogdanii is described for the first time, and each repetitive sequence is physically mapped. A high degree of chromosome variation, including aneuploidy and structural changes, was observed in H. brevisubulatum. Although the distribution of repeats in the chromosomes of H. brevisubulatum is different from that of H. bogdanii, similar patterns between the two species imply that the autopolyploid origin of H. brevisubulatum is from a Hordeum species with an I genome. A comparison of the I genome and the other Hordeum genomes, H, Xa and Xu, shows that colocalization of motifs AAC, ACT and CAT and colocalization of motifs AAG and AGG are characteristic of the I genome. In addition, we discuss the evolutionary significance of repeats in the genome during genome differentiation.
Hordeum bogdanii , Hordeum brevisubulatum , autopolyploid, repetitive sequence, FISH
Species in Triticeae have large genomes, 75% of which consists of repetitive sequences (
The genus Hordeum Linnaeus, 1753 in the tribe Triticeae is divided into 32 species and is distributed in southern South America, South Africa, and the northern hemisphere (
Hordeum bogdanii Wilensky, 1918, and H. brevisubulatum , should include the I genome based on the description of
In this paper, the molecular karyotype of the I genome is described in detail based on physical mapping of all possible dinucleotide and trinucleotide SSRs along with 5S rDNA, 45S rDNA, pSc119.2, and pAs1 repeats on mitotic chromosomes in H. bogdanii and H. brevisubulatum. The results provide more information on the genomic differentiation in Hordeum at the chromosomal level and will also help elucidate the functional and evolutionary implications of different repetitive sequences as genomes differentiate during speciation.
Hordeum bogdanii was collected in Germu, Qinghai, China. Hordeum brevisubulatum ssp. brevisubulatum was collected in Tongde, Qinghai, China. More than 50 or more than 100 individuals of both species were collected in the field. Samples used for cytogenetic investigation were randomly selected from different individuals. Approximately 20 individuals were used for chromosome preparation. Only the investigated samples that displayed clear FISH patterns were present in this study.
Seeds of H. bogdanii and H. brevisubulatum were germinated on moist filter paper in petri dishes at room temperature. Root tips with a length of approximately 1–2 cm were excised, pretreated in N2O gas for 2 h as described by
Synthetic dinucleotide SSRs (AT)15, (AG)15, (AC)15, and (GC)15 and trinucleotide SSRs (AAG)10, (AAC)10, (AAT)10, (ACG)10, (ACT)10, (AGG)10, (CAC)10, (CAG)10, (CAT)10 and (GGC)10 were end-labelled with fluorescein amidate (FAM, green, Sangon Biotech Co., Ltd., Shanghai, China). For the repetitive sequences pAs1 (
FISH experiments were conducted using the method of Dou et al. (
A stable chromosome number of 2n = 14 was detected in all tested samples of H. bogdanii. The repetitive sequence pAs1 produced multiple sites that were subtelomeric, intercalary, or pericentromeric on all chromosomes. The hybridization pattern of pAs1 was informative enough to distinguish each chromosome of H. bogdanii. Thus, chromosome localization of other repeats was conducted using pAs1 as a landmark (Fig.
FISH patterns of mitotic metaphase chromosomes of H. bogdanii detected by pAs1 (red) combined with the several other repeats (green): a (AC)15b (AG)15c (AAC)10d (AAG)10e (AAT)10f (ACG)10g (ACT)10h (AGG)10i (CAC)10j (CAG)10k CAT)10l (GCC)10m 5S rDNA n 45SrDNA o pSc119.2. Bar = 10 µm.
Molecular karyotypes of H. bogdanii probed by pAs1 (red) combined with the other several repeats (green). Seven pairs of chromosomes are designated from A–G for distinguishing them from the numerals of the 7 homologue groups used in barly. Bar =10 µm.
The four possible dinucleotide SSR probes (AG)15, (AC)15, (AT)15, and (GC)15 were used to characterize the chromosomes of H. bogdanii. Only (AC)15 produced detectable hybridization signals, which appeared in subtelomeric regions on all chromosomes and in pericentromeric regions on a few chromosomes. The signals were strongest in subtelomeric regions of chromosomes B, C and D (Fig.
All 10 trinucleotide SSRs produced detectable hybridizations (Fig.
Three 45S rDNA sites were detected in two pairs of chromosomes. One carried a distinct site on the subtelomeric region of the short arm; the other harboured two faint hybridization sites in the ends of both arms. 5S rDNA was distributed in all chromosomes except for one. Nine distinct 5S rDNA sites were exclusively determined and were localized on centromeric, pericentromeric, intercalary, or subtelomeric regions. Three pairs of chromosomes carried only one 5S rDNA site, and another three harboured two 5S rDNA sites. Two 45S rDNA and three 5S rDNA sites were reported in an accession of H. bogdanii (
A chromosome number of 2n = 28 was detected in nearly all tested individuals of H. brevisubulatum. However, chromosome numbers of 2n = 26 and 2n = 27 (Fig.
FISH patterns of mitotic metaphase chromosomes of H. brevisubulatum detected by pAs1 (red) combined with several other repeats (green): a (AC)15b (AG)15c (AAC)10d (AAG)10e (AAT)10f (ACG)10g (ACT)10h (AGG)10i (CAC)10j (CAG)10k (CAT)10l (GCC)10m 5S rDNA n 45SrDNA o pSc119.2. Bar = 10 µm.
Molecular karyotypes of H. brevisubulatum probed by pAs1 (red) combined with several other repeats (green). Chromosome types are designated by lowercase roman letters from a to t to distinguish them from the symbols designating H. bogdanii. Bar = 10 µm.
All possible di- and trinucleotide SSR probes except for (AG)15, (AT)15 and (GC)15 produced hybridizations on chromosomes of H. brevisubulatum. The (AC)15 probe was primarily detected in subtelomeric regions. Nearly half of the total chromosomes carried detectable signals, and signals of seven or eight chromosomes appeared more distinct. The chromosomal distribution of (AAC)10, (ACT)10, and (CAT)10 was similar to that in H. bogdanii. The (AAC)10, (ACT)10, and (CAT)10 repeats were still colocalized and were detected on 22–26 chromosomes. (AAG)10 and (AGG)10 appeared to be colocalized and were primarily distributed in pericentromeric regions on 13–16 chromosomes. (CAC)10 and (CAG)10 had similar distributions to those observed in H. bogdanii. Signals of (CAC)10 and (CAG)10 were detectable in 19–22 chromosomes and were stronger on eight or nine chromosomes. The FISH pattern of (AAT)10 in H. brevisubulatum was distinctly different from that in H. bogdanii. (AAT)10 produced a stronger hybridization signal in H. brevisubulatum than in H. bogdanii, primarily in subtelomeric regions on nearly all chromosomes. Signals of (ACG)10 were detectable in 13 chromosomes. As with (AAT)10, stronger hybridization signals of (GCC)10 were detected in H. brevisubulatum than in H. bogdanii.
45S rDNA produced hybridization signals in subtelomeric regions on seven or eight chromosomes. 5S rDNA was detected in centromeric, pericentromeric, intercalary, or subtelomeric regions on 24 chromosomes. The present karyotypes of 5S rDNA and 45S rDNA in H. brevisubulatum differed from those of H. brevisubulatum based on the accessions reported by
Furthermore, the technique of genomic
Fifteen of the 18 repetitive sequences produced detectable hybridization in mitotic metaphase chromosomes in H. bogdanii. A few repeats, including highly polymorphic sites, can be used to uniquely identify each chromosome. The satellite DNA pAs1 and 5S rDNA can be used to distinguish each chromosome in H. bogdanii. The SSRs AAC, ACT, CAT, AAG and AGG are also ideal markers for chromosome identification because of their abundance and their large number of polymorphic sites across individual chromosomes (Fig.
Idiogram of chromosomes of H. bogdanii (I genome) and H. vulgare (H genome) showing the distributions of the SSRs AAC, ACT, CAT, AAG and AGG. Chromosomal information of H. vulgare is taken from Cuadrado et al. (2007a)
Most of the repeats produced multiple hybridizations. However, high-intensity hybridizations were always observed on pericentromeric or subtelomeric regions. This implies that the H. bogdanii genome contains more repetitive sequences in subtelomeric and pericentromeric parts of the chromosome than in interstitial regions. The distributions of AAC, ACT, and CAT were revealed to be colocalized and were identical in intensity in this study, suggesting that AAC, ACT, and CAT may be evenly distributed in an intermingled way. The same was true for AAG and AGG. Although CAC and CAG were found to be colocalized, different hybridization intensities suggest that their distribution was close rather than intermingled.
Differences between the I and H genomes can be observed by comparing their distribution of their repetitive sequences. A distinct difference can be observed in a few trinucleotide SSR motifs. In H. vulgare (H genome), the AAC, AAG, and AGG motifs are colocalized around the centromere in all chromosomes; ACT produces multiple signals in six chromosome pairs; and CAT produces a strong signal and a weak signal in 4H and 5H, respectively (
Thus, the genomic composition of the I genome in H. bogdanii revealed by the distribution of several repeats was highly different from that of the H, Xa, and Xu genomes in Hordeum.
The Hordeum brevisubulatum species complex has a range of cytotypes and can occur in diploid, tetraploid, and hexaploid forms (
Several examples of structural rearrangements of chromosomes at the population level in the H. brevisubulatum complex have been reported (
Major genetic changes, including the loss of homologs and DNA sequences, have been documented in recently formed polyploid species (
Repetitive DNA sequences are the main components of heterochromatin and are subject to rapid change. Such changes in the distribution of repetitive DNA sequences are one of the driving forces of genome evolution and speciation. One proposed function of repetitive sequences may be related to higher-order molecular structure (
The SSR motif AAC was shown to be distributed mainly in the pericentromeric regions of the I, H, Xa, and Xu genomes. In addition, the wide distribution of AAC has been detected in wheat and the genus Secale (Cuadrado et al. 2000
AC repeat sites are remarkably similar and have been shown to exhibit uniformly dispersed hybridization along the euchromatic portion of metaphase chromosomes in humans and barley and in the metaphase and polytene chromosomes of Drosophila melanogaster (Meigen, 1830) (
The sequence pAs1 contains Afa family sequences. The Afa family sequence was abundant in the four basic genomes of the genus Hordeum, and the hybridization patterns differed among the diploid species (
Variation in the abundance and distribution of repetitive sequences and the shared distribution of the same repeats between different genomes suggest that repetitive sequences play a key role in both the structure and function of the genomes of higher eukaryotes (
Fifteen repetitive sequences, including SSR motif AC and all possible trinucleotide motifs and satellite DNAs pAs1, 5S rDNA, 45s rDNA, and pSc119.2, were accurately physically mapped on individual chromosomes in the I genome in H. bogdanii. High genome instability was revealed in tetraploid H. brevisubulatum. The similar distribution of the repeats in both species suggests an autopolyploid origin of H. brevisubulatum from an I genome species. Comparative cytogenetic analysis between the I genome and other genomes in Hordeum showed that the distribution of a few repeats differed. Colocalization of motifs AAC, ACT, and CAT and colocalization of motifs AAG and AGG is characteristic of the I genome.
This work was supported by the Natural Science Foundation of Qinghai Province (no. 2015-ZJ-903).