draft - university of toronto t-space · draft chromosomal distribution patterns of the (ac)10...

Draft

Chromosomal distribution patterns of the (AC)10

microsatellite and other repetitive sequences, and their use in chromosome rearrangement analysis of Avena species

Journal: Genome

Manuscript ID gen-2016-0146.R2

Manuscript Type: Article

Date Submitted by the Author: 30-Sep-2016

Complete List of Authors: Fominaya, Araceli; Universidad de Alcalá de Henares, Biomedicina y Biotecnología Loarce, Yolanda; Universidad de Alcala de Henares, Biomedicina y Biotecnología Montes, Alexander; Universidad de Alcalá de Henares, Biomedicina y Biotecnología Ferrer, Esther; Universidad de Alcalá de Henares, Biomedicina y Biotecnología

Keyword: Avena, chromosome structure, fluorescence in situ hybridization (FISH), repetitive DNA, microsatellite

https://mc06.manuscriptcentral.com/genome-pubs

Genome

Draft

Chromosomal distribution patterns of the (AC)10 microsatellite and other

repetitive sequences, and their use in chromosome rearrangement

analysis of Avena species

Araceli Fominaya1, Yolanda Loarce, Alexander Montes, and Esther Ferrer.

Department of Biomedicine and Biotechnology, University of Alcalá, Alcalá de Henares,

Spain.

Complete address: Department of Biomedicine and Biotechnology, University of Alcalá,

Campus Universitario, Ctra. Madrid-Barcelona Km 33,600, 28871 Alcalá de Henares, Madrid,

Spain.

Corresponding author1: Araceli Fominaya; [email protected]

Page 1 of 40


Genome

Draft

2

Abstract

Fluorescence in situ hybridization (FISH) was used to determine the physical location of

the (AC)10 microsatellite in metaphase chromosomes of six diploid species (AA or CC

genomes), two tetraploid species (AACC genome), and five cultivars of two hexaploid

species (AACCDD genome) of the genus Avena, a genus in which genomic relationships

remain obscure. A preferential distribution of the (AC)10 microsatellite in the

pericentromeric and interstitial regions was seen for both the A- and D-genome

chromosomes, while in C-genome chromosomes the majority of signals were located

in the pericentromeric heterochromatic regions. New large chromosome

rearrangements were detected in two polyploid species: an intergenomic translocation

involving chromosomes 17AL and 21DS in A. sativa cv. Araceli, and another involving

chromosomes 4CL and 21DS in all the analyzed cultivars of A. byzantina. The latter

4CL-21DS intergenomic translocation differentiates clearly between A. sativa and A.

byzantina. Searches for common hybridization patterns on the chromosomes of

different species revealed chromosome 10A of A. magna and 21D of hexaploid oats to

be very similar in terms of the distribution of 45S and Am1 sequences. This suggests a

common origin for these chromosomes and supports a CCDD rather than an AACC

genomic designation for this species.

Keywords: Avena, chromosome structure, fluorescence in situ hybridization (FISH),

repetitive DNA.

Page 2 of 40


Genome

Draft

3

Introduction

The genus Avena contains approximately 30 species, two of which are important crop

species: Avena sativa L. and A. byzantina C. Koch. These species are also described as

homotypic subspecies (Rodionova et al. 1994; Diederichsen 2008). A. strigosa Schreb

is also cultivated in some areas, and is an important resource of resistance for

economically important diseases of oats such as cereal cyst nematode (caused by

Heterodera avenae Woll) and crown rust (caused by Puccinia coronata) (Loskutov and

Rines 2011; Rines et al. 2007). Many wild Avena species are also of economic interest

given their possession of genes potentially useful in crop improvement (Loskutov and

Rines 2011). Research into the differentiation of chromosome structure may provide

important information for the management of useful Avena genes via interspecific

hybridization.

Avena species can be divided into diploid, tetraploid and hexaploid groups with

a base chromosome number of seven. Diploid species have either an A or C genome,

tetraploids have either A, B or A, C genomes, and hexaploids have three genomes i.e.,

A, C and D. A-genome diploid species have five minor genomes - As, Al, Ad, Ap and Ac -

whereas C genome diploids have two minor genomes - Cv and Cp (Thomas 1992;

Loskutov and Rines 2011). Information on genome structure differences among related

species has been provided by C-banding (Fominaya et al. 1988a, 1988b; Linares et al.

1992; Jellen et al. 1993a, 1993b; Shelukhina et al. 2007; Shelukhina et al. 2008a,

2008b; Badaeva et al. 2011; Chaffin et al. 2016), genomic in situ hybridization (GISH)

(Chen and Armstrong, 1994; Jellen et al. 1994; Leggett and Markhand, 1995; Katsiotis

et al. 1996), and fluorescence in situ hybridization (FISH) of homologous repetitive

Page 3 of 40


Genome

Draft

4

sequences (Fominaya et al. 1995; Linares et al. 1996, 1998; Irigoyen et al. 2001; Sanz

et al. 2010; Luo et al. 2014, 2015).

The cytogenetic positions of specific repetitive sequences have been useful in

revealing the structural evolution of chromosomes in Avena species. For example,

information on the relative locations of ribosomal 45S and 5S DNA in diploid and

polyploid species has produced evidence regarding the structural evolution of the

chromosomes carrying these sequences (Linares et al. 1996; Badaeva et al. 2010, 2011;

Tomás et al. 2016), while nucleotide similarity among diploid and polyploid rDNA

copies has revealed some of their phylogenetic and genomic relationships (Peng et al.

2010). A small number of satellites, such as As120a in A. strigosa, and Am1 in A.

murphyi, have also been useful in revealing chromosome structure. As120a is

dispersed on the chromosomes of the A genome (Linares et al. 1998) while obvious

signals of Am1 repeats have been detected on those of the C genome (Fominaya et al.

1995). Other tandem repeats appear in more restricted locations in pericentromeric

positions. For example, CCS1 Avena-700 appears all around the centromeric regions

(Tomás et al. 2016) whereas A336 (isolated from chromosome 18D) appears in the

centromeric regions of A- and D-genome chromosomes (Luo et al. 2015). Low copy

genes represented by families of resistance gene analogues (Sanz et al. 2012), or which

code for endosperm proteins (Fominaya et al. 2016), have also been located on

specific chromosomes of diploid and hexaploid species, even allowing for the

identification of certain homeologous regions in hexaploids. However, the above

findings are insufficient to clarify the complex homology relationships within Avena.

New sequences with chromosome distributions that give further clues in this respect

are much needed.

Page 4 of 40


Genome

Draft

5

Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated sets

of a few base pairs (1-6) that can vary extensively in terms of the number of times they

are repeated. They form a large part of non-coding regions, but are also found in

transcribed regions (see a review by Kalia et al. 2011). Their repeated nature has

facilitated in situ hybridization studies, and many papers report differences in their

density, distribution and composition (Pinto et al. 2003; Oliveira et al. 2006; Fuentes et

al. 2009; Cuadrado et al. 2008; Cuadrado and Jouve, 2010; Molnár et al. 2011;

Carmona et al. 2013; Kejnovský et al. 2013; Han et al. 2015). In oats, SSRs have been

used in: (i) the construction of genetic maps (Pal et al. 2002; Jannink and Gardner

2005; Becher 2007; Wight et al. 2010; Oliver et al. 2010, 2011a, 2011b; Song et al.

2015), (ii) to examine the association of microsatellites with other repetitive elements

(Li et al. 2000), (iii) in population genetics studies and genotyping analyses (He and

Bjørnstad 2012), (iv) to identify relationships among oat species (Li et al. 2000; 2009),

and (v) to determine the transferability of A. sativa SSR to A. strigosa germplasm (Da-

Silva et al. 2011). However, no report discusses the use of FISH to determine the

distribution of any microsatellite on the Avena chromosomes.

Since the (AC)10 motif has been described as common in oats (Li et al. 2000; Pal

et al. 2002; Becher 2007; He and Bjørnstad 2012), diploid and polyploid species of

Avena were subjected to (AC)10-FISH to reveal the overall distribution of this repetitive

element in their chromosomes. The resulting patterns were compared in order to

detect homology among the examined species. Repetitive sequences such As120a and

Am1, in combination with 45S and 5S rDNA sequences, were also used to identify

chromosomes. Karyotypes for the different Avena species, including all the above

Page 5 of 40


Genome

Draft

6

sequences, were analyzed, and differences in the chromosome structure of polyploid

species examined.

Material and Methods

Plant materials

Table 1 shows the Avena species used in the present study. These include wild diploid

and tetraploid forms, as well as several cultivars of two hexaploid species (A. sativa

and A. byzantina). These were kindly provided by different germplasm resource

centres.

Mitotic chromosome preparation

Mitotic chromosomes were prepared as described in earlier work (Fominaya et al.

1995) with some modifications. Briefly, seeds of all the examined species/cultivars

were germinated on moistened filter paper for 48 h at 25o C. To synchronize cell

division and accumulate metaphases, the germinated seed were then exposed to a

temperature of 4o C for 72 h followed by 24 h at 25o C. Oat root tips were excised and

placed in ice-cold water for 24 h and fixed for at least a further 24 h in ethanol-acetic

acid (3:1). They were then kept in that mixture at -20o C until use. Chromosomes were

prepared using enzymatically (cellulose, peptinase) digested root tips prior to

squashing the meristematic cells in a drop of 60% acetic acid on a clean microscope

slide. After removal of the coverslips by freezing, the slides were air dried for further

processing.

DNA probes

Page 6 of 40


Genome

Draft

7

The microsatellite sequence (AC)10, with biotin-11-dUTP incorporated at both ends,

was produced (automated synthesis) by TIB MOLBIOL. Four repetitive DNA probes

were used for chromosome identification: (1) pAs120a, a satellite DNA sequence

specific to the oat A genome, containing an insert of 114 bp isolated from A. strigosa

(Linares et al. 1998); (2) pAm1, a satellite DNA sequence specific to the oat C genome,

containing an insert of 464 bp isolated from A. murphyi (Solano et al. 1992); (3) p45S, a

ribosomal probe derived from A. strigosa (Sanz et al. 2010), and (4) pTa794, a satellite

DNA sequence containing a 410 bp 5S rDNA gene and the intergenic spacer isolated

from T. aestivum (Gerlach and Dyer 1980). These clones were amplified and labelled by

PCR with digoxigenin11-dUTP or biotin16-dUTP (Roche). These repetitive probes were

used in FISH both simultaneously and sequentially after hybridization with the (AC)10

probe.

Fluorescence in situ hybridization

Chromosome preparations were treated with RNase, fixed with 4% (w/v)

paraformaldehyde, and dehydrated in an ethanol series before air drying, according to

Fominaya et al. (1995). Between the RNAse and paraformaldehyde treatments, the

preparations were washed in 2xSSC (saline-sodium citrate). Hybridization mixture (30

µl) containing 50% (v/v) formamide, 2 x SSC, 10% (w/v) sodium dodecyl sulphate (SDS),

10% (w/v) dextran sulphate, 50 µg/ml of Escherichia coli DNA, and 2.6 pmol of the

microsatellite (AC)10 sequence, was denatured for 15 min at 75o C and then applied to

each chromosome preparation. The preparations were then covered with plastic

coverslips and the chromosomes and probe denatured at 75o C for 7 min using a

programmable thermal controller (PT-100, M.J. Research Inc.). Hybridization was

Page 7 of 40


Genome

Draft

8

performed at 37o C in a humidity chamber overnight. Post-hybridization washing was

performed in 4 x SSC/0.2% Tween-20, agitating for 10 min at room temperature (RT).

The detection of biotin was performed at 37o C in a humidity chamber, incubating the

chromosome preparations for 1 h in streptavidin-Cy3 (Sigma) in 5% (W/V) BSA. The

preparations were then rinsed for 10 min in 4 x SSC/0.2% Tween-20 at RT. 4´,6´-

diamidine-2´-phenylindole dihydrochloride (DAPI) was used as a counterstain. The

preparations were the mounted in Vectashield (Vector). Reprobing FISH and probe

detection were performed as described in Linares et al. (1998).

Images were obtained using a Zeiss Axiophot microscope equipped with a

fluorescent lamp and appropriate filters, and recorded using a CCD camera (Nikon DS).

The images were pseudo-coloured in blue (chromosomes) and pink or green (signals),

merged, and optimized for brightness and contrast using Adobe Photoshop.

Results

(AC)10-FISH signal patterns in diploid species

The karyotypes of the present diploid species were previously described in studies

involving conventional staining (Rajhathy and Thomas 1974) and C-banding (Fominaya

et al. 1988a, 1988b; Badaeva et al. 2005; Shelukhina et al. 2008a, 2008b). FISH has

previously been used to reveal the number and physical locations of 45S and 5S

ribosomal loci in the chromosomes of the examined diploid species (Linares et al.

1996; Shelukhina et al. 2008a; Tomás et al. 2016). The nomenclature used in the above

papers is followed here to designate both the A-genome and C-genome chromosomes.

Page 8 of 40


Genome

Draft

9

To identify individual chromosomes labelled with (AC)10 repeats, the slides were

reprobed with 45S and 5S ribosomal sequences.

When the (AC)10 oligonucleotide probe was hybridized with metaphases of the

diploid species, two signals patterns were clearly identified (Fig. 1). One showed

discrete, well-defined bands in the pericentromeric and interstitial regions of the A-

genome chromosomes (Fig. 1a, 1c, 1e, 1g, and 1m); the other showed very strong

bands in the pericentromeric regions and dispersed signals throughout the arms of the

C-genome diploid species chromosomes (Fig. 1i, 1k, and 1m).

In A. strigosa, the (AC)10 probe produced signal patterns in each chromosome

(Fig. 1a and 1m) except for chromosome 2. Distinct signal patterns were observed in

the pericentromeric regions of each chromosome, with chromosomes 1, 3, and 4

showing very strong signals. In chromosomes 1 and 4 they covered a large region

around the centromeres. Chromosome 1 also showed a strong signal in the distal

region of the long arm. Chromosomes 6 and 7 showed only weak signals in the

pericentromeric region, while 5 and 6 showed very strong signals in the interstitial

region of the long arms. Chromosomes 2 and 3 were identifiable by their 45S and 5S

rDNA signals (Fig. 1b and 1m).

For A. damascena, all chromosomes possessed obvious (AC)10 signals except for

3 and 5 (Fig. 1c and 1m). Chromosomes 1, 4, and 6 showed signals on both sides of the

centromeric region, while 2 and 7 showed signals on only one side. 45S and 5S rDNA

was used to identify chromosomes 3 and 6 (Fig. 1d and 1m).

For A. longiglumis, the (AC)10 probe produced signals in the interstitial region of

the long arms as well as close to the pericentromeric region in all chromosomes except

Page 9 of 40


Genome

Draft

10

3 and 6 (Fig. 1e and 1m). Chromosomes 1 and 3 showed signals with the 45S and 5S

rDNA probes (1f and 1m). For A. canariensis, the (AC)10 probe produced strong signals

in the interstitial regions of all chromosomes except number 6 (Fig. 1g and 1m). One

strong signal on chromosome 1 was produced near the 45S rDNA loci (Fig. 1h and 1m).

In contrast, no signals were detected near the 45S rDNA loci of chromosome 3.

Chromosome 7 also returned a strong signal at the end of the short arm. On the

whole, signals in A. longiglumis and A. canariensis were less intense than those

observed in the other two A-genome species.

A. eriantha (Fig. 1i and 1m) and A. ventricosa (Fig. 1k and 1m) showed strong

similarity in their signal patterns. The (AC)10 probe produced clear, bright signals in the

pericentromeric regions of all the chromosomes of these C-genome species. Scattered

signals were evident in most of the chromosomes, especially on the short arms of

chromosomes 6 and 7.

(AC)10-FISH signal patterns in tetraploid species

Sequential FISH analysis was performed on the AACC-tetraploid species A. magna and

A. murphyi, first using the (AC)10 probe on mitotic metaphase chromosomes (Fig. 2a

and 2c), and then using the 45S and Am1 probes simultaneously on the same cells

(Figs. 2b and 2d). This allowed the chromosome of these two species to be assigned to

either the A or C genome. The chromosomes were numbered 1-14 according to the

nomenclature of Linares et al. (1996). This was based on the chromosome arm ratio,

relative length, and FISH patterns for the four repetitive probes. The resulting

karyotype was taken as the reference karyotype (Fig. 2e).

Page 10 of 40


Genome

Draft

11

In both A. magna and A. murphyi, the A-genome chromosomes showed

discrete (AC)10 signals in the interstitial regions, whereas the C-genome chromosomes

showed strong (AC)10 signals in the pericentromeric regions. Comparison of the A-

genome chromosomes of these tetraploid species revealed differences in the number

of signals and in the intensity of some of these. In the karyotype of A. magna more

discrete signals were seen than in that of A. murphyi. Several chromosomes, such as

6A, 11A and 13A, each showed two signals of similar intensity. In contrast, no A-

genome chromosome of the A. murphyi showed more than one signal, and the

intensity of these signals was highly variable across chromosomes. Very conspicuous

signals were recorded in the short arm of chromosomes 12A and 13A. It should be

noted that the signal patterns of the A-genome chromosomes of these two species are

more similar in their intensity to those of A. longiglumis and A. canariensis than to

those of A. strigosa and A. damascena. Very strong signals were seen on all C-genome

chromosomes of both species, covering a large area of the pericentromeric regions.

Clear signals were also seen at the ends of the long arms of chromosomes 2C and 4C of

A. magna, and on those of 2C and 6C of A. murphyi. These chromosomes also showed

strong Am1 signals (related to heterochromatin) in similar positions.

(AC)10-FISH signal patterns in hexaploid species

The usefulness of the (AC)10 probe in karyotyping the two AACCDD hexaploid species

of cultivated oats - i.e., three cultivars of A. sativa (Figs. 3a, 3d, and Fig. 4) and two of

A. byzantina - was tested (Fig. 4). Both simultaneous and sequential FISH was then

performed using on the same cells, As120a or Am1, in combination with a ribosomal

Page 11 of 40


Genome

Draft

12

probe (45S or 5S) (Fig. 3b, 3e, 3f and 4). This allowed all the chromosomes to be

assigned to the A, C or D genome. The nomenclature used for the reference hexaploid

karyotype (Fig. 4) was that proposed by Sanz et al. (2010) who used the same

repetitive probes as in the present work.

Most of both the A- and D-genome chromosomes showed discrete signals of

(AC)10 sequences in the interstitial regions. The signals in the C-genome

chromosomes, however, covered a large region either side of the centromeres. For all

the cultivars analyzed, the (AC)10 hybridization signals were less intense on the D-

genome chromosomes than the A-genome chromosomes (Fig. 4). Indeed, the

hybridization of this chromosome marker differentiated every chromosome of each

cultivar (Fig. 4). The chromosomes of all five cultivars were further examined in an

attempt to identify diagnostic signals common to them all. In A. sativa, chromosomes

8A, 13A, 15A, 16A and 19A showed either interstitial, pericentromeric or terminal

signals common to all three of its cultivars, although differences in intensity were

evident (e.g., in the interstitial signal on chromosome 8A, and in the pericentromeric

signal on chromosome 15A). Chromosomes 11A, 15A and 19A of A. byzantina showed

identical signal patterns in both the cultivars analyzed. Moreover, the signals seen on

chromosomes 15A and 19A were the same in both A. byzantina and A. sativa.

The C-genome chromosomes showed strong (AC)10 signals in the

pericentromeric region of every chromosome (Fig. 4). In addition, distinct signals were

observed in the interstitial and terminal regions of the long arms of some

chromosomes, with some intra- and interspecific variation observed. Common signals

in the interstitial regions of the long arms appeared on chromosome 5C of all five

Page 12 of 40


Genome

Draft

13

analyzed cultivars, and more faintly on chromosome 4C of the A. sativa cultivars. The

(AC)10–FISH distribution pattern on the D-genome chromosomes was similar to that

observed on the A-genome chromosomes (Fig. 4), although the signals were less

intense. Among the three A. sativa cultivars, similar patterns were found on

chromosomes 10D, 14D, and 20D. The two A. byzantina cultivars showed similar

patterns for chromosomes 10D, 20D and 21D. A comparison of the A. sativa and A.

byzantina signal patterns revealed strong similarities between chromosomes 10D and

20D. By contrast, an important difference between the two species affected

chromosome 21D. A. byzantina showed a conspicuous signal on the short arm of this

chromosome which was absent in A. sativa. Other minor differences affecting other

chromosomes were also seen between the two species; for example, hybridization

signals were terminally located on chromosome 9D of A. byzantina but interstitially

located on that of A. sativa.

Discussion

The (AC)10 regions in the diploid and polyploid species of Avena mainly appeared as

sharp, thin bands in the pericentromeric and interstitial positions of the A- and D-

genome chromosomes. These regions have been characterized as euchromatin

(Fominaya et al. 1988a; Linares et al. 1992; Jellen et al. 1993a, 1993b; Shelukhina et al.

2008b; Badaeva et al. 2011). This agrees with observations in barley and Rumex

acetosa, in which the distribution of (AC)10-FISH signals and euchromatic regions

appear closely correlated (Cuadrado et al. 2008; Kejnovský et al. 2013). This suggests

that blocks of this microsatellite lie within regions containing single copy sequences. In

the present work, however, the location of the (AC)10 hybridization signals in the C-

Page 13 of 40


Genome

Draft

14

genome chromosomes was very similar to the C-banding of the centromeric and

pericentromeric regions. This indicates that (AC)10 is either a component of these

regions or of similar sequences of C-banded heterochromatin (Fominaya et al. 1988b;

Linares et al. 1992; Jellen et al. 1993a, 1993b; Shelukhina et al. 2008b; Badaeva et al.

2011). However, no strict correlation was seen between (AC)10 and the C-bands in

terminal positions. Thus, the terminal C-bands of 3C and 5C in hexaploid species must

be composed of sequences others than (AC)10.

The somatic chromosomes of A. sativa and A. byzantina have been

characterized by C-banding (Linares et al. 1992; Jellen et al. 1993a, 1993b; Badaeva et

al. 2011). FISH and GISH techniques have also been used to make detailed descriptions

of them (Chen and Armstrong 1994; Jellen et al. 1994; Leggett and Markhand 1995;

Linares et al. 1998; Linares et al. 2000, 2001; Sanz et al. 2010). However, these studies

failed to detect chromosome polymorphism among cultivars, except where large

chromosome arrangements had occurred (Jellen and Beard 2000; Sanz et al. 2010).

The existance of intra-cultivar variation detected could raise doubts on the validity of

using information provided by SSR-FISH to make inferences on inter-species

differentiation. Only highly conserved signal patterns in chromosomes of different

cultivars of one species should be used for detect interspecific polymorphism. In the

present study, contrasted interspecific polymorphisms were observed fairly frequently

(Fig. 4). The (AC)10 pattern on chromosome 21D, with a strong signal in the terminal

region of the short arm in the cultivars of A. byzantina but absent from those of A.

sativa, differentiates these species. In addition, this chromosome showed a unique

(AC)10 pattern in the non-terminal region of the short arm of A. sativa cv. Araceli.

Moreover, changes were evident in the position of several hybridization signals in A.

Page 14 of 40


Genome

Draft

15

byzantina cv. Kanota compared to A. sativa. These seem to follow a pattern in which

the Kanota signals lie in a terminal position, whereas those of the others cultivars are

in interstitial positions (e.g., on the long arm of chromosome 17A and both arms of 8A

and 9D chromosomes). The polymorphisms observed could have been caused by

changes in the number of copies of the microsatellite (e.g., via amplifications or

deletions) during the differentiation of the species and less likely in that affecting

cultivars (Kalia et al. 2011). However, determining the chromosome distribution of

SSRs in different cultivars allowed to evaluate the cytogenetic diversity within each

Avena species. This is especially significant in species where chromosome

rearrangements have played an important role in the differentiation within and

between species (Jellen and Beard 2000; Sanz et al. 2010).

The existance of translocations and inversions between parents used in genetic

mapping have complicated the obtention of a robust consensus genetic map in oats

(Oliver et al. 2013; Chaffin et al. 2016). Detection of changes in signal position of SSRs

in those parental lines, migth contribute to establish a more complete assignments of

linkage groups to specific chromosomes and explain some uneven distribution of

markers along the linkage groups. The present work using (AC)10-FISH provides

evidence of new large translocations. A. sativa cv. Araceli carries a translocation

involving the long arm of chromosome 17A and the short arm of 21D (Figs. 3e, 3f, and

4). This translocation was detected using C-genome specific sequences (Am1) and

ribosomal 45S, but the location of the (AC)10 hybridization signals helped to outline the

interchange (Figs. 4 and 5). This cultivar has not taken part of any mapping population

but if a similar translocation affected to a parental line the consensus linkage groups

merged 18 and merged 28 would be likely altered (Chaffin et al. 2016; Yan et al. 2016).

Page 15 of 40


Genome

Draft

16

A second intergenomic translocation detected by (AC)10-FISH affected the satellite of

chromosome 21D and the long arm of chromosome 4C in the two cultivars of A.

byzantina (Fig. 6). This 4CL-21DS translocation might explain the lack of markers and

the short genetic distances observed for chromosome 21D in the map for the cross

between A. sativa cv. Ogle and A. byzantina cv. Kanota (Oliver et al. 2013). Thus, It is

noteworthy that (AC)10–FISH allowed the detection of the 4CL-21DS intergenomic

translocation, but the detection of other putative chromosome rearrangements

between these two cultivars is no less interesting. For example, that likely affecting

chromosome 9D might explain the reduced genetic distance observed in the

corresponding linkage group KO17 (Oliver et al. 2013).

Sanz et al. (2010) classified the different types of intergenomic translocations

seen in the cultivars of A. byzantina and A. sativa into three classes: (i) common

translocations, present in homologous chromosomes of all closely related hexaploid

oat species; (ii) species-specific translocations involving certain chromosomes,

exclusive to each hexaploid species; and (iii) cultivar-specific, exclusive to some

cultivars of crop oats. Since the new intergenomic translocation 17AL-21DS (Fig. 5) is

seen only in A. sativa cv. Araceli, its origin must lie within the breeding programme for

this cultivar, and should therefore be identified as cultivar-specific. In contrast, the

intergenomic translocation 4CL-21DS detected in A. byzantina should be identified as

species-specific.

Based on molecular cytogenetic studies, a genomic designation of CCDD has

been suggested for the tetraploid species involved in the formation of the AACCDD-

genome hexaploid species (see Fominaya et al. 2006). The change was proposed since

Page 16 of 40


Genome

Draft

17

the species thought to be the donor of the A genome, A. strigosa, had repetitive

sequences that failed to hybridize with the chromosomes of AACC tetraploids, but did

hybridize with those of the AACCDD hexaploids (Linares et al. 1998). In the present

work, the similar degree of (AC)10 signalling among chromosomes of the reference A

genome of A. magna and the D-genome chromosomes of the hexaploid species may

indicate close proximity between these genomes, even though no individual

chromosome maintained an (AC)10 pattern in common with those seen in the

tetraploid and hexaploid species. This was likely due to the many rearrangements that

have occurred during the evolution of the genus (Chen and Armstrong 1994; Jellen et

al. 1994; Leggett and Markhand 1995; Linares et al. 1998; Linares et al. 2000, 2001;

Sanz et al. 2010). However, the hybridization patterns observed for the 45S and Am1

repetitive sequences on chromosome 10A of the tetraploid species A. magna (Fig. 2)

were identical to those on chromosome 21D of the hexaploid oat species, reinforcing

the idea that a close relationship exists between the A genome of the reference A.

magna genome and the D genome of the reference A. sativa genome (Fig. 4). In

contrast, no similar hybridization patterns were observed in the other tetraploid

species A. murphyi. The above observation regarding A. magna is supported by

comparisons of molecular linkage maps (Oliver et al. 2011b). For example, a region

containing several crown rust resistant QTLs, which are anchored to chromosome 9D

of hexaploid oats (Jackson et al. 2009), was found in the genetic map constructed from

an A. magna recombinant inbred line population. One A. magna SNP has been mapped

to chromosome 20D of the physically-anchored consensus map of hexaploid oats

(Oliver et al. 2013).

Page 17 of 40


Genome

Draft

18

Indirect observations argue against the involvement of A. strigosa in the

formation of the ancestral tetraploid AACC. Molecular studies have revealed the

dissimilarity between the A. strigosa genome and the A genome of tetraploid oat

species (Linares et al. 1998; Peng et al. 2010; Oliver et al. 2011a). Chromosome pairing

studies of the hybrid between a synthetic allotetraploid (involving A. strigosa and A.

clauda) and A. magna showed highly irregular pairing, indicating that the two A.

magna genomes differed considerably from those of the diploid species involved in the

tetraploid (Ladizinsky 2012). Genotypic by sequencing (GBS) has recently returned a

large SNP dataset for numerous accessions belonging to species with different

genomes and levels of ploidy. Comparing genetic distances between in silico

tetraploids (obtained by combining information for the A. strigosa SNPs and two C-

genome species) and A. magna, Chew et al. (2016) raised serious doubts about the

involvement of A. strigosa in the ancestral tetraploid AACC. If it is not involved, the D-

genome donor of the hexaploid species must have been the tetraploid species A.

magna or a closely related species. Thus, the hexaploid forms might came into

existence via the allopolyploid convergence of species with the CCDD genome and

others with the A genome. In addition, Chew et al. (2016) came to the conclusion that

both the tetraploidization and hexaploidization events may have occurred in

northwest Africa, followed by the radiation of the hexaploid oats to their current

worldwide distribution. The results of the above authors' analyses revealed strong

genetic similarity between the in silico and extant hexaploids, suggesting an ancestral

hexaploid oat participated in the creation of different hexaploid Avena species. More

recently, Yan et al. (2016) extent substantially the Avena GBS studies and present

strong evidences to justify the CCDD genome identity of the tetraploids species A.

Page 18 of 40


Genome

Draft

19

insularis, A. magna (A. maroccana) and A. murphyi. These authors examine the genetic

similarities of fifty-one possible combinations of diploids with tetraploids using GBS

markers and demonstrate that all in silico hexaploid comprised of CCDD genome

tetraploid plus A-genome diploids clustered closely with the extant hexaploids. The

closest in silico matches to the extant hexaploids included the CCDD tetraploids plus A.

longiglumis which led to the authors to suggest a possible role of these species in the

formation of the hexaploids. The similarity of intensity in (AC)10-FISH signals of the A.

longiglumis accession studied here and the A (D) genome of the tetraploids would not

agree with that proposal. However, more A. longiglumis accessions should be studied

study to clarify this.

In conclusion, (AC)10-FISH patterns have allowed differenciate all the

chromosomes of the Avena species analyzed. This has resulted in more complete and

specific karyotypes than the obtained using C-banding and FISH with the so far

available repetitive probes. However, it will be interesting to extent this study to

several accessions of diploid and tetraploid species for quantifying the magnitude of of

intra- and interspecific variation. Moreover, the analysis carried out on hexaploid

species shows the utility of this SSR to identify new translocations undetected by the

other karyotyping methods. Increasing the number of microsatellites used in FISH-

based studies on oats might provide valuable information on the structure of the

genomes and chromosomes of this genus, as well as help explain the incongruities of

genetic oat maps. The economical and easy applicability of this technique compared to

conventional FISH involving cloned sequences will facilitate this task.

Page 19 of 40


Genome

Draft

20

Acknowledgements

The authors thank the Ministerio de Ciencia e Innovación of Spain (AGL210-17042) and the

Universidad de Alcalá (UAH GC2014-002 and UAH CCG2014/EXP-068) for their support of

this work.

References

Badaeva, E.D., Loskutov, I.G., Shelukhina, O.Y., and Pukhalsky, V.A. 2005. Cytogenetic

analysis of diploid Avena L. species containing the As genome. Russ J Genet 41:

1428-1433.

Badaeva, E.D., Shelukhina, O.Y., Diederichsen, A., Lokustov, I.G., and Pukhalskiy, V.A.

2010. Comparative cytogenetic analysis of Avena macrostachya and diploid C-

genome Avena species. Genome 53: 125–137.

Badaeva, E.D., Shelukhina, O.Y., Dedkova, O.S., Loskutov, I.G., and Pukhalsky, V.A.

2011. Comparative cytogenetic analysis of hexaploid Avena L species. Russ J

Genet 47: 691-702.

Becher, R. 2007. EST-derived microsatellites as a rich source of molecular markers for

oats. Plant Breeding 126: 274—278.

Carmona, C., Friero, E., de Bustos, A., Jouve, N., and Cuadrado, A. 2013. Cytogenetic

diversity of SSR motifs within and between Hordeum species carrying the H

genome: H. vulgare L. and H. bulbosum L. Theor Appl Genet 126: 949–961.

Chaffin, A.S., Huang, Y.F., Smith, S., Bekele, W.A., Babiker, E., Gnanesh, B.N., Foresman,

B.J., Blanchard, S.G., Jay, J.J., Reid, R.W., Wight, C.P., Chao, S., Oliver, R.,

Islamovic, E., Kolb, F.L., McCartney, C., Mitchell Fetch, J.W., Beattie, A.D.,

Bjornstad, Å., Bonman, J.M., Langdon, T., Howarth, C.J., Brouwer, C.R., Jellen,

Page 20 of 40


Genome

Draft

21

E.R., Klos, K.E., Poland, J.A., Hsieh, T-F, Brown, R., Jackson, E., Schlueter, J.A.,

and Tinker, N.A. 2016. A consensus map in cultivated hexaploid oat reveals

conserved grass synteny with substantial sub-genome rearrangement. Plant

Gen. 2016; doi: 10.3835/plantgenome2015.10.0102

Chen, Q., and Armstrong, K. 1994. Genomic in situ hybridization in Avena sativa.

Genome 37: 607–612.

Chew, P., Meade, K., Hayes, A., Harjes, C., Bao, Y., Beattie, A.D., Puddephat, I., Gusmini,

G., and Tanksley, S.D. 2016. A study on the genetic relationships of Avena taxa

and the origins of hexaploid oat. Theor Appl Genet 129:1405–1415.

Cuadrado, A., Cardoso, M., and Jouve, N. 2008. Physical organization of simple

sequence repeats (SSRs) in Triticeae: structural, functional and evolutionary

implications. Cytogenet Genome Res 120: 210-219.

Cuadrado, A., and Jouve, N. 2010. Chromosomal detection of simple sequence repeats

(SSRs) using nondenaturing FISH (ND-FISH). Chromosoma 119: 495-503.

Da-Silva, P.R., Milach, S.C.K., and Tisian, L.M. 2011. Transferability and utility of White

oat (Avena sativa) microsatellite markers for genetic studies in black oat (Avena

strigosa). Genet. Mol. Res. 10: 2916-2923.

Diederichsen, A. 2008. Assessments of genetic diversity within a world collection of

cultivated hexaploid oat (Avena sativa L.) based on qualitative morphological

characters. Genet. Resour. Crop. Evol. 55: 419-440.

Fominaya, A., Vega, C., and Ferrer, E. 1988a. Giemsa C-Banded karyotypes of Avena

species. Genome 30: 627–632.

Fominaya, A., Vega, C., and Ferrer, E. 1988b. C-banding and nucleolar activity of

tetraploid Avena species. Genome 30: 633–638.

Page 21 of 40


Genome

Draft

22

Fominaya, A., Hueros, G., Loarce, Y., and Ferrer, E. 1995. Chromosomal distribution of

a repeated DNA from C-genome heterochromatin and the identification of a

new locus ribosomal DNA locus in the Avena genus. Genome 38: 548-557.

Fominaya, A., Loarce, Y., Irigoyen, M.L., and Ferrer, E. 2006. Molecular genetic and

cytogenetic evidences supporting the genomic relationships of the genus

Avena. In Plant genome: biodiversity and evolution, Vol 1, Part D Phanerogams

(Gymnosperm) and (Angiosperm-Monocotyledons). Edited by A.K. Sharma and

A. Sharma. ISBN 978-1-57808-402-3, Science Publishers (USA) pp. 305-324.

Fominaya, A., Loarce, Y., González, J.M., Ferrer, E. 2016. Tyramide Signal

Amplification-Fluorescence in situ hybridization for identifying homoeologous

chromosomes. In Plant Cytogenetics, Methods and Protocols. Methods in

Molecular Biology vol. 1429. Edited by S.F. Kianian and P.M.A. Kianian. ISBN 978-

1-4939-3620-5, Springer Science + Business Media, New York (USA) pp. 35-48.

Fuentes, F.F., Martinez, E.A., Hinrichsen, P.V., Jellen, E.N., and Maughan, P.J. 2009.

Assessment of genetic diversity patterns in Chilean quinoa (Chenopodium

quinoa Willd.) germplasm using multiplex fluorescent microsatellite markers.

Conservation Genetics 10: 369–377.

Gerlach, W.L., and Dyer, T.A. 1980. Sequence organization of the repeated units in the

nucleus of the wheat which contains 5S-rRNA genes. Nucleic Acids Res 8: 4851-

4865.

Han Y., Zhang, T., Thammapichai, P., Weng, Y., and Jiang, J. 2015. Chromosome-specific

painting in Cucumis species using bulked oligonucleotides. Genetics 200: 771-

779.

Page 22 of 40


Genome

Draft

23

He, H., and Bjørnstad, Å. 2012. Diversity of North European oat analyzed by SSR, AFLP

and DArT markers. Theor Appl Genet 125: 57–70.

Irigoyen, M.L., Loarce, Y., Linares, C., Ferrer, E., Leggett, J.M., and Fominaya, A. 2001.

Discrimination of the closely related A and B genomes in tetraploid species of

Avena. Theor Appl Genet 103: 1160-1166.

Jackson, E., Jellen, E., Oliver, R., Lazo, G., Tinker, N., Rossnagel, B., Anderson, J., and

Bonman, J.M. 2009. Resolving the oat mapping story by weaving together a

consensus map. In: Proc Plant Animal Gen Conf XVII. Edited by N.A. Tinker. San

Diego, W337.

Jannink, J.L., and Gardner, S.W. 2005. Expanding the pool of PCR based markers for

oat. Crop Sci. 45: 2383—2387.

Jellen, E.N., Phillips, R.L., and Rines, H.W. 1993a. C-banded karyotypes and

polymorphisms in hexaploid oat accessions (Avena spp.) using Wright´s stain.

Genome 36: 1129-1137.

Jellen, E.N., Rooney, W.L., Phillips, R.L., and Rines, H.W. 1993b. Characterization of the

hexaploid oat Avena byzantina cv. Kanota monosomic series using C-banding

and RFLPs. Genome 36: 962-970.

Jellen, E.N., Gill, B.S., and Cox, T.S. 1994. Genomic in situ hybridization differentiates

between A/D- and C-genome chromatin and detects intergenomic

translocations in polyploid oat species (genus Avena). Genome 37: 613–618.

Jellen, E.N., and Beard, J. 2000. Geographical distribution of a chromosome 7C and 17

intergenomic translocation in cultivated oat. Crop Sci 40: 256-263.

Kalia, R.K., Rai, M.K., Kalia, S., Singh, R., and Dahwan, A.K. 2011. Microsatellite markers:

an overview of the recent progress in plants. Euphytica 177: 309-334.

Page 23 of 40


Genome

Draft

24

Katsiotis, A., Schmidt, T., and Heslop-Harrison J.S. 1996. Chromosomal and genomic

organization of Ty1-copia-like retrotransposon sequences in the genus Avena.

Genome 39:410–417.

Kejnovský, E., Michalovova, M., Steflova, P., Kejnovska, I., Manzano, S., Hobza, R.,

Kubat, Z., Kovarik, J., Jamilena, M., and Vyskot, B. 2013. Expansion of

Microsatellites on Evolutionary Young Y Chromosome. Plos One 8 (1) e45519.

Ladizinsky, G. 2012. Studies in oat evolution. Springer briefs in agriculture. ISBN 978-3-

642-30546-7. Springer New York. pp 1-87.

Leggett, J.M., and Markhand, G.S. 1995. The genomic structure of Avena revealed by

GISH. In Kew Chromosome Conference IV, Edited by P.E. Brandham and M.D.

Bennett. London, pp. 133–139.

Li, C.D., Rossnagel, B.G., and Scoles, G.J. 2000. The development of oat microsatellite

markers and their use in identifying relationships among Avena species and oat

cultivars. Theor Appl Genet 101:1259-1268.

Li, W.T., Peng, Y.Y., Wei, Y.M., Baum, B.R., and Zheng, Y.L. 2009. Relationship among

Avena species as revealed by consensus chloroplast simple sequence repeat

(ccSSR) markers. Genet. Resour. Crop. Evol. 56: 465–480.

Linares, C., Vega, C., Ferrer, E., and Fominaya, A. 1992. Identification of C-banded

chromosomes in meiosis and the analysis of nucleolar activity in Avena

byzantina C. Koch cv. ‘Kanota’. Theor Appl Genet 83:650–654.

Linares, C., González, J., Ferrer, E., and Fominaya, A. 1996. The use of double

fluorescence in situ hybridization to physically map the positions of 5S rDNA

genes in relation to the chromosomal location of 18S-5.8S-26S rDNA and a C

genome specific DNA sequence in the genus Avena. Genome 39: 535–542.

Page 24 of 40


Genome

Draft

25

Linares, C., Ferrer, E., and Fominaya, A. 1998. Discrimination of the closely related A

and D genomes of the hexaploid oat Avena sativa L. Proc Nat Acad Sci USA 95:

12450–12455.

Linares, C., Irigoyen, M.L., and Fominaya, A. 2000. Identification of C-genome

chromosomes involved in intergenomic translocations in Avena sativa L., using

cloned repetitive DNA sequences. Theor Appl Genet 100: 353-360.

Linares, C., Loarce, Y., Serna, A., and Fominaya, A. 2001. Isolation and characterization

of two novel retrotransposon of the Ty1-copia group in oat genomes.

Chromosoma 110: 115-123.

Loskutov, I.,and Rines, H.W. 2011. Avena. In: Wild crop relatives: genomic and

breeding resources: cereals. Edited by Springer, New York, pp 109–184

Luo, X., Zhang, H., Kang, H., Fan, X., Wang, Y., Sha, L., and Zhou, Y. 2014. Exploring the

origin of the D genome of oat by fluorescence in situ hybridization. Genome 57:

469–472.

Luo, X., Tinker, N.A, Zhang, H., Wight, C.P, Kang, H., Fan, X., Wang, Y., Sha, L., and Zhou,

Y. 2015. Centromeric position and genomic allocation of a repetitive sequence

isolated from chromosome 18D of hexaploid oat, Avena sativa L. Genet Resour

Crop Evol 62:1–4

Molnár, I., Cifuentes, M., Schneider, A., Benavente, E., and Molnár-Lang, M. 2011.

Association between simple sequence repeat-rich chromosome regions and

intergenomic translocation breakpoints in natural populations of allopolyploid

wild wheats. Annals of Botany 107: 65-76.

Page 25 of 40


Genome

Draft

26

Oliveira, E.J., Padua, J.G., Zucchi, M.I., Vencovsky, R., and Vieira, M.L.C. 2006. Origin,

evolution and genome distribution of microsatellites. Genet Mol Bio 29: 294–

307.

Oliver, R.E., Obert, D.E., Hu, G., Bonman, J.M., O’Leary-Jepsen, E., and Jackson, E.W.

2010. Development of oat-based markers from barley and wheat

microsatellites. Genome 53: 458–471.

Oliver, R.E., Lazo, G.R., Lutz, J.D., Rubenfield, M.J., Tinker, N.A., Anderson, J.M.,

Morehead, N.H.W., Adhikary, D., Jellen, E.N., Maughan, P.J., Guedira, G.L.B.,

Chao, S., Beattie, A.D., Carson, M.L., Rines, H.W., Obert, D.E., Bonman, J.M., and

Jackson, E.W. 2011a. Model SNP development for complex genomes based on

hexaploid oat using highthroughput 454 sequencing technology. BMC

Genomics 12: 77.

Oliver, R.E., Jellen, E.N., Ladizinsky, G., Korol, A.B., Kilian, A., Beard, J.L., Dumlupinar, Z.,

Wisniewski-Morehead, N.H., Svedin, E., and Coon, M. 2011b. New diversity

arrays technology (DArT) markers for tetraploid oat (Avena magna Murphy et

Terrell) provide the first complete oat linkage map and markers linked to

domestication genes from hexaploid A. sativa L. Theor. Appl. Genet. 123:

1159–1171.

Oliver, R.E., Tinker, N.A., Lazo, G.R., Chao, S., Jellen, E.N., Carson, M.L., Rines, H.W.,

Obert, D.E., Lutz, J.D., Shackelford, I., Korol, A.B., Wight, C.P., Gardner, K.M.,

Hattori, J., Beattie, A.D., Bjørnstad, Å., Bonman, J.M., Jannink, J-L., Sorrells,

M.E., Guedira, G.L.B., Fetch, J.W.M., Harrison, S.A., Howarth, C.J., Ibrahim, A.,

Kolb, F.L., McMullen, M.S., Murphy, J.P., Ohm, H.W., Rossnagel, B.G., Yan, W.,

Miclaus, K.J., Hiller, J., Maughan, P.J., Hulse, R.R.R., Anderson, J.M., Islamovic,

Page 26 of 40


Genome

Draft

27

E., and Jackson, E.W. 2013. SNP discovery and chromosome anchoring provide

the first physically anchored hexaploid oat map and reveal synteny with model

species. PLoS ONE 8: e58068.

Pal, N., Sandhu, J.S., Domier, L.L., and Kolb, F.L. 2002. Development and

characterization of microsatellite and RFLP derived PCR markers in oat. Crop Sci

42: 912—918.

Peng, Y-Y., Baum, B.R., Ren, C-Z., Jiang, Q-T., Chen, G-Y., Zheng, Y-L., and Wei, Y-M.

2010. The evolution pattern of rDNA ITS in Avena and phylogenetic relationship

of the Avena species (Poaceae: Aveneae). Hereditas 147: 183–204.

Pinto, L.R., Vieira, M.L.C., Souza, Jr. C.L,. and Souza, A.P. 2003. Genetic-diversity

assessed by microsatellites in tropical maize populations submitted to a high-

intensity reciprocal recurrent selection. Euphytica 134: 277-286.

Rajhathy, T., and Thomas, H. 1974. Cytogenetics of oats (Arena L.). Misc Publ Genet

Can 2:1-90.

Rines, H.W., Porter, H.L., Carson, M.L., and Ochocki, G.E. 2007. Introgression of crown

rust resistance from diploid oat Avena strigosa by two methods: direct crosses

and through and initial 2x_4x synthetic hexaploid. Euphytica 158: 67-79.

Rodionova, N.A., Soldatov, V.N., Merezhko, V.E., Jarosh, N.P., Kobyljanskij, V.D. 1994.

Flora of cultivated plants, Vol. 2, Part 3, Oat. Kolos, Moscow.

Sanz, M., Jellen, E., Loarce, Y., Irigoyen, M.L., and Fominaya, A. 2010. A new

chromosome nomenclature system for oat (Avena sativa L. and A. byzantina C.

Koch) based on FISH analysis of monosomic lines. Theor Appl Genet 12: 1541–

1552.

Page 27 of 40


Genome

Draft

28

Sanz, M.J., Loarce, N., Ferrer, E., and Fominaya, A. 2012. Use of Tyramide-Fluorescence

in situ hybridization and chromosome microdissection for ascertaining

homology relationships and chromosome linkage group associations in oats.

Cytogenet Genome Res 136: 145-156.

Shelukhina, O.Y., Badaeva, E.D., Loskutov, I.G., and Pukhalsky, V.A. 2007. A

comparative cytogenetic study of the tetraploid oat species with the A and C

genomes: Avena insularis, A. magna, A. murphyi. Genetika 43: 747–761.

Shelukhina, O.Y., Badaeva, E.D., Brezhneva, T.A., Loskutov, I.G., and Pukhalsky, V.A.

2008a. Comparative analysis of diploid species of Avena L. using cytogenetic

and biochemical markers: Avena canariensis Baum et Fedak and A. longiglumis

Dur. Russ J Genet 44: 694–701.

Shelukhina, O.Y., Badaeva, E.D., Brezhneva, T.A., Loskutov, I.G., and Pukhalsky, V.A.

2008b. Comparative analysis of diploid species of Avena L. Using cytogenetic

and biochemical markers: Avena pilosa M. B. and A. clauda Dur. Russ J Genet

44: 1087–1091.

Solano, R., Hueros, G., Fominaya, A., and Ferrer, E. 1992. Organization of repeated

sequences in species of the genus Avena. Theor Appl Genet 83: 602-607.

Song, G., Huo, P., Wu, B., and Zhang, Z. 2015. A genetic linkage map of hexaploid naked

oat constructed with SSR markers. The Crop Journal 3: 353-357.

Thomas, H. 1992. Cytogenetics of Avena. In: Oat science and technology. Agronomy

Monographs No. 33. Edited by H.G. Marshall and M.E. Sorrells. ASA, CSSA, SSSA,

Madison, WI, USA, pp 473–507.

Tomás, D., Rodrigues, J., Varela, A., Veloso, M.M., Viegas, W., and Silva, M. 2016. Use

of Repetitive sequences for molecular and cytogenetic characterization of

Page 28 of 40


Genome

Draft

29

Avena species from Portugal. Int. J. Mol. Sci. 7, 203. DOI:

10.3390/ijms17020203.

Wight, C.P., Yan, W., Mitchell Fetch, J., Deyl, J., and Tinker, N.A. 2010. A set of new

simple sequence repeat and avenin DNA markers suitable for mapping and

fingerprinting studies in oat (Avena spp.). Crop Sci 50: 1207-1218.

Yan, H., Bekele, W.A., Wight, C.P., Peng, Y., Langdon, T., Latta, R.G., Fu, Y-F.,

Diederichsen, A., Howarth, C.J., Jellen, E.N., Boyle, B., Wei, Y., and Tinker, N.A.

2016. High‑density marker profiling confirms ancestral genomes of Avena

species and identifies D‑genome chromosomes of hexaploid oat. Theor Appl

Genet DOI 10.1007/s00122-016-2762-7.

Page 29 of 40


Genome

Draft

30

Figure 1.- FISH microphotographs showing the distribution of repetitive sequences on

metaphase chromosomes of diploid species. A. strigosa (a, and b), A. damascena (c,

and d), A. longiglumis (e, and f), A. canariensis (g, and h), A. eriantha (i, and j), and A.

ventricosa (k, and l), afther DAPI staining and in situ hybridization with biotin-labelled

probes (pink) or digoxigenin-labelled probes (green): (a), (c), (e), (g), (i) and (k)

fluorescence in situ hybridization (FISH) with (AC)10 probe; (b), (d), (f), (h), (j), and (l)

simultaneous FISH with p45S and p5S probes. (m) Karyotypes of each diploid species

analysed showing a single chromosome of each homologous group chosen from the

metaphases in a-l. Note that chromosomes carried signals from p45S and p5S are

shown in duplicate on karyotypes at the right side of chromosomes with (AC)10 signals.

Scale bar = 10 µm.

Figure 2.- FISH performed on mitotic metaphase of tetraploid species A. magna (a, and

b), and A. murphyi (c, and d). (a) FISH of biotin-labelled (AC)10 (pink) probe. (b) The

same cell as in a after simultaneous FISH with the biotin-labelled pAm1 (pink) and

digoxigenin-labelled p45S (green) probes. (c) FISH of biotin-labelled (AC)10 (pink) probe.

(d) The same cell as in c after simultaneous FISH with the biotin-labelled pAm1 (pink)

and digoxigenin-labelled p45S (green) probes. (e) Karyotypes showing a single

chromosome of each homologous group chosen from the metaphases in a and c. The

reference karyotypes was made from the metaphases b and d for A. magna and A.

murphyi, respectively. They are based on the hybridization patterns of Am1 (pink) and

45S (green) sequences and following the nomenclature of Linares et al. (1996). Scale

bar = 10 µm.

Page 30 of 40


Genome

Draft

31

Figure 3.- FISH performed on metaphase chromosomes of hexaploid species A. sativa

cvs. Ogle (a) and Araceli (d) using (AC)10 probe (pink). Chromosomes are

counterstained with DAPI. (b) The same cell as in a after double FISH with pAs120a

(green) and p45S (pink) probes. (c) The same cell as in a after rehybridizing with pAm1

(green) and p5S (pink) probes. (e) The same cell as in d after double FISH with pAs120a

(green) and p5S (pink) probes. (f) The same cell as d after rehybridizing with pAm1

(pink) and p45S (green) probes. In e, arrows indicate the translocated chromosome

pair 21D; asterisks indicate the translocated chromosome pair 17A. Scale bar = 10 µm.

Figure 4.- (AC)10-FISH (pink) karyotypes from each of three cultivars of hexaploid

species A. sativa and two cultivars of hexaploid species A. byzantina. Each

chromosome probed with (AC)10 was identified by the hybridization patterns of

repetitive sequences after simultaneous and sequential FISH with pAs120a, pAm1,

p45S and p5S probes and reprobing of the same cell. According to Sanz et al. (2010),

the reference karyotype was based on the hybridization patterns of pAs120a (green),

p45S (pink) and p5S (pink) probes which identified the A-genome chromosomes; the

hybridization patterns of both pAm1 (green) and p5S (pink) wich identified the C-

genome chromosomes; and the hybridization patterns of pAm1 (green), p45S (pink)

and p5S (pink) probes which identified the D-genome chromosomes. Chromosomes of

reference karyotype were chosen from the same metaphase and corresponding to cv.

Ogle (Figs. 3 b, and 3c).

Figure 5.- Diagram showing the formation of a intergenomic translocation detected in

cv. Araceli compared to A. sativa cv. Ogle and affecting to chromosomes 17A and 21D.

Page 31 of 40


Genome

Draft

32

FISH signals using different probes are illustrated. In (a) probes pAs120a, pAm1 and

p45S. In (b) probe (AC)10.

Figure 6.- Diagram showing the formation of the intergenomic translocation in A.

byzantina (compared to A. sativa). (AC)10 signals are illustrated.

Page 32 of 40


Genome

Draft

Table 1. Avena species used in this study, including ploidy level, genomic composition,

accession numbers (or cultivar names) and provider.

Species Ploidy Genomic

composition

Accession Provider

A. strigosa Schreb 2x AsAs PI 258727 National Small Grains Collection, Betsville, USA.

A. damascena 2x AdAd CAV 0258 Plant Research Centre, Ottawa, Canada.

A. longiglumis Dur. 2x AlAl PI 367390 National Small Grains Collection, Betsville, USA.

A. canariensis Baum 2x AcAc WIR-292 N.I. Vavilov Research Institute, Russia.

A. eriantha 2x CpCp PI 657579 National Small Grains Collection, Betsville, USA.

A. ventricosa 2x CvCv PI 657338 National Small Grains Collection, Betsville, USA.

A. magna Murph et Terr 4x AACC PI 659402 National Small Grains Collection, Betsville, USA.

A. murphyi Ladiz. 4x AACC PI 657382 National Small Grains Collection, Betsville, USA.

A. sativa L. cv Ogle 6x AACCDD Plant Research Centre, Ottawa, Canada.

A. sativa L. cv Cobeña 6x AACCDD Cobeña National Institute of seeds, Madrid, Spain.

A. sativa L. cv Araceli 6x AACCDD Araceli National Institute of seeds, Madrid, Spain.

A. byzantina C Koch cv Culta 6x AACCDD WIR 5206 N.I. Vavilov Research Institute, Russia.

A. byzantina C Koch cv Kanota 6x AACCDD 2265 Plant Research Centre,Ottawa, Canada.

Page 33 of 40


Genome

Draft

Page 34 of 40


Genome

Draft

238x340mm (300 x 300 DPI)

Page 35 of 40


Genome

Draft

244x390mm (300 x 300 DPI)

Page 36 of 40


Genome

Draft

158x137mm (300 x 300 DPI)

Page 37 of 40


Genome

Draft

245x163mm (300 x 300 DPI)

Page 38 of 40


Genome

Draft

17A 21D

A.sativa

cv Ogle

A.sativa

cv Araceli

17AL-21DS 21DS-17AL

a

17A 21D

A.sativa

cv Ogle

A.sativa

cv Araceli

b

120a

Dapi

45S Am1 (AC)

10

Page 39 of 40


Genome

Draft

4C 21D

21DS-4CL

A.sativa

A.byzantina

(AC)10

Dapi

Page 40 of 40


Genome

draft - university of toronto t-space · draft chromosomal distribution patterns of the (ac)10...

Documents