Fluorescence in situ hybridization with multiple

Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics

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Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of chromosomal and genomic relationship levels between faba bean (Vicia faba) and its close wild relatives Raina Soom Nath , Khandavilli Kesavacharyulu , Behrouz Shiran & Arman Mahmoudi To cite this article: Raina Soom Nath , Khandavilli Kesavacharyulu , Behrouz Shiran & Arman Mahmoudi (2010) Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of chromosomal and genomic relationship levels between faba bean (Vicia faba) and its close wild relatives, Caryologia, 63:3, 215-222, DOI: 10.1080/00087114.2010.10589730 To link to this article: http://dx.doi.org/10.1080/00087114.2010.10589730

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CARYOLOGIA

Vol. 63, no. 3: 215-222, 2010

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of chromosomal and genomic relationship levels between faba bean (Vicia faba) and its close wild relatives Raina1* Soom Nath, Khandavilli Kesavacharyulu2, Behrouz Shiran3 and Arman Mahmoudi4


1

Laboratory of Cellular and Molecular Cytogenetics, Department of Botany, University of Delhi, Delhi 110 007, India. 2 Present address: Central Sericultural Research and Training Institute, Manandavadi Road, Mysore 570008, India. 3 Department of Crop Science, Faculty of Agriculture, University of Shahr-e-Kord, Shahr-e-Kord 88186-3-4141, Iran. 4 Department of Biology , University of Mazandaran , Babolsar , Iran.

Abstract — The organisation of DNA sequences at chromosomal level in twelve Vicia species, more particularly among V. faba and its close wild relatives (V. narbonensis, V. hyaeniscyamus, V. johannis, V. galilaea, V. bithynica), has been performed using fluorescence in situ hybridization (FISH). The four BamHI discrete size classes of 850, 900, 990 and 1750-bp of highly repetitive DNA isolated from V. faba, and the probes pTa 71 and pTa 794 containing the 18S-5.8S-26S rDNA and 5S rDNA, respectively, were used. The chromosomal location of the two ribosomal gene families distinguished V. faba from its close relatives. FISH to metaphase chromosomes indicates the presence of BamHI family sequences interspersed in both euchromatin and heterochromatin of the V. faba, V. narbonensis, V. hyaeniscyamus, V. galilaea, V. johannis and V. bithynica of taxonomic section Faba, and V. hybrida of section Hypechusa genomes; however, the level of hybridization clearly distinguished the V. faba genome from other species genomes. This analysis provides further evidence that V. faba should be considered as the most distinct. In the context of possible sources of germplasm for V. faba breeding, it is argued that none of the species can be considered as putative allies of faba bean. The repetitive sequences did not hybridize in situ with the chromosomal DNA of five Vicia species of section Vicia and section Cracca. The quantitative variation and/or complete absence of BamHI family repeats enabled differentiation among Vicia genomes. Key words: genome evolution, in situ hybridization, repetitive DNA sequences, 18S/26S and 5S rRNA gene loci, Vicia faba, Vicia species.

INTRODUCTION The important grain legume and forage plant, Vicia faba (subgenus Vicia, section Faba), is one of the cytogenetically best characterized plant

*Corresponding author: phone +91-120-4392838; email: [email protected] [email protected]

species (see FUCHS et al. 1998). The astonishingly large amount of DNA (1C = 13.5 pg) is distributed among only six pairs of one metacentric and five pairs of acrocentrics (RAINA and REES 1983; RAINA and BISHT 1988). Because of many advantages for chromosomal related analysis, many cytogenetic phenomena were observed for the first time in this species (see FUCHS et al. 1998). However, information about the chromosomes, genomes and genetics of V. faba vis-à-vis its close wild relatives and other Vicia species is conflicting (RAINA and REES 1983; RAINA and BISHT 1988;


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RAINA , KESAVACHARYULU , SHIRAN

RAINA et al. 1989; RAINA 1990, 2000; see MAXTED et al. 1991; VAN DE VEN et al. 1993; RAINA and OGIHARA 1994, 1995; BISHT et al. 1998; KOUL et al. 1999). Section Faba has indeed been considered to be the genus Faba but has been recently classified as a section of Vicia. Several others, on the other hand, are of the view that V. faba could be either considered as a separate genus (Faba bona Medikus/Faba vulgaris Moerich) or given subgeneric rank placing faba bean as the only species of the subgenus Faba of genus Vicia (see STANKEVICH 1978). Fluorescence in situ hybridization (FISH) enables physical mapping of repetitive DNA sequences and multicopy gene families on the chromosomes. In situ hybridization of repetitive probes is proving not only to be valuable method for examining chromosome evolution but it also uniquely enables the detailed physical analysis of large scale chromosome structure that underpins such studies (see SHARMA and RAINA 2005). Several types of cloned repetitive DNA sequences derived from V. faba (KATO et al. 1984, 1985; YAKURA et al. 1984; MAGGINI et al. 1991; NOUZOVA et al. 1999) have not so far been utilized for the assessment of chromosomal organisation among species of faba bean gene pools. In the present study, we used FISH with four highly repeated DNA sequences of the BamHI family to analyze genome organization of twelve Vicia species. As an experimental model, we have chosen close wild relatives of the faba bean which could potentially be used in wide crosses with faba bean, and other Vicia species, both wild and cultivated, which are distantly or not so distantly related to V. faba as revealed by morphological features. Further, we physically localized 5S and 18S–5.8S–26S rRNA genes by FISH to enable further comparisons of chromosomal organisation to be made between V. faba and the morphologically closest wild relative of faba bean, V. narbonensis. MATERIALS AND METHODS Twelve Vicia species, belonging to the section Faba sensu KUPICHA (1976), section Hypechusa and section Vicia of subgenus Vicia, and the section Cracca of subgenus Vicilla were selected (Table 1) for the present study. The methods used for chromosome preparations and FISH were described previously (RAINA et al. 1998; RAINA and MUKAI 1999). The six DNA probes used were: (i) BamHI repeat family sequences

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of 850, 900, 990 and 1750-bp isolated from Vicia faba and cloned into the plasmid pBR 322 (KATO et al. 1985); (ii) pTa 71, a highly repeated sequence containing a 8.95-kb EcoRI fragment of the 18S-5.8S-26S rDNA isolated from Triticum aestivum and cloned into pUC 19 (GERLACH and BEDROOK 1979); and (iii) pTa 794, a highly repeated sequence containing a 410-bp BamHI fragment of 5S rDNA isolated from T. aestivum and cloned into pBR 322 (GERLACH and DYER 1980). The hybridization sites of the repetitive DNA sequence (850, 900, 990, 1750-bp) and pTa 794 digoxigenin-labeled probes were detected with sheep antidigoxigenin-FITC (fluorescein isothiocyanate, Boehringer Mannheim) and antidig rhodamine, respectively. The rDNA probe (pTa 71) with directly labeled nucleotide (FITC-dUTP) did not need detection steps. RESULTS Although the double target in situ hybridization with pTa 71 and pTa 794 probes revealed two sites each for both 5S and 18S-5.8S-26S rDNA in V. faba and V. narbonensis, their distribution on the chromosomes explicitly distinguished the genomes of the two species (Fig. 1a). Two 18S5.8S-26S loci with intense signals were located at the NORs of the largest chromosome pair in V. faba, and the smallest one in V. narbonensis. Using image-analysis software IPLAB SPECTRUM H (signal analytics), the chromosomes were identified by size and arm ratio, and FISH sites for 5S rDNA and 18S-5.8S-26S rDNA. Two 5S rDNA sites in V. faba appeared in the satellite of the large metacentric chromosome pair distal to the secondary constriction. In V. narbonensis two sites were located in the short arm of the second chromosome pair. The present finding of number and location of 5S and 18S-5.8S-26S rDNA loci in V. faba, and 5S rDNA loci in V. narbonensis correspond to the results of radioactive in situ patterns observed by KNÄLMANN and BURGER (1977, 1986). Also, the chromosomal location of two ribosomal gene families distinguished V. faba from other species (RAINA et al. 2001) considered to be close wild relatives. All the four DNA probes derived from BamHI family sequences yielded no specific signals on the chromosome regions of Vicia species after FISH. Instead to this, intense labeling to no labeling of all chromosomes was observed (Table 1, Fig. 1bd), indicating that BamHI family sequences are interspersed all over the chromosomes. V. faba

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constriction regions in the five species were devoid of hybridization signals and they characteristically fluoresced red with propidium iodide. V. bithynica and V. hybrida differed from V. faba, V.


had the strongest signals with all the four probes (Fig. 1b). Weak levels of signals were detected in V. narbonensis (Fig. 1c), V. hyaeniscyamus, V. galilaea and V. johannis. The prominent secondary

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Fig. 1 — Fluorescence in situ hybridization of Vicia species with the 18S-5.8S-26S rDNA (green fluorescence) and 5S rDNA (red fluorescence) probes (a); and BamHI repeat family 1750-bp sequence (yellow fluorescence) probe (b-d). (a) Karyograms of V. faba (upper row) and V. narbonensis (lower row); (b) V. faba; (c) V. narbonensis; (d) V. sativa. Note chromosomes of V. sativa are devoid of hybridization signals. Chromosomes were counterstained with DAPI (a) and PI (b-d). The position of secondary constrictions is indicated in b and c. Bar = 10µm.

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narbonensis, V. hyaeniscyamus, V. galilaea and V. johannis in their very weak and inconsistent sites for BamHI repeated sequences. Careful analysis of many metaphase plates showed that non homogeneity of labeling which appeared in a few plates was accidental, because no chromosomal regions showed preferential labeling in a repeatable way. The five species within Sativa and Villosa species complexes were characterized by the absence of hybridization sites for the four probes (Table 1, Fig. 1d). By DAPI staining, it was possible to visualize prominent bands mainly at centromeric and telomeric regions, and at few places in interstitial regions of several Vicia species. The FISH site patterns indicated that these regions did not remain free of hybridization signals. DISCUSSION Nuclear genomes of plant species include repeated DNA sequences, and there is often conservation of these sequences between differ-

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ent species (GRELLET et al. 1986; HALLDEN et al. 1987; RAINA et al. 2005; see RAINA and RANI 2001; see SHARMA and RAINA 2005). In some cases, the presence of repeated sequences from one species in others can be used as an indicator of relationships (GUPTA et al. 1989; ANAMTHAWAT-JONSSON and HESLOP-HARRISON 1992; KAMM et al. 1995; BADAEVA et al. 1996; TSUJIMOTO et al. 1997; see SHARMA and RAINA 2005). FISH of highly repetitive DNA sequences of BamHI family isolated from V. faba has been used in the present study to study the organisation of DNA sequences at chromosomal level in Vicia species. It has been estimated that 21% (3% each of 250, 850, 900, 990, 1150, 1500 and 1750-bp) (~ 2.4 pg) of the total 85% of the repeated DNA sequences in V. faba (FLAVELL et al. 1974) is constituted by BamHI family sequences alone (KATO et al. 1985). These repeats devoid of any long-range internal subrepeats show sequence heterogeneity not only between the various family repeats but also within the single repeat. The 250-bp BamHI family, for example, showed 13-25% sequence

TABLE 1 — The frequency of hybridization signals on chromosomes of Vicia species with Bam HI family repetitive sequence probes. Section (subgenus)

Species

Faba (Vicia)

V. faba V. narbonensis

Species complex

2n

Narbonensis

12 14

2C DNA* amount (× 10–12 g) 27.07 16.11

Probe**

V. hyaeniscyamus

Narbonensis

14

15.8

++

++

++

++

V. johannis

Narbonensis

14

14.14

++

++

++

++

V. galilaea

Narbonensis

14

++

++

++

++

850-bp 900-bp 990-bp 1750-bp + + +*** + + + + + + + + + ++ ++ ++ ++

V. bithynica

14

9.98

+

+

+

+

Hypechusa (Vicia)

V. hybrida

12

16.46

+

+

+

+

Vicia (Vicia)

V. sativa

Sativa

12

4.5

–

–

–

–

V. macrocarpa

Sativa

12

5.4

–

–

–

–

V. amphicarpa

Sativa

14

5.1

–

–

–

–

V. villosa

Villosa

14

4.67

–

–

–

–

V. varia

Villosa

14

3.7

–

–

–

–

Cracca (Vicilla)

* Data from Raina and Rees (1983), Maxted et al. (1991), Bennett and Leitch (1995). ** Bam HI highly repetitive DNA sequences. *** Strong (+ + +), weak (+ +), very weak (+) and no (-) labeling.


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variation among six cloned sequences (KATO et al. 1985). The present results clearly demonstrate that BamHI family sequences of 850, 900, 990 and 1750-bp are dispersed all over the chromosomes of V. faba, V. narbonensis, V. hyaeniscyamus, V. galilaea, V. johannis, V. bithynica and V. hybrida genomes. It is also clear that these sequences do not discriminate between euchromatin, and heterochromatin regions revealed by various cytological techniques (SCHWEIZER 1973; TAKEHISA and UTSUMI 1973; PERRINO and PIGNONE 1981; SINGH and LELLEY 1983; RAMSAY 1984, present study). Previous in situ hybridization with H3 labeled DNA probes of 250 and 1500-bp BamHI family sequences have shown that these repeats are as well dispersed throughout all the metaphase chromosomes of V. faba (YAKURA et al. 1987). In the case of V. faba genome, other types of interspersed highly repetitive sequences in both euchromatin and heterochromatin regions have also been reported (TIMMIS et al. 1975; ROWLAND 1980; CIONINI et al. 1985; NOUZOVA et al. 1999). Despite the abundance of published information on V. faba, there is still considerable uncertainity and speculation concerning its origin. There are four different opinions: (1) V. narbonensis is considered by many authors (BALL 1968; DAVIS and PLITMANN 1970; HANELT et al. 1972; SCHÄFER 1973; KUPICHA 1976) to be the morphologically closest wild relative of V. faba. It is one species of a morphologically closely related group of less common species (V. galilaea, V. hyaeniscyamus, V. johannis, V. serratifolia, V. kalakhensis, V. eristalioides) referred to collectively as Narbonensis species complex (RAINA and OGIHARA 1994, 1995; BENNETT and MAXTED 1997; RAINA et al. 2001). So the putative allies of V. faba include all the taxa within the complex (MAXTED et al. 1991). (2) Other evidences, however, bring out startling differences between V. faba and the taxa within the complex (LADIZINSKY 1975; CUBERO 1981; RAINA and REES 1983; PERRINO et al. 1989; VAN DE VEN et al. 1993; RAINA and OGIHARA 1994, 1995; KOUL et al. 1999; RAINA 2000; JAASKA 1997; LEHT and JAASKA 2002), and the authors conclude that none of the complex species can be considered to be wild progenitor of the faba bean. (3) PERRINO and PIGNONE (1981), POTOKINA et al. (1999) and many Vicia taxonomists suggest that V. bithynica is the closest ally of V. faba in section Faba. The results of RADZHI (1971), MAXTED et al. (1991) and MAXTED (1993),

219

however, indicate that V. bithynica is sufficiently distinct from both V. faba and V. narbonensis. (4) VAN DE VEN et al. (1993) suggest that V. faba is more closely aligned to species from the sections Peregrinae and Hypechusa than to those in Narbonensis species complex. Consequently, in terms of explicit elucidation of wild ancestry of the cultivated pulse, these extensive analyses ended in disappointment. Hybridization to V. faba chromosomes with the four BamHI family DNA sequences in the present study showed strong signals, indicating that these sequences are present as a major class of interspersed repeats. The same DNA probes resulted in a similar distribution pattern in V. narbonensis, V. hyaeniscyamus, V. galilaea and V. johannis under similar stringency conditions but clearly with more scarcely distributed signals. Hybridization to V. bithynica and V. hybrida chromosomes showed even much weaker signals. It seems to us likely that these sequences probably originate from a common ancestral sequence in the genome of a progenitor species of V. faba, the Narbonensis species complex, V. bithynica and V. hybrida; and V. faba genome is more aligned to species from the complex rather than to V. bithynica in section Faba. The wild progenitor or a bridging species could either be extinct, not recognized for what it is or as indicated by ZOHARY (1977) is yet to be collected. It is suggested by MAXTED et al. (1991) that efforts to locate a possible faba bean progenitor should be concentrated in two areas; the Near East, which is centre of diversity of section Faba, and Afghanistan, where most primitive forms of V. faba occur. The distinct quantitative variation of FISH sites for BamHI family sequences among taxa of section Faba could be the result of extensive divergence among these sequences due to mutations, deletions, base substitutions within the genomes of Narbonensis species complex and V. bithynica. No hybridization sites for BamHI family sequences were detected on the chromosomes of the section Vicia taxa belonging to Sativa species complex (V. sativa, V. macrocarpa, V. amphicarpa) and section Cracca taxa belonging to Villosa species complex (V. villosa, V. varia) suggesting that these sequences are restricted to only some species of the genus Vicia; and indicates that the quantitative variation and/or complete absence of BamHI family repeats offers a reliable means of discriminating chromosome complements of Vicia species.

220


Acknowledgements — We thank Dr. K. Yakura, Kanagwa University, Japan, and International Centre for Agricultural Research in Dry Areas (ICARDA), Aleppo, Syria for the supply of BamHI family sequences and seed samples, respectively.


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