Identification of Intergenomic Translocations Involving Wheat, Hordeum Vulgare and Hordeum

Identification of intergenomic translocations involving wheat, Hordeum vulgare and Hordeum chilense chromosomes by FISH.

Prieto, P.1, Ramírez, M.C. 1, Ballesteros, J. 1 and Cabrera, A. 2

1Instituto de Agricultura Sostenible (CSIC), Apdo. 4084, 14080 Córdoba (Spain)

Phone +34 957 499210, Fax +34 957 499252, E-mail:

2Departamento de Genética, ETSIAM, 14080 Córdoba (Spain)

ABSTRACT

Intergenomic translocations between wheat, Hordeum chilense and Hordeum vulgare have been obtained in tritordeum background. Advanced lines from the crosses between three disomic chromosome addition lines for chromosome 2Hv, 3Hv, and 4Hv of barley (Hordeum vulgare) in Triticum aestivum cv. Chinese Spring (CS) and hexaploid tritordeum (2n=6x=42, AABBHchHch) were analyzed. Multicolour FISH using both genomic DNA from H. chilense and H. vulgare were used to establish the presence and numbers of H. vulgare introgressions into tritordeum. Interspecific H. vulgare-H. chilense and intergeneric wheat-H. vulgare and wheat-H. chilense translocations were identified. Frequencies of plants containing different kinds of intergenomic translocations between chromosome arms are presented. These lines can be useful for introgressing into tritordeum characters of interest from H. vulgare.

INTRODUCTION

Interspecific and intergeneric hybrids in the Triticeae are valuable for transfers of genes from one species to another and to extend the range of genetic variation available to plant breeders. For a long time, one goal of cereal breeders has been the production of amphiploids between wheat and barley in order to combine desirable features from both species. Since the first success achieved by KRUSE (1973) crossing cultivated barley and wheat (Hordeum vulgareTriticum aestivum, T. turgidum, and T. monococcum), numerous hybrid combinations between the two genera have been reported (see FEDAK 1992, for a review), mainly involving non-cultivated Hordeum species. Among these, H. chilense is a wild barley from Chile and Argentina with high cross-ability with other Triticeae genera (FEDAK 1992). Crosses between H. chilense and diploid, tetraploid and hexaploid wheats give rise to tetraploid, hexaploid and octoploid tritordeums, respectively (MARTÍN and SÁNCHEZ-MONGE LAGUNA 1982, MARTÍN et al. 1987 CABRERA and MARTÍN 1991). Hexaploid tritordeum (2n=6x=42, AABBHchHch) is a subject for a breeding program with the goal of creating a new cereal crop (MARTÍN 1988) and in this way we intent to incorporate the genetic variability of cultivated barley, H. vulgare, into the gene pool of tritordeum. For achieving this objective hybrids between barley and tritordeum at the three ploidy levels have been produced (MARTÍN et al. 1995). Another different approach is to cross barley addition lines in T. aestivum with hexaploid tritordeum. We present here the identification by fluorescence in situ hybridization (FISH) of intergenomic translocations involving barley, wheat and H. chilense chromosomes obtained from crosses between barley addition lines in hexaploid wheat and tritordeum.

MATERIALS AND METHODS

Root tips were collected from germinating seeds of advanced lines from the cross between three disomic chromosome addition lines for chromosomes 2Hv, 3Hv, and 4Hv of barley (Hordeum vulgare) in Triticum aestivum cv. Chinese Spring (CS) (ISLAM et al. 1975) and hexaploid tritordeum (2n=6x=42, AABBHchHch). These root tips were pre-treated for 3h in a 0.05% colchicine solution at 25ºC and fixed in 100% ethanol-acetic acid, 3:1 (v/v), for at least a week at room temperature. Root tips were then stained in acetocarmine for 3 min, scraped out the meristems and squashed in 45% acetic acid on ethanol-cleaned slides. The preparations were frozen in liquid nitrogen, the cover slips subsequently removed and then dehydrated for 3 min each in 70% and absolute ethanol at room temperature. The air-dried slides were stored at 4ºC until used.

Probes used were total genomic H. vulgare DNA and total genomic H. chilense DNA and they were labelled by nick translation with biotin-11-dUTP (Roche Corporate, Postfach, Basel, Switzerland) and digoxigenin-11-dUTP (Roche Corporate, Postfach, Basel, Switzerland), respectively. Both probes were mixed to a final concentration of 5ng/l in the hybridization mixture. The in situ hybridization protocol was performed according to CABRERA et al. (2002).

Biotin-labelled H. vulgare DNA and digoxigenin-labelled H. chilense DNA were detected with Streptavidin-Cy3 conjugate (Sigma, St. Louis, MO, USA) and anti-digoxigenin-FITC (Roche Corporate, Postfach, Basel, Switzerland), respectively. Chromosomes were counterstained with DAPI (4´,6-diamidino-2-phenylindole) and mounted in Vectashield. Signals were visualized using a Leica epifluorescence microscope. Images were captured with a SPOT CCD camera using the appropriate SPOT 2.1 software (Diagnostics Instruments, Inc., Sterling Heights, Michigan, USA) and processed with PhotoShop 4.0 software (Adobe Systems Inc., San Jose, California, USA). Images were printed on a Hewlett Packard Deskjet HP 840C Color Printer.

RESULTS AND DISCUSSION

Double in situ hybridization experiments using both total genomic DNA from H. vulgare and H. chilense as probes enabled the identification of different genomes in the material analysed. H. vulgare chromosomes were labelled by red fluorescence and H .chilense by yellow-green fluorescence whereas the remaining wheat chromosomes appeared as blue DAPI fluorescence (Fig. 1a, b and c). This differential pattern allowed to distinguish three types of chromosome translocations at somatic metaphase, namely those involving wheat-H. vulgare (Fig. 1a), wheat-H. chilense (Fig.1a and c) and H. chilense-H. vulgare (Fig. 1b and c) chromosomes. The translocation break-points were clear and appeared as centric break-fusion products. Interchanges between A-genome chromosomes and both Hordeum species could not be distinguished from those between B and both Hordeum species owing the identical pattern of DAPI fluorescence shown by all wheat chromosomes; for this reason, both were pooled into the wheat type.

Translocations occurred spontaneously in the progeny of crosses between H. vulgare addition lines for chromosomes 2Hv, 3Hv and 4Hv in T. aestivum with hexaploid tritordeum. Plants with simple, double or triple intergenomic translocations were found, and the relative frequencies of these translocations were different for the three barley chromosomes involved (Table 1). The highest frequency of plants with translocations were found in the offspring of crosses involving barley addition line for chromosome 3Hv with a 44.6% compared to a 10.7% and 4.8% for barley chromosomes 2Hv and 4Hv, respectively. Translocations between wheat and barley chromosomes were only found in the progeny of crosses involving barley chromosome 2Hv, although at low frequency. However, a high percentage of plants with translocations involving barley chromosome 3Hv and H. chilense was found in the descendants analyzed. These results indicated those chromosome arms from barley 2Hv and 3Hv can be introgressed into tritordeum background. No translocation involving barley chromosome 4Hv and both wheat and H. chilense chromosomes was obtained. Only one wheat-H. chilense translocation was found in the progeny of barley addition line for chromosome 4Hv in T. aestivum with tritordeum (Table 1).

Considering all single, double and triple translocations obtained in the offspring of crosses involving the three barley chromosomes, the most frequent translocation obtained was the inter-specific H. vulgare-H. chilense (52.54%) following by wheat-H. chilense (40.7%) and wheat-H. vulgare (6.8%) intergeneric translocations. The successful transfer of useful genes to tritordeum from alien species such as barley requires that the target genes or a segment of chromosome carrying them can be incorporated into the tritordeum chromosomes as recombinant segments or translocations. Earlier reports of hybrids involving Hordeum with wheat suggest a lack of pairing of Hordeum chromosomes with chromosomes of wheat (FEDAK 1985). Also, no chromosome pairing has been observed in the H. chilense x H. vulgare hybrid (THOMAS and PICKERING 1985). The material developed in the present work offers the opportunity for introgressing either parts or entire barley chromosomes in tritordeum background. As has been previously found in wheat-rye translocations (e.g. 1BL/1RS and 1AL/1RS), centric-break fusion events are an important source of introgression and they have significantly contributed to global wheat production (PEÑA et al. 1990). Most of the translocation lines obtained in the present work were fertile and could be also useful for introgression into wheat of genetic material from both H. vulgare and H. chilense genomes. Further effort are carried out to identify the wheat and H. chilense chromosomes as well as barley chromosome arms involved in the translocations.

The use of total genomic DNA as species-specific probes resulted in an efficient procedure to distinguish between different genomes in the material analyzed. Previous workers have successfully applied fluorescence in situ hybridization, including the multi-colour type, for wheat and its relatives and to identify alien chromosomes and chromosome segments in wheat (LAPITAN et al. 1986, LEE et al. 1989, MUKAI and GILL 1991, SCHWARZACHER et al. 1992, SÁNCHEZ-MORÁN et al. 1999). In most of these experiments, total genomic DNA from the introgressed alien species was used as probe, together with excess amounts of unlabelled blocking from wheat, for DNA:DNA in situ hybridization (ANAMTHAWAT-JONSSON et al. 1990, HESLOP-HARRISON et al. 1990). In all our samples analyzed the quality of in situ hybridization was highly satisfactory and clearly differentiate between wheat, H. vulgare and H. chilense genomes without the need of blocking DNA. The use of genomic H. chilense DNA as probe without blocking produced hybridization bands on the A- and B-genome chromosomes from wheat as a result of cross-hybridization (Fig. 1c). The pattern of hybridization obtained is similar to that obtained with N-banding and allowed the identification of all A- and B-genomes chromosomes present in tritordeum and wheat (GONZÁLEZ and CABRERA 1999).

ACKNOWLEDGEMENTS

Financial support from CICYT, Spain, projects AGF98-0945-CO2-01 and AGF99-1036-CO2-02 is gratefully acknowledged. The first author acknowledges the Spanish Ministeriode Ciencia y Tecnología for a FPI grant.

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Figure 1. Multicolor GISH using double genomic DNA from H.chilense (green) and H. vulgare (red) as probes to somatic spreads of advanced lines from crosses between disomic barley addition lines for chromosomes 2Hv, 3Hv and 4Hv in T.aestivum and tritordeum. Somatic metaphase spreads showing: (a) wheat/Hv and wheat/Hch double translocations; (b) Hv/Hch single translocation; (c) wheat/Hch and Hv/Hch double translocations. Translocations are indicated by arrows. Chromosomes were counterstained with DAPI. Scale bar = 10 m.

Table 1. Intergenomic translocations obtained in the descendence from the crosses between barley addition lines for chromosomes 2Hv, 3Hv and 4Hv in T.aestivum cv. Chinese Spring and hexaploid tritordeum, respectively analyzed by FISH.

------

Barley No. of plants No. of plants with intergenomic translocations

chromosome analyzed ------

wheat-Hv wheat-Hch Hv-Hch double triple

------

2I 56 3 (5.3%) 1 (1.8%) 0 1 (1.8%)* 1 (1.8%)

3I 65 0 0 9 (13.8%) 20 (30.8%)# 0

4I 21 0 1 (4.8%) 0 0 0

* wheat/Hv and wheat/Hch

# wheat/Hch and Hv/Hch