1 Development and Cytogenetic Characterisation of Non-Brittle Rachis

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Development and cytogenetic characterisation of non-brittle rachis tritordeum lines.

P. Prieto, C. Ramírez, A. Cabrera, J. Ballesteros & A. Martín

P. Prieto1, C.Ramírez, J. Ballesteros & A. Martín Instituto de Agricultura Sostenible (CSIC), Apartado 4084, 14080 Córdoba, Spain

A. Cabrera Departamento de Genética ETSIAM, Apartado 3048, 14080 Córdoba, Spain

1Author for correspondence should be addressed P. Prieto Instituto de Agricultura Sostenible (CSIC), Apartado 4084, 14080 Córdoba, Spain (E-mail: [email protected])

1 ABSTRACT ×Tritordeum (Ascherson et Graebner, an amphiploid between Triticum turgidum conv. durum and Hordeum chilense), and a set of chromosome substitution lines of tritordeum, where 2Hch or 3Hch H. chilense chromosomes were replaced with 2D chromosome of T. aestivum or 3Hv chromosome of H. vulgare, were used to assess the effect of the specific chromosome on brittle rachis in tritordeum. ×Tritordeum has brittle rachis. In contrast, the 2D(2Hch) and 3Hv(3Hch) substitution lines showed non- brittle rachis, and they also have tenacious glumes and compact spikes which are excellent characters to improve the tritordeum threshing. We have also developed different combinations between 2D and 3Hv translocation lines in tritordeum. We present in this paper the identification and characterisation of all these introgression lines in tritordeum by fluorescent in situ hybridisation.

Key words in situ hybridisation, rachis tenacity, tritordeum, wheat

INTRODUCTION Since the beginning of the last century, plant breeders have been interested in the production of amphiploids between wheat and barley (Farrer 1904). Martín and Chapman (1997) got the first fertile amphiploid between a Hordeum species (Hordeum chilense Roem. et Schult.) and the cultivated wheat (Triticum aestivum). The amphiploid between H. chilense and Triticum turgidum conv. durum has also been obtained and named ×Tritordeum. Tetra, hexa and octoploid amphiploids have been synthesised from crosses between H. chilense and diploid, tetraploid and hexaploid wheats, respectively, to increase the variability of the tritordeum. Hexaploid tritordeum (AABBHchHch, 2n = 6x = 42) has some favourable agronomic traits including good seed size and high grain protein content which indicate the potential of this amphiploid as a possible new crop (Martín and Cubero 1981, Martín et al. 1996). After several years of breeding, its potential yield has been confirmed (Martín et al. 1999). Tritordeum can also be used as animal feed and tritordeum flour has good characteristics to be used in bread or biscuit instead of wheat flour (Álvarez et al. 1992). Tritordeum is also very important as a bridge species to introgress the genetic variability of H. chilense into wheat and to detect agronomic characters of interest that appear from the interaction of H. chilense and wheat. Early studies in plant chromosomes showed that whole chromosomes carried similar group of genes in different species (Darlintong and LaCour 1942, Sears 1941). Today molecular markers show that gene orders are conserved over substantial evolutionary distances (Ahn et al. 1993, Devos and Gale 1993, 1997), with the number of chromosomal and genetic differences between species generally increasing with evolutionary distance (Bennetzen et al. 1998, Tikhonov et al. 1999). Barley (Hordeum vulgare L.) carries potentially useful genes for tritordeum improvement and H. vulgare substitution lines provide excellent material for the study of alien gene expression in tritordeum background. For example, cultivated barley (H. vulgare ssp. vulgare) has non-brittle rachis. Hexaploid tritordeum has brittle rachis, which makes impossible to thresh it in a mechanical harvester. The concept of rachis fragility can be simply defined as a spike having a rachis which can be easily broken. The genetics of brittle rachis has been extensively studied. Non-brittle rachis was a key character in the evolution of cultivated barley (Harlan 1968, Bothmer and Jacobsen 1985). The brittle rachis is formed by complementary genes at two tightly linked loci, btr1 and btr2, located on H. vulgare 3HvS chromosome arm (Takahashi and Hayashi 1964, Franckowiak and Konishi 1997a, b). Position of non-brittle rachis loci btr1 and 3 btr2 on the short arm of chromosome 3 has been recently mapped using molecular markers (Komatsuda and Mano 2002, Kandemir et al. 2004, Komatsuda et al. 2004). Wild barley (H. vulgare ssp. spontaneum) has brittle rachis as a consequence of possessing the dominant forms of both genes (Btr1Btr1 Btr2Btr2) (Takahashi, 1964). Cultivated barley has a mutation in one of these loci: most Occidental cultivars are recessive homozygous at the btr1 locus (btr1btr1Btr2Btr2), whereas most Oriental cultivars are recessive homozygous at the btr2 locus (Btr1Btr1btr2btr2) resultanting both resulting in a non-brittle rachis (Takahashi 1955, 1963, Takahashi et al. 1983). In situ hybridisation (ISH) techniques applied to plant mitotic chromosomes effectively combine specificity and resolution for screening a high number of plants in an early developmental stay. Genomic in situ hybridisation (GISH) and fluorescent in situ hybridisation (FISH) are well-established and highly efficient techniques for cytogenetical identification. The physical size of introgressed segments can be assessed by in situ hybridisation, which readily distinguishes H. chilense and H. vulgare chromosomes from those of wheat (Cabrera et al. 1995, Prieto et al. 2001). The genetic background of tritordeum has been previously expanded by the synthesis of new amphiploids using additional accessions of H. chilense collected in Chile and Argentina (Tobes et al. 1995, Giménez et al. 1997). In the present report, 3Hv substitution lines in hexaploid tritordeum have been developed and analysed by in situ hybridisation with the aim of introgressing the non-brittle rachis character into tritordeum.

MATERIAL AND METHODS Plant material. Advanced lines of the cross between disomic chromosome addition line for chromosome 3Hv of Hordeum vulgare in T. aestivum cv. Chinese Spring (CS) (AABBDD+ pair 3Hv) (Islam et al. 1975) with hexaploid tritordeum (AABBHchHch) have been analysed. H. vulgare addition line was kindly supplied by Dr. Islam (Waite Agricultural Research Institute, Glen Osmond, South Australia). Cytogenetic analysis. Root tips were collected from germinating seeds and were pre-treated for 3 hours 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. Preparations were as described by Prieto et al. (2001). GAA-satellite sequence (Pedersen et al. 1996) and pAs1 probe (Rayburn and Gill, 1986) were used to identify substituted chromosomes and chromosomes involved in translocations. The barley clone pHvG38 containing the GAA-satellite sequence was kindly provided by Dr. Rasmussen from the Risø National Laboratory, Roskilde (Denmark) and the pAs1 probe isolated from T. tauschii was kindly provided by the Wheat Genetics Resource Centre, University of Kansas, USA. The GAA-satellite sequence and the pAs1 probes were labelled by nick translation with biotin-11-dUTP (Roche Corporate, Basel, Switzerland) and digoxigenin-11-dUTP (Roche Corporate, Basel, Switzerland) , respectively, and mixed to a final concentration of 5 ng μl-1 in the hybridisation solution. After examination of nuclei hybridised with the repetitive probes, preparations were reprobed using total genomic DNA of H. chilense and H. vulgare as probes. Total genomic H. chilense and H. vulgare DNA were also labelled by nick translation with digoxigenin-11-dUTP and biotin-11-dUTP, respectively. Both probes were mixed to a final concentration of 5 ng μl-1 in the hybridisation mixture. The in situ protocol was performed according to Cabrera et al. (2001). Digoxigenin-labelled and biotin-labelled probes were detected with antidigoxigenin- FITC (Roche Corporate) and streptavidin-Cy3 conjugate (Sigma, St. Louis, MO, USA), 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

5 Jose, California, USA). Images were printed on a Hewlett Packard Deskjet HP 840C Colour Printer.

RESULTS AND DISCUSSION Crosses between disomic chromosome addition line for chromosome 3Hv of barley (H. vulgare) in Triticum aestivum conv. Chinese Spring (CS) (AABBDD + pair 3Hv, Islam, 1975) and hexaploid tritordeum (2n = 6x = 42, AABBHchHch) were developed with the aim to introgress the non-brittle rachis character (located on chromosome 3Hv) into tritordeum. Initial backcrosses with the hexaploid tritordeum were developed to reduce the number of D chromosomes and to increase the number of H. chilense chromosomes in the tritordeum background. After several backcrossing and selfing generations we have obtained more than one hundred plants and analysed the 70% of the plants obtained. More than the 50% of the plants obtained were fertile (Table 1). Fluorescent in situ hybridisation using genomic H.vulgare and H.chilense DNA as probes was used to identify and select H. vulgare introgressions along these several plant generations and to analyse the tritordeum lines obtained. The use of total genomic DNA as species-specific probes resulted in an efficient procedure to distinguish between different genomes in the material analysed. Previous workers have successfully applied fluorescence in situ hybridization, including the multi-colour type, for wheat and its relatives (Schwarzacher et al. 1992, Prieto et al. 2001). In these experiments, total genomic DNA from H. chilense and H. vulgare were used as probe for DNA:DNA in situ hybridization. In all our samples analysed the quality of in situ hybridization was highly satisfactory and clearly differentiated between wheat, H. vulgare and H. chilense genomes without the need of blocking DNA. Table 1 shows the number of plants with 3Hv chromosome substitution and the number of plants with intergenomic translocations. GISH (genomic in situ hybridisation) results show different chromosome combinations between H. vulgare and H. chilense chromosomes (Table 1, Figure 1) and it has been especially useful in the breeding program of the tritordeum because we had to analyse a very high number of tritordeum lines. GISH technique allowed detecting H. vulgare introgressions not only in somatic metaphase but also in somatic interphase (Figure 1). We have detected more than 25% monosomic or disomic substitution lines for the chromosome 3Hv substituted. We have also found a high number (63%) of intergenomic translocations by FISH analysis using total genomic DNA from both H. chilense and H. vulgare (Table 1). Translocations involving wheat - 3Hv, wheat - H. chilense and H. chilense - 3Hv have been detected (Table 1). Translocations between H. chilense and wheat are the most frequent translocations. FISH analysis using pAs1 or GAA-satellite probes prior to GISH in the same metaphases makes it possible to determine the exact 7 chromosomal composition of every tritordeum line and which arm of the 3Hv, H. chilense or wheat chromosomes are involved in translocations (Figure 1). A 3Hv chromosome introgression was identified in tritordeum lines with non-brittle rachis. Plants with non-brittle rachis had a 3Hv monosomic or disomic introgression, 3Hv telocentric chromosome or as a 3Hv translocation chromosome (Table 2). We have also obtained several substitution lines in tritordeum with different combinations involving chromosome 2D of T. aestivum and chromosome 3Hv of H. vulgare (Tables 1 and 2). The 3Hv(3Hch) and 2D(2Hch) double substitution line has been developed. This double chromosome substitution is disomic for both chromosomes substituted and it has been fixed along several self-pollinated generation in more than 15 tritordeum lines. All these lines presented tough rachis, tenacious glumes and compact spike, characters controlled by genes located on 3HvS, 2DS and 2DL chromosome arms, respectively (Takahashi and Hayashi 1964, Franckowiak and Konishi 1997a, b, Jantasuriyarat et al., 2004). These characters are extremely important to get the tritordeum as a new cultivated cereal. The progeny of the 2D(2Hch) and 3Hv(3Hch) lines is also homogenous and fertile. We don’t expect meiotic recombination because no meiotic pairing has been detected between H. chilense, wheat and H. vulgare chromosomes (Martín and Sánchez-Monge, 1980; Martín et al. 1995). Cytogenetic analysis of 2D-3Hv double substitution line is shown in Figure 1. The 2D(2Hch) chromosome substitution occurs spontaneously in the progeny of the crosses between the 3Hv addition line in T. aestivum with hexaploid tritordeum. We have fixed the 2D(2Hch) chromosome substitution in more than 20 tritordeum lines. All these plants were fertile and presented tough rachis. They also present tenacious glumes and compact spike, character located on chromosome 2DS and 2DL by Tg and C genes, respectively. It has been recently described that coincident QTL on the short arm of 2D, probably representing the effect of Tg, explained 44% of the variation in threshability, 17% of the variation in glume tenacity, and 42% of the variation in rachis fragility (Jantasuriyarat et al., 2004). Translocations occur spontaneously in the descendence of the crosses between H. vulgare addition lines in T. aestivum with the hexaploid tritordeum (Prieto et al. 2001). 3Hv-Hch translocation lines have been also developed with the chromosome 2D introgression and without it. These lines also show non-brittle rachis. Most of the translocation lines obtained in the present work were fertile and could be also useful for introgression into wheat from both H. vulgare and H. chilense genomes. 8

Barley genes could be introgressed into tritordeum by genetic transformation. This technique was successfully developed in tritordeum (Barceló, 1994) and transgenic plants expressing genes for wheat storage proteins have been developed (Barro, 2002). Unfortunately we don’t have the cloned barley genes related with the rachis brittleness (btr1 and btr2). We haven’t found tough or non-brittle rachis tritordeum line or it hasn’t been obtained by mutagenesis in H. chilense or tritordeum either. In contrast, the first genetic map of H. chilense has been developed and the overall structure of the H. chilense linkage groups is similar to that of the B and D genomes of wheat and the Hv genome of barley (Hernandez et al. 2001). Based on this, H. chilense must have at least one homeologous of the btr H. vulgare genes on chromosome 3Hch. The material developed in the present work offers the opportunity to obtain non-brittle rachis tritordeum lines by the introgression of the 3Hv H. vulgare chromosome in tritordeum background.

9 ACKNOWLEDGEMENTS This research was supported by a Spanish, MCYT, Subdirección General de Proyectos de Investigación, Grants AGL2003-00720 and AGL2001-2419-CO2-01.

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13 Schwarzacher, T., Anamthawat-Jónsson, K., Harrison, G.E., Islam, A.K., Jia, J.Z., King, I.P., Leitch, A.R., Miller, T.E., Reader, S.M., Rogers, W.J., Shi, M. & Heslop-Harrison, J.S. 1992. Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat. Theor Appl Genet 84: 778- 786.

Progeny of the cross wheat 3Hv addition line × tritordeum Number of plants obtained 117 Number of plants analysed 81 Fertile plants 53% Number of plants with 3Hv chromosome substitution 30 (37%) Monosomic substitution 11 (13,6%) Disomic substitution 12 (14,8%) Ditelosomic substitution 7 (8,6%) Number of plants with intergenomic translocations 51 (63%) Hv-Hch 9 (11,1%) wheat-Hch + Hch-Hv 39 (48,1%) wheat-Hv + Hch-Hv 3 (3,7%) Table 1. Progeny of the cross between disomic chromosome addition line for chromosome 3Hv of barley (H. vulgare) in Triticum aestivum conv. Chinese Spring (CS) with hexaploid tritordeum.

Tritordeum line Chromosomal composition Rachis Genotype 2DL 3HvS HT623 (n=42) 28 wheat +14 Hch brittle no no

2D(2Hch) and 3Hv(3Hch) 30 wheat +10 Hch +2 Hv non-brittle yes yes substitution line (n=42)

2D(2Hch) and 3HvL substitution 30 wheat +10 Hch +2 telos 3HvL non-brittle yes no line (n=40 + 2 telos)

3HvS-Hch and 3HvL-Hch 2 wheat + 10 Hch + 2 translocations D-Hch + 1 non-brittle no yes translocation line (n=42) translocation 3HvS-Hch + 1 translocation 3HvL-Hch

2DL-2HchS and 3Hv-3Hch 28 wheat +10 Hch +2 translocations 2DL-2HchS + non-brittle yes yes translocation line (n=42) 2 translocations 3HvS-3HchL Table 2. Effect of the 3Hv and 2D chromosome on the rachis brittleness.

Figura 1. GISH using both H. chilense (detected with fluorescein, green) and H. vulgare (detected with rhodamine, red) genomic DNA probes in interphase cells of tritordeum lines with a) two H. vulgare chromosomes, b) no H. vulgare introgresion, c) one H. vulgare chromosome and d) two H. vulgare telocentric chromosomes. Cytological analysis using H. chilense (detected with fluorescein, green) and H. vulgare (detected with rhodamine, red) genomic DNA in metaphase cells of tritordeum e) HT623, g) 2D(2Hch) and 3Hv(3Hch) substitution line, i) 3HvL telocentric substitution line, k) 2DL-2HchS and 3Hv-3Hch translocation line; (f), (h), (j) and (l) double FISH signals with the pAs1 (green) and GAA-satellite (red) sequences hybridized to the same metaphase as in (e), (g), (i) and (k), respectively. Bar = 10 µm.