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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 554-560© TÜBİTAKdoi:10.3906/biy-1503-30

Molecular cytogenetic identification of a novel hexaploid wheat–Thinopyrum interme-dium partial amphiploid with high protein content

Mariyana GEORGIEVA1, Klaudia KRUPPA2, Nedyalka TYANKOVA1, Márta MOLNÁR-LÁNG2,*1Department of Molecular Genetics, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria

2Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Martonvásár, Hungary

* Correspondence: molnar.marta@agrar.mta.hu

1. IntroductionThe use of alien germplasm has increased intensively in cereal breeding programs for wheat improvement (Whitford et al., 2013). The alien species involved in the development of new amphiploids and wheat–alien chromosome addition, substitution, and translocation lines include the genera Aegilops (Schneider et al., 2005; Kumar et al., 2010; Farkas et al., 2014), Thinopyrum (Sepsi et al., 2008; Georgieva et al., 2011b; Zeng et al., 2013), Dasypyrum (Chen et al., 1995; Yang et al., 2005), Secale (Tang et al., 2008), Hordeum (Munns et al., 2011; Molnár-Láng et al., 2014), Psathyrostachys (Wang et al., 2011), etc. Despite major progress in plant production, the development of salt- and drought-tolerant crops remains elusive in the case of every important crop (Priya et al., 2015). Crosses between Thinopyrum spp. and Triticum aestivum L. contribute to disease resistance and tolerance for soil acidity, drought, salinity, and waterlogging in wheat.

The diversity of hybrids can also genetically  enrich the wheatgrass germplasm used for pasture, hay, and soil erosion control (Turner et al., 2013). Wheat–wheatgrass lines produced via crosses with Th. intermedium, Th. ponticum, and Th. junceum provide an excellent opportunity for the transfer of a range of genes responsible

for resistance to pathogens and abiotic stress. However, various levels of disturbance in the chromosome pairing of newly formed hybrids may influence their genome stability, seed set, and perenniality (Turner et al., 2013).

Since the initial hybridization between wheatgrasses and wheat about 70 years ago (Armstrong, 1936), a number of genes for resistance against wheat diseases have been transferred into wheat in the form of wheat–alien chromosome translocations. Thinopyrum intermedium (Host) Barkworth and Dewey (2n = 6x = 42) [syn. Agropyron intermedium (Host) Beauvoir; syn. Elytrigia intermedia (Host) Nevski] has frequently been used in crosses aimed at the transfer of genes for efficient resistance to rusts (Lr38: Friebe et al., 1992; Sr44: Friebe et al., 1996; Turner et al., 2013), powdery mildew (Pm40: Luo et al., 2009; Pm43: He et al., 2009), barley yellow dwarf virus (Bdv2: Zhang et al., 1992; Bdv3: Ohm et al., 2005), and wheat streak mosaic virus (Wsm1: Friebe et al., 2009), as summarized by Li and Wang (2009). Based on in situ hybridization data, this allohexaploid species was found by Chen et al. (1998) to consist of the genomes JJJsJsSS. Although this formula varies in the work of different authors, there is general agreement that it consists of 2 very similar (J = E and St) genomes plus a third genome of unknown origin. Chen’s genomic formula was contested by other authors,

Abstract: Genomic in situ hybridization and fluorescence in situ hybridization were used to define the cytogenetic constitution of the intergeneric introgression line 29(1-57) obtained from the wheat × Thinopyrum intermedium (wheatgrass) cross. This line is a high-protein line resistant to leaf rust, yellow rust, and powdery mildew under field conditions and has 42 chromosomes. GISH analysis using a total genomic DNA probe from Thinopyrum intermedium resulted in the identification of 14 chromosomes of wheatgrass origin. The sequential GISH patterns obtained using St-genomic and J-genomic DNA as probes and ABD-genomic DNA from wheat as a blocker led to a better determination of the genomic type of wheatgrass chromosomes. Rearrangements involving Thinopyrum chromosomes and the A, B, and D genomes of wheat were not observed. FISH using the repetitive DNA probes pSc119.2, Afa-family, and pTa71 allowed for identification of all the wheat chromosomes present.

Key words: FISH, GISH, wheat–Thinopyrum intermedium introgression line

Received: 10.03.2015 Accepted/Published Online: 09.06.2015 Final Version: 18.05.2016

Research Article

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including Mahelka et al. (2011), who analyzed chloroplast trn L-F and partial nuclear GBSSI sequences, performed multicolor GISH (mcGISH), and revealed new aspects of the genomic composition of Th. intermedium. The authors concluded that while Pseudoroegneria is the most probable maternal parent of the accessions analyzed, nuclear GBSSI sequences suggested the contribution of distinct lineages corresponding to the following present-day genera: Pseudoroegneria, Dasypyrum, Taeniatherum, Aegilops, and Thinopyrum. These results showed that a high level of heterogeneity existed at the genomic level in this species.

Sequential fluorescence and genomic in situ hybridization (FISH and GISH) on Th. intermedium chromosomes using the DNAs of microdissected chromosomes indicated that the Jst genome is closer to the J/E genomes from Th. bessarabicum and Th. elongatum than to the introgressed St genome centromeric sequences (Deng et al., 2013).

Several bread wheat–Th. intermedium amphiploids have been obtained for leaf rust, yellow rust, and powdery mildew resistance (Friebe et al., 1996; Bao et al., 2009; He et al., 2009; Luo et al., 2009; Chang et al., 2010; Zeng et al., 2013), as well as high protein content (Han et al., 2004; Georgieva et al., 2011b). The aim of the present work was to combine the FISH and GISH techniques in order to characterize a new wheat–Th. intermedium introgression line 29(1-57) with the ultimate goal of utilizing it for wheat improvement.

2. Materials and methods2.1. Plant material and chromosome preparationLine 29(1-57) was obtained from a cross between T. aestivum cv. Russalka as the female parent and Th. intermedium (accession of unknown origin) as the male parent, followed by backcrossing with the hexaploid wheat cv. Sadovo. Line 29(1-57) is fertile and intermediate in morphology between both original parents. Root-tip chromosome spreads of the line were prepared as described by Lukaszewski et al. (2004). 2.2. DNA probes and labelingSeveral DNA probes were used for in situ hybridization: (1) total genomic DNA of Th. intermedium; (2) total genomic DNA of Th. bessarabicum (J genome) and Pseudoroegneria spicata (St genome); (3) clone pTa71, containing the 18S-5.8S-26S rDNA repeat unit from T. aestivum (Gerlach and Bedbrook, 1979); (4) Afa-family (Nagaki et al., 1995), a subclone of the pAs1 tandem repetitive sequences originating from Ae. squarrosa L. (Rayburn and Gill, 1986); and (5) clone pSc119.2, containing the 120 bp repeat unit of a tandemly arranged DNA family derived from Secale cereale (McIntyre et al., 1990).

The probes were labeled with digoxigenin-11-dUTP or biotin-11-dUTP using nick translation (pTa71), random

priming (genomic DNA), or the polymerase chain reaction (Afa-family and pSc119.2) with universal forward and reverse primers.

For GISH analysis, the genomic DNA probes of Th. intermedium and J and St genome were added to the hybridization mixture at ratios of 1:35 and 1:50, respectively, with the nonlabeled blocking genomic wheat DNA. 2.3. In situ hybridizationIn situ hybridization was performed as described by Sepsi et al. (2008). Digoxigenin-labeled probes were detected using antidigoxigenin-rhodamine Fab fragments (Roche Diagnostics, GmbH), while biotin-labeled probes were detected using streptavidin-fluorescein thiocyanate (FITC) (Roche Diagnostics, GmbH). All the preparations were counterstained with 1 µg/mL 4’,6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield antifade solution (Vector Laboratories, Ltd). The best cells were captured with a Zeiss Axioscope 2 Epifluorescence microscope equipped with a Spot CCD camera (Diagnostic Instruments, Inc., USA) and Image Pro Plus 5.1 software (Media Cybernetics, USA). 2.4. Determination of crude protein content (CPC) The seed protein content was assessed according to the Kjeldahl procedure described by Georgieva et al. (2011b). The summarized crude protein data are reported as mean values ± standard error (SE) for each group investigated [Sadovo and 29(1-57)]. Differences between the 2 groups were assessed for statistical significance with the parametric Student’s t-test. Data analysis was performed with SPSS v. 15.0. A P-value less than 0.05 was considered statistically significant.

3. Results Thinopyrum intermedium is commonly used for hay production and pasture. Crude protein measurement plays a role in livestock feed, and it estimates the amount of protein calculated from the determined nitrogen content. Overall, the introgression line 29(1-57) showed a tall wheatgrass-like phenotype (Figure 1). Follow-up data evaluations of crude protein content were available for 29(1-57) (7-year data). Significant differences (P < 0.001) in crude protein content were found between the control wheat and the introgression line. The crude protein content in the seeds was about 19.13 ± 2.4% for 29(1-57), compared with 12.39 ± 2.09% for the standard cultivar, Sadovo; in the introgression line the crude protein content increased significantly.

The chromosome number of 29(1-57) varied from 40 to 42. The GISH technique was used to determine the number of Th. intermedium chromosomes in the introgression line. By probing with digoxigenin-labeled total genomic DNA of Th. intermedium and blocking with

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wheat genomic DNA, 14 Th. intermedium chromosomes and 28 wheat chromosomes were distinguished (Figure 2A). The mcGISH technique, using J and St genomic DNA as probes, allowed us to examine the chromosome composition of the alien species. Six chromosomes (Al2-Al7) were labeled at the terminal regions with a stronger red fluorescence signal and were weakly hybridized over the remaining parts of the chromosomes (Figure 2B). J and St genomic DNA also hybridized to the other parts, resulting in yellowish hybridization signals; therefore, they were more likely to belong to the J genome than St or Jst.

There were strong red hybridization signals in 2 chromosomes (Al1), and they were identified as St-genome chromosomes (Figure 2B). There were no Jst chromosomes (where the centromeric region was hybridizing with St genomic DNA) present in 29(1-57). No translocation events involving wheat and Th. intermedium chromosomes were observed.

The use of sequential three-color FISH made it possible to distinguish and characterize the wheat and Th. intermedium chromosomes in 29(1-57). The simultaneous hybridization of the repetitive DNA probes pSc119.2, Afa- family, and pTa71 allowed the wheat chromosome to be identified unequivocally (Figure 3A). The chromosomes 3A, 4A, 1B, 3B, 4B, 5B, 6B, 7B, 1D, 2D, 3D, 5D, 6D, and 7D were identified; the wheat chromosome pairs 1A, 2A, 5A, 6A, 7A, 2B, and 4D were not detected (Figure 3A).

The 7 alien chromosome pairs in 29(1-57) were distinguished with sequential FISH/GISH (Figure 3) based on the pattern and intensity of the Afa-family, pSc119.2, Figure 1. Spike and seed morphology of wheat–wheatgrass line

29(1-57).

Figure 2. GISH patterns of 29(1-57). (A) Total genomic DNA of Th. intermedium was used for probing. Fourteen wheatgrass chromosomes fluoresced in red (marked Al as alien), wheat chromosomes were unlabeled. (B) Multicolor genomic in situ hybridization with St (Pseudoroegneria spicata) and J (Thinopyrum bessarabicum) genomic DNA. Scale bar: 10 µm.

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and pTa71 hybridization sites, chromosome size, and arm ratio. To facilitate identification, the alien chromosomes in 29(1-57) were designated as Al1-7. All the Al chromosome pairs were metacentric to nearly submetacentric (Figure 3B). A karyogram was constructed based on the metaphase images of the wheatgrass chromosomes (Figure 3B), and it provided an overview of the specific FISH patterns of the Th. intermedium chromosomes (Figure 3C). Probe pTa71 gave an atypical hybridization signal on alien chromosome Al2, which was located near consecutive pSc119.2 and Afa-family signals.

4. DiscussionWild relatives of bread wheat have been extensively used as valuable sources for the introgression of useful traits for the purpose of wheat improvement. Thinopyrum intermedium is a donor of a number of useful genes for wheat improvement, such as resistance to pests, rust

diseases, and tolerance to abiotic stresses (e.g., salinity and drought). Like its progenitor, the introgression line 29(1-57) displayed increased resistance to leaf rust, yellow rust, and powdery mildew compared to the wheat cultivar and other wheat–Thinopyrum stabilized hybrids under field conditions (Tyankova, 2011).

Partial amphiploids between wild species and wheat are often cytologically stable at 2n = 42 or 56 (Brasileiro-Vidal et al., 2005). This is due to normal meiotic pairing and recombination between genomes in the F1 hybrids or in their subsequent selfed or back-crossed progenies (Cifuentes and Benavente, 2009). Partial amphiploids have been reported to contain 42 (38–42) wheat and 14 (14–18) alien chromosomes (Fedak and Han, 2005). In the present study the introgression line possesses 42 chromosomes: 28 from wheat and 14 from Th. intermedium. This chromosome number has been detected in other wheat lines with introgressed Thinopyrum chromatin but usually refers to

Figure 3. (A) FISH karyotype of metaphase chromosomes of amphiploid 29(1-57). The repetitive DNA probes were labeled using biotin-11-dUTP and digoxigenin-11-dUTP and detected with streptavidin-FITC and antidigoxigenin-rhodamine, respectively: pScll9.2 (green), Afa-family probe (red), pTa71 (yellow). Scale bar: 10 µm. (B) FISH pattern of the seven types of Th. intermedium chromosomes in the wheat–Th. intermedium amphiploid 29(1-57). (C) Comprehensive FISH ideogram representation of alien introgressions.

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substitution lines containing 1 alien pair, such as P12, P29, and Zhong 199 (Sharma et al., 1999; Li et al., 2014).

Usually, efficient gene transfer between wheat and Thinopyrum species in the form of chromosome substitutions or translocation lines involves the D genome, and only rarely the A or B genomes (Qi et al., 2007), because the wheatgrass genome shares more homology with the D genome (Liu et al., 2007). In the present study, the 29(1-57) introgression line differs from those obtained so far, because it contains 14 alien chromosomes involving the replacement of five chromosome pairs from the A genome and 1 chromosome pair each from the B or D genomes of the wheat parent. Compared with previous studies the atypical FISH pattern observed for alien chromosome Al2, especially the pattern of pTa71 probe (Li et al., 2004; Georgieva et al., 2011a, 2011b), suggests that reciprocal translocations may exist between the J and St genomes.

GISH has been used to characterize genomes and chromosomes in hybrid polyploids, hybrid lines, partial allopolyploids, and recombinant breeding lines and to localize and detect alien introgressions (Chen et al., 2003; Tek et al., 2004; Fedak and Han, 2005; Bao et al., 2009; Chang et al., 2010; Georgieva et al., 2011b; Xie et al., 2013; Książczyk et al., 2015). In the present study, GISH using Thinopyrum intermedium, St, and J genomic DNA as probes provided information about the number and type of introgressed alien chromosomes and demonstrated the presence of intergenomic chromosome rearrangements. The results obtained with GISH were in agreement with those of the FISH analysis and indicated that chromosomes with different, unknown patterns were of alien origin. St genomic DNA also hybridized to the NOR region of the wheat chromosomes (Figure 2B), while Th. intermedium DNA did not hybridize to the satellite regions of wheat (Figure 2A), despite the fact that Th. intermedium also contains St genomes. Tang et al. (2000) also found that it is possible to distinguish the 3 satellited chromosomes of Th. intermedium by GISH analysis using genomic DNA from Ps. strigosa as a probe (Ps. spicata and Ps. strigosa also have St genomes). In their study the probe hybridized to the inner border of the satellite on the short arm.

Compared to the FISH karyotype of E genome published by Linc et al. (2012), Al2 had a similar FISH pattern to 6E, Al3 to 3E, and Al7 to 7E chromosomes of different Elytrigia elongata accessions. Al1 was the shortest alien chromosome. This was in good agreement with other publications (Hsiao et al., 1986; Chen et al., 1999), as St chromosomes were shorter than J chromosomes.

Chen et al. (1999) noted that the J genome chromosomes present in TAF46 wheat × Th. intermedium partial amphiploid displayed a unique GISH hybridization pattern with the St genomic DNA probe, in which St genomic DNA strongly hybridized at the terminal regions and hybridized weakly over the remaining parts of the chromosomes. The authors suggested this phenomenon as a diagnostic marker for distinguishing J genome chromosomes from Jst or St genome or wheat ABD genome chromosomes.

In the present study sequential GISH and FISH were used to discriminate the constituent genomes and to identify the wheat and wheatgrass chromosomes in an introgression line containing 42 chromosomes. Fourteen wheat chromosomes (mainly A genome) were substituted by 14 wheatgrass chromosomes. GISH revealed the number of introgressed Th. intermedium chromosomes, whereas FISH-banding allowed the chromosomes belonging to both parents to be accurately identified. As a genetic source for improving disease resistance and quality, the 29(1-57) line could be a promising crossing partner in wheat breeding programs. However, further insight into the detailed genetic constitution of this line will be required before initiation of a crossing program.

AcknowledgmentsThis work was supported by grant no. BG051PO001-3.3.06-0025, financed by the European Social Fund and Operational Program Human Resources Development (2007–2013), and co-financed by the Bulgarian Ministry of Education and Science and the Hungarian National Scientific Research Fund (OTKA K104382, OTKA K108555). Thanks are due to B Hooper for revising the manuscript linguistically.

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