Intergenomic Crossover Formation in Newly Synthesized Trigeneric Hybrids Involving Wheat, Rye and Barley ^†

Abstract

Polyploidization, or whole genome duplication (WGD), has an important role in evolution and speciation. One of the biggest challenges faced by a new polyploid is meiosis, particularly, discriminating between multiple related chromosomes, so that only homologs recombine, to ensure regular chromosome segregation and fertility. Here, we report the production of two new hybrids that are formed by the genomes of species from three different genera, as follows: a hybrid between Aegilops taushii (DD), Hordeum chilense (H^chH^ch), and Secale cereale (RR), with the haploid genomic constitution DH^chR (n = 7x = 21); and a hybrid between Triticum turgidum spp. durum (AABB), H. chilense, and S. cereale, with the constitution ABH^chR (n = 7x = 28). We used genomic in situ hybridization to establish the chromosome composition of the new hybrids, and to study their meiotic behavior. Interestingly, there were multiple chromosome associations at metaphase I in both hybrids, indicating the presence of a crossover formation between the different genomes. We tried to duplicate the genome of both hybrids, to obtain the corresponding amphiploid, achieving success with the ABH^chR hybrid. Several amphiploids AABBH^chH^chRR were obtained and characterized. These results indicate that recombination between the genera of three economically important crops is possible.

1. Introduction

Many of the world’s most important crops, including wheat, rapeseed, sugarcane, and cotton, are recent allopolyploids. This is not a coincidence; allopolyploidy often show higher adaptability, can grow over larger geographical areas, and show better adaptation to the local environment than their diploid progenitors [1]. Despite their obvious advantages in adaptation, newly formed allopolyploids face the challenge of organizing two or more genomes that have evolved independently, within a single nucleus. Many will be the challenges, but probably, the biggest of all will be meiosis. Meiosis is the specialized cell division that generates haploid gametes for sexual reproduction. During meiosis, at least one crossover (CO) needs to be formed between every pair of homologs, to ensure accurate chromosome segregation and balanced gametes. In newly formed allopolyploids, apart from the two identical homologs present in diploid species, there are also very similar chromosomes (homeologs), which will complicate the process of recognition and synapsis between homologs. Consequently, allopolyploidization is frequently accompanied by irregular meiosis, unbalanced gametes and sterility.

In this study, we describe the production of two new intergeneric hybrids and one amphiploid, formed by the genome of species from three different genera inside the Triticeae tribe. We then used genomic in situ hybridization (GISH) to establish their chromosome composition and to study their meiotic behavior at metaphase I.

2. Materials and Methods

The plant material used in this study includes the following: the wild barley Hordeum chilense accession H7 (2n = 2x = 14; genome H^chH^ch); Aegilops tauschii (2n = 2x = 14; genome DD); Secale cereale (2n = 2x = 14; genome RR); and ×Tritordeum martinii lines HT377 (with the translocation T1RS·1BL) and HT474 (2n = 6x = 42; genome AABBH^chH^ch).

Genomic in situ hybridization (GISH) was performed as previously described [2]. Briefly, genomic DNA from T. durum (A and B genomes), H. chilense (H^ch genome) and S. cereale (R genome) were used as probes. H. chilense and S. cereale genomes were labeled with biotin-16-dUTP and digoxigenin-11-dUTP using the biotin- or the DIG-nick translation mix, respectively (Sigma). Biotin-labeled probes were detected with streptavidin-Cy3 or streptavidin-Cy5; digoxigenin-labeled probes were detected with anti-digoxigenin-FITC (Sigma). T. durum genomic DNA was labeled by nick translation with tetramethyl-rhodamine-5-dUTP.

3. Results and Discussion

3.1. Production and Chromosome Constitution of the Trigeneric Hybrids and Amphyploids

3.1.1. Hybrid H^chDR

H. chilense and Ae. taushii were duplicated after colchicine treatment. Tetraploid H. chilense was pollinated with tetraploid Ae. Taushii, and H^chH^chDD hybrid plants were established after embryo rescue. Finally, H. chilense × Ae. taushii hybrids were pollinated with rye (S. cereale), and out of 200 florets that were pollinated, 2 adult hybrids H^chDR were recovered by embryo rescue. The H^chDR hybrids showed vigorous vegetative growth and tillered profusely. They showed a similar morphology to the female parent H. chilense × Ae. squarrosa hybrid. All the florets were sterile.

The root-tip metaphase spreads were analyzed by multicolour GISH, to verify the genome constitution of the H^chDR hybrid (Figure 1a). The three recovered hybrids had 21 chromosomes, comprising 7 chromosomes from H. chilense (magenta), 7 chromosomes from rye (green), and 7 chromosomes from Ae. taushii (grey), confirming that they are true trigeneric hybrids (n = 3x = 21).

3.1.2. Hybrid ABH^chR

To produce the trigeneric hybrid ABH^chR, we used hexaploid ×Tritordeum martinii lines HT377 (with the translocation T1RS·1BL) and HT474. Tritordeum was crossed as the female parent, with rye as the male parent. The ABH^chR hybrids all showed similar morphology to the tritordeum female parent, with an erect vegetative development and a smaller number of tillers than the DH^chR. This hybrid displayed a morphology that was more characteristic of a crop, and not the one of a wild species as the H^chDR does. All the florets were sterile.

The root-tip metaphase spreads were analyzed by GISH, to verify the genome constitution of the ABH^chR hybrids obtained (Figure 1b). All the recovered hybrids possessed 28 chromosomes, containing 14 chromosomes from the AB genome (grey), 7 from H. chilense (magenta), and 7 from rye (green). This confirmed that they were true trigeneric hybrids (n = 4x = 28; ABH^chR).

3.1.3. Amphiploid AABBH^chH^chRR

Several trigeneric hybrids have been obtained in the last decades, to be used as bridges in the transference of genes from wild species into wheat, with varying degrees of success [3,4,5,6,7]. However, apart from triticale and tritordeo, the production of the stable amphiploid is rare. Here, we treated the H^chDR and the ABH^chR hybrids with colchicine, to induce whole genome duplication and obtain the corresponding amphiploids. Only the ABH^chR, using tritordeum HT377, was duplicated. The chromosome number of the obtained amphiploids AABBH^chH^chRR ranged from 49 to 54, none of them having the complete set of 56 chromosomes. Only the following two amphiploids produced progeny: AABBH^chH^chRR-1 and AABBH^chH^chRR-2. The morphology of the partial amphiploids was similar to ABH^chR hybrids, but shorter. Each individual amphiploid was slightly different from each other, due to the different chromosome compositions. Unfortunately, all the florets were sterile.

We used the same combination of genomic GISH probes as with the ABH^chR hybrid, to establish the genome constitution of the amphiploids. All the individuals were aneuploids, with chromosome numbers ranging from 46 + 2 telosomic chromosomes (46 + 2 t) to 51 chromosomes (Figure 1c). The H. chilense genome was the most affected, with only seven chromosomes in some of the individuals. All the amphiploids showed at least one T1RS·1BL translocation coming from tritordeum HT377. There were numerous telosomic chromosomes from all the genomes.

3.2. Analysis of Meiotic Metaphase I Configuration in the Trigeneric Hybrid H^chDR

In diploid species, only homologous chromosomes recombine during meiosis, to ensure accurate chromosome segregation. However, no homologous chromosomes are present in a haploid hybrid. We used GISH to study the meiotic behavior of the three genome’s chromosomes at metaphase I, using the same labelling conditions as for the somatic cells (

Table 1. Number of chromosome associations observed in the trigeneric hybrid H^chDR.

	No. and Type of Chromosome Associations			Total % of Associations
	Rod Bivalent	Ring Bivalent	Trivalent
Hordeum-Hordeum	3	0	0	1.7
Aegilops-Aegilops	1	0	0	0.6
Secale-Secale	15	0	0	8.5
Hordeum-Aegilops	73	5	0	47.2
Hordeum-Secale	13	0	0	7.4
Aegilops-Secale	51	0	0	29.0
Hordeum-Aegilops-Secale	0	0	4	4.5
Hordeum-Aegilops-Aegilops	0	0	1	1.1
Total No. of associations	156	10	10

). Surprisingly, 76.7% of the cells showed some chromosome associations. Most of these associations were rod bivalents between two chromosomes, but several ring bivalents and trivalents were also observed (Figure 2a–d). One might expect that most of these associations were between chromosomes belonging to the same genera; however, that was not the case, with the higher number of associations being observed between Hordeum and Aegilops (47.2%), followed by Aegilops and Secale associations (29%). We could even detect several trivalents, where chromosomes from the three genera were involved. These results highlight the potential use of this material to promote or stimulate recombination between genomes that would not normally recombine in a wild-type situation. We are not able to demonstrate that all the associations observed are quiasmatic and produce a recombination event, because this hybrid is sterile, and we cannot recover the results of these recombination events in the next generation. However, even if some of the more end-to-end associations could be non-chiasmatic, there are some very clear examples where the crossover structure is clearly observed between two pairs of chromosomes (Figure 2a,d), confirming that there is recombination between the different genera.

Analysis of Meiotic Metaphase I Configuration in the Trigeneric Hybrid ABH^chR and Its Corresponding Amphiploid AABBH^chH^chRR

A total of 100 meiotic metaphase I cells were analyzed, out of which 38 cells showed some chromosome association, frequently a single association (

Table 2. Number of chromosome associations observed in the trigeneric hybrid ABH^chR.

	Rod Bivalent	Total % of Associations
Triticum-Triticum	4	8.5
Hordeum-Hordeum	5	11.9
Secale-Secale	4	8.5
Triticum-Hordeum	15	35.7
Triticum-Secale	10	23.8
Hordeum-Secale	4	8.5
Total No. of associations	42

). Although associations were observed between all the different genomes (Figure 2e–h), the number was much lower than in H^chDR hybrids, and, moreover, only rod bivalent structures were detected. Rod bivalents have one association per bivalent, instead of the two associations per trivalent and ring bivalent structures, which emphasizes the lower number of associations observed in the ABH^chR hybrid compared with the H^chDR. This could be explained due to the present of the ph1 locus on the 5B genome. Due to its polyploid nature, polyploid wheat had to develop a mechanism to ensure than only homologous chromosomes recombine, ensuring accurate chromosome segregation and fertility. Thus, wheat behaves as a diploid during meiosis, with every chromosome recombining only with its true homolog; this is a phenotypic behavior that has been mostly attributed to Ph1, a dominant locus on chromosome 5B [8,9]. Moreover, not only the number of associations is smaller, but, also, contrary to the associations in H^chDR, they are extremely distal, and thus, it is possible that they are non-chiasmatic. Unfortunately, since ABH^chR is also sterile, we cannot check the progeny to determine the output of the meiotic associations.

Finally, we analyzed the meiotic behavior of the following two of the amphiploids: AABBH^chH^chRR-1-1 and AABBH^chH^chRR-2-1 (Figure 2i–k). A total of 125 and 112 meyocites were analyzed from each genotype, respectively. Interestingly, despite being aneuploids and presenting multiple chromosome reorganizations, the presence of associations between the different genera was only anecdotical, as shown in Figure 2i. There were multiple rod and ring bivalents, as expected in an amphiploid, but also trivalents and quatrivalents were present (Figure 2j), due to their aneuploidy. No difference, in terms of associations, was observed between AABBH^chH^chRR-1-1 and AABBH^chH^chRR-2-1.

4. Conclusions

Due to their economic importance, Triticeae species, such as wheat, rye, and barley, have been bred intensively in the past hundreds of years, resulting in massive improvements in yield and quality, but also in a huge decrease in their genetic diversity. Thus, the incorporation of genetic variability, from related and wild species into cultivated ones, is a priority, but also a challenge, due to the presence of reproductive barriers that hinder genetic transfer among them. The results presented here demonstrate that recombination between three cultivated species as distant as wheat, rye, and barley, is possible. The next challenge will be to recover these recombination events, so they can be transferred into crops.