Thinopyrum ponticum Chromatin-Integrated Wheat Genome Shows Salt-Tolerance at Germination Stage

A wild wheatgrass, Thinopyrum ponticum (2n = 10x = 70), which exhibits substantially higher levels of salt tolerance than cultivated wheat, was employed to transfer its salt tolerance to common wheat by means of wide hybridization. A highly salt-tolerant wheat line S148 (2n = 42) was obtained from the BC3F2 progenies between Triticum aestivum (2n = 42) and Th. ponticum. In the cross of S148 × salt-sensitive wheat variety Chinese Spring, the BC4F2 seeds at germination stage segregated into a ratio of 3 salt tolerant to 1 salt sensitive, indicating that the salt tolerance was conferred by a dominant gene block. Genomic in situ hybridization analysis revealed that S148 had a single pair of Th. ponticum–T. aestivum translocated chromosomes bearing the salt-tolerance. This is an initial step of molecular breeding for salt-tolerant wheat.


Introduction
Salinity is a major external factor affecting plant growth and productivity. Of the world's 230 million ha of all irrigated lands, 19.5% are seriously affected by salinity (http://www.fao.org/ OPEN ACCESS nr/land/en/). Wheat is grown under irrigated and rain-fed conditions; both types of agriculture are threatened by salinization [1,2]. Therefore, the development of salt-tolerant wheat is an important breeding objective. To date, however, little progress has been made towards improving the salt tolerance of wheat. Shalaby et al. [3] indicated that breeding salt tolerant wheat by exploiting naturally occurring variation is difficult.
Fortunately, amongst halophytic members of Triticeae, tall wheatgrass species (e.g., Thinopyrum spp.) have received the most attention as a potential gene source for improving salt tolerance in wheat [4,5]. A perennial grass, Th. ponticum (Podp.) Barkworth and D.R.Dewey (2n = 10x = 70, JJJJsJs) was used as forage on salt-affected lands [6]. Several accessions of Th. ponticum survived 750 mM NaCl [7] and some maintained reasonable growth at an EC of 13.9 dS·m -1 [8]. Thus, Th. ponticum is a promising material to transfer its salt tolerance into common wheat by chromosome engineering through wide hybridization. Towards the improvement of salt tolerance of wheat, wheat-tall wheatgrass amphiploids was developed as a new salt-tolerant cereal, named Tritipyrum (derived from Triticum spp. × Thinopyrum spp.) [9][10][11]. Although the resulting crop would not be expected to produce grain quality of bread or durum wheat, it could likely be used as feed wheat.
The second step is to develop recombinant wheat lines containing small segments of tall wheatgrass chromosomes without linkage drag, such as deleterious effects on yield or grain quality. In this study, Th. ponticum chromosomes was transferred to T. aestivum by back crossing onto the amphiploids of T. aestivum × Th. ponticum, and that the salt-tolerance small chromatin was successfully inherited at germination stage in the BC4F2.

Transfer of Th. ponticum Genetic Material with Salt Tolerance to Wheat by Wide Hybridization
Firstly, an amphiploid TAe68 (2n = 56) developed from the hybrid of T. aestivum salt-sensitive variety Jinmai 24 × Th. ponticum showed salinity tolerance. TAe68 was backcrossed to "Jinmai 24" twice. Until the BC2 crossing, the hybrid embryo was excised 15-20 days after pollination and cultured on the media for callus induction and then plantlets were differentiated. Through the BC2F2 to BC2F6 generation, plants grown in a greenhouse were selected according to the salt tolerance test. Chromosome numbers were examined using 2% (w/v) aceto-carmine, and Th. ponticum chromatins were identified by C-banding analysis according to Gill et al. [12]. In the BC2F6 generation, a highly salt-tolerance with 2n = 42 was selected and backcrossed twice with T. aestivum salt-sensitive cv. Chinese Spring. The BC4F1 line showed 21 bivalents in pollen mother cells. These results indicated that BC3F2 line S148 is a Th. ponticum-T. aestivum translocation line.
Grains of a salt-resistant line S148, Chinese Spring, and the BC4F2 progeny of Chinese Spring × S148 were used for genetic analysis for salt tolerance under NaCl conc. 0.1, 0.2, 0.3 and 0.4 M, respectively. Both genotypes germinated in the presence of 0.1 and 0.2 M NaCl, but subsequent coleoptile and root extension was retarded. Notably, only S148 was able to germinate in the presence of 0.4 M NaCl and showed the least reductions in coleoptile and root lengths (Figure 1). In the cross of S148 × Chinese Spring, the BC4F2 seeds segregated into a ratio of 72 salt tolerant to 27 salt sensitive. In salt tolerant plants, the averaged coleoptile and root length were 17.5 and 21.4 mm, respectively. Whereas, in salt sensitive plants, averaged coleoptile and root length were 3.5 and 6.8 mm, respectively. This segregation ratio was well fitted to a ratio of 3:1 (χ 2 = 0.273; 0.50 < p < 0.75), indicating that the salt tolerance was conferred by a dominant gene block derived from Th. ponticum.

Identification of Th. ponticum Chromatin in Salt-Tolerant Recombinant Wheat
Fluorescence GISH method according to Tomita et al. [13] was applied to detect the Th. ponticum chromatin in the wheat background. The total genomic DNA of Th. ponticum labeled with biotin-16-dUTP or Digoxigenin-11-dUTP was hybridized as the probe to the denatured chromosomes. In the double-exposed photographs, the in situ hybridization sites on Th. ponticum chromosome segments were visualized using FITC or rhodamine, and the other chromosomes were counter-stained with PI or DAPI ( Figure 2). Using this approach, two alien chromosome segments were detected in S148 and some salt resistant individuals in BC4F2. GISH analysis verified that S148 had a single pair of Th. ponticum-T. aestivum translocated chromosomes including the salt tolerance gene block of Th. ponticum. The observed single genotypic ratio of salt tolerance means that this alien translocation transmitted normally and did not have segregation distortion.
Soil salinity is a major abiotic constraint to agricultural productivity. Introgression of genes conferring salt tolerance from Th. ponticum (2n = 10x = 70) into Triticum aestivum is one of the few methods by which salt-tolerant wheat varieties can be developed. The modern breeding strategy is developing recombinant lines of wheat containing small segments of tall wheatgrass chromosomes without deleterious effects on yield or grain quality. Such an approach has enabled the use of tall wheatgrass to confer disease resistance to wheat [14][15][16][17][18]. However, Th. ponticum-T. aestivum translocation lines with the salt tolerance have not been developed, and the salt tolerance of Th. ponticum has not been employed in wheat varieties. In this study, we attempted to identify alien chromatin in the progenies resulting from T. aestivum-Th. ponticum hybrids using genomic in situ hybridization method. Here, salt tolerance of Th. ponticum had been introgressed into bread wheat as a stably inherited translocated chromatin. This study is an initial step to developing the salt tolerant wheat plant. The future studies for line S148 will be further characterized by molecular cytogenetic approaches to reveal the specific chromosome arms of wheat and tallgrass which are involved to the translocation, as in the case of wheat-Lophopyrum elongatum recombinant lines [19]. Molecular genetic analysis of salt tolerance is also important [18,20]. Salt tolerance in decaploid tall wheatgrass was associated with a capacity to restrict the rate of accumulation of sodium ion. Obtained Th. ponticum-T. aestivum translocation line with salt stress tolerance could potentially lead to identification of salt tolerance determinants.

Salinity Test
The seeds were sown in Petri dishes on filter paper soaked with distilled water and 0.1, 0.2, 0.3 and 0.4 M NaCl and the dishes were placed in an incubator at 25 °C. After 7 days, coleoptile and main root lengths were measured. Electroconductivity was measured daily and the salinity was adjusted as needed to maintain the NaCl.