In situ hybridization of the six substitution lines
By GISH analysis of somatic cells, alien chromosomes derived from Th. ponticum or Th. intermedium were able to be traced. It was showed that all the six lines, ES-9, ES-10, ES-23, ES-24, ES-25 and ES-26 contained 42 chromosomes (Fig. 1). ES-9 and ES-10 both carried two Th. ponticum chromosomes with a bright-green hybridization signal by using Th. ponticum genome DNA as a probe (Fig. 1, b1 and b2). Whereas ES-23 (Fig. 1, b3), ES-24 (Fig. 1, b4), ES-25 (Fig. 1, b5) and ES-26 (Fig. 1, b6), each of them carried two Th. intermedium chromosomes with a bright-green hybridization signal, by using the GISH probe of Th. intermedium. Therefore, ES-9 and ES-10 were wheat- Th. ponticum disomic substitution lines, and ES-23, ES-24, ES-25, as well as ES-26 were wheat- Th. intermedium disomic substitution lines.
Two Oligonucleotide probes of pTa535 and pSc119.2 were combined for a sequential FISH–GISH to simultaneously examine the elimination of wheat chromosomes in the six substitution lines. Pairwise comparisons for the FISH results between substitution lines and the corresponding parent lines, Abbondanza, Zhong4 and Xiaoyan784, were conducted. It was revealed that chromosome 2A was eliminated in ES-9 and substituted by one pair of Th. ponticum chromosomes with three specific signal bands, including the terminal pTa535 hybridization sites detected on short arms and long arms as well as an interstitial pTa535 signal on the long arms, which was different from the FISH patterns of other wheat chromosomes (Fig. 1, a1). ES-10 lost chromosome 3D and contained one pair of Th. ponticum chromosomes carrying terminal pSc119.2 hybridization sites on short arms with terminal pTa535 hybridization segments on the long arms and short arms (Fig. 1, a2). Wheat chromosome 2A, chromosome 2B, and wheat chromosome 2D were eliminated in ES-23 (Fig. 1, a3), ES-25 (Fig. 1, a5) and ES-26 (Fig. 1, a6), respectively, and replaced by the same pair of Th. intermedium chromosomes with the identical FISH patterns of the alien chromosomes presenting in ES-9. Moreover, the telomeric region of chromosome 5B carrying a bright-green fluorescence signal was eliminated in ES-25 compared with other related materials. In terms of ES-24, chromosome 3D was substituted by a pair of Th. intermedium chromosomes with the FISH patterns almost consistent with the alien chromosomes detected in ES-10 (Fig. 1, a4).
In addition, according to the mc-GISH results, each of the six derived lines contained two alien chromosomes carrying a bright-red fluorescence signal originating from P. spicata (St) genome DNA (Fig. 1, c1-c6). It was suggested that ES-9 and ES-10 carried two different pairs of St chromosomes derived from Th. ponticum. While ES-23, ES-25 and ES-26 contained the same pair of St chromosomes from Th. intermedium which was distinguished from the pair of St chromosomes in ES-24.
Wheat 15K SNP array analysis of the six substitution lines
The chromosomal composition of the six substitution lines were determined based on genotype data by using a wheat 15K SNP array (Table S1-6). Generally, the common SNP sequences detected between the substitution lines and the same wheat parent line Abbondanza were much higher than between the substitution lines and Th. ponticum or Th. intermedium. However, obvious point of intersection was found in each of the substitution lines (Fig. 2 a-f). As shown in ES-9 (Fig. 2a), an intersection point was distinctly observed in chromosome 2A, where ES-9 had the most of the same SNP marker loci as Th. ponticum but few SNP marker loci as Abbondanza. According to the same genotype SNP loci number in chromosome 2A, ES-9 contained more of the same genotype SNP loci as Th. ponticum rather than Abbondanza. It suggested that chromosome 2A in ES-9 were replaced by the pair of Th. ponticum chromosome, which was consistent with the FISH result. In ES-10 (Fig. 2b), the intersection point was detected in chromosome 3D where ES-10 had the most of the same SNP marker loci as Th. ponticum but few SNP marker loci compared with Abbondanza, which was consistent with the FISH result, suggesting that chromosome 3D of ES-10 were substituted by the pair of Th. ponticum chromosomes. While in ES-24, the intersection point was also detected in chromosome 3D, but the most of the same SNP marker loci was obtained from the comparison between Th. intermedium and ES-24, which meant that chromosome 3D of ES-24 was replaced by the pair of Th. intermedium chromosomes (Fig. 2d). It was consistent with the FISH analysis of ES-24. In terms of ES-23, ES-25 and ES-26, the intersection point of each material was undoubtedly identified in chromosome 2A (Fig. 2c), chromosome 2B (Fig. 2e), as well as chromosome 2D (Fig. 2f). Combined with the FISH results, it was revealed that chromosome 2A in ES-23, chromosome 2B in ES-25, as well as chromosome 2D in ES-26 were substituted by the same pair of Th. intermedium chromosomes.
PLUG marker analysis of the six substitution lines
The 135 PLUG markers were screened to further validated the homoeologous groups for the alien chromosomes. There were four PLUG markers (TNAC1142-HaeIII, TNAC1142-TaqI, TNAC1132-TaqI, TNAC1140-TaqI) mapped to the second homoeologous group in ES-9, ES-23, ES-25 and ES-26 (Table S7, Fig. 3a-d). While three pairs of primers (TNAC1326-HaeIII, TNAC1326-TaqI, TNAC1359-TaqI) were distributed in the third homoeologous group in ES-10 and ES-24 (Table S7, Fig. 3e-g). Combined with the mc-GISH results of each substitution lines, it was showed that 2St-chromosome-specific bands could be amplified in ES-9, ES-23, ES-24, ES-25, ES-26, Th. intermedium and Th. ponticum, in addition, 3St-chromosome-specific bands were identified in ES-10, ES-24, as well as, Th. intermedium and Th. ponticum, whereas the above polymorphic bands could not be amplified in Abbondanza.
The FISH karyotypes of Th. intermedium chromosomes 2St/3St, as well as, Th. ponticum chromosomes 2St/3St were characterized by in situ hybridization combined with wheat 15K SNP array analyses and a further PLIG marker screening. The genome composition of ES-25 (Fig. 4d) was 14A + 12B + 14D + 2(2St), while that of ES-26 (Fig. 4f) was 14A + 14B + 12D + 2(2St). Remarkably, chromosome 2St contained in ES-23 (Fig. 4b) were derived from Th. intermedium whereas the chromosome 2St of ES-9 (Fig. 4a) were derived from Th. ponticum, but they were for the same genome composition of 12A + 14B+ 14D+ 2(2St). In addition, chromosome 3St of ES-24 (Fig. 4e) and ES-10 (Fig. 4c) derived from Th. intermedium and Th. ponticum, respectively, were for the same genome composition of 14A+ 14B+ 12D+ 2(3St).
Evaluation of resistance to stripe rust and agricultural performance of the six substitution lines
The agronomic traits of the six substitution lines as well as their parents Abbondanza and Xiaoyan784 (Table 1, Fig 5) or Zhong4 (Table 2, Fig 5) were compared. On average, the tiller number of ES-9 was higher and the spikes exhibited longer than those of Abbondanza. In terms of the other substitution lines derived from Zhong4, both ES-23 and ES-26 showed much more tillers, and the spikelets per spike number of ES-26 was higher than that of Abbondanza as well as Zhong4. Surprisingly, the average thousand kernel weight of the alien lines containing chromosome 2St (ES-9, ES-23, ES-25 and ES-26) were more than 43g. It was indicated that the chromosome 2St whether originating from Th. ponticum or Th. intermedium increased thousand-kernel weight.
At the adult stage, stripe rust reaction test of the six substitution lines was conducted by comparisons with the susceptible control (HXH). Sequentially, the IT score of the six substitution lines, Abbondanza, Xiaoyan784, Zhong4, as well as Th. ponticum and Th. intermedium were recorded under field conditions. The IT score of the above-mentioned materials were as follows: Th. ponticum, IT = 0, Th. intermedium, IT = 0, Xiaoyan784, IT = 0, Zhong4, IT = 0, ES-9, IT = 1, ES-10, IT = 0, ES-23, IT = 1, ES-24, IT = 0, ES-25, IT = 1, ES-26, IT = 1, Abbondanza, IT = 3, HXH, IT = 4 (Fig 5c). Furthermore, the seedling stage stripe rust infection was conducted in the greenhouse, and the IT scores were recorded at 24 days post-inoculation (Fig 5d). With an IT score of 0, Zhong4 and Xiaoyan784 were immune to the disease. Additionally, ES-10 and ES-24 were nearly immune (IT score of 1). In contrast, Abbondanza, ES-9, ES-23, ES-25 and ES-26 were susceptible (IT score of 3). The results suggested that ES-9, ES-23, ES-25 and ES-26 carried chromosome 2St of Th. ponticum or Th. intermedium showed highly resistant to stripe rust at the adult stage. While ES-10 and ES-24 contained chromosome 3St of Th. ponticum or Th. intermedium were highly resistant at all stages.
Meiotic chromosome pairing analysis of F1 hybrids
Based on molecular cytogenetic identification of the six substitution lines, crosses were made between the alien lines with the same genome compositions, respectively. There were 15 F1 plants obtained from the cross between ES-9 and ES-23, and 11 F1 plants obtained from the cross between ES-10 and ES-24. Meiotic chromosome pairing analysis of the F1 hybrids was conducted to further validate the related genome constitution (Table 3). More than half of the pollen mother cells (PMCs) of ES-9×ES-23 and ES-10×ES-24 had 21 bivalents at metaphase I, and there was no trivalents or quadrivalents, as well as lagging chromosomes observed at meiosis anaphase I. It was indicated that chromosome 2St originating from Th. ponticum and Th. intermedium exhibited the close homologous relationship between each other, so did the Thinopyrum chromosome 3St.
Pairwise comparisons of genomic polymorphism analyses and St-chromosomes-specific molecular markers development
After high-throughput sequencing, SLAF library was constructed with the sequencing details (Supplementary table 8). A total of 1,055,234 (ES-9), 938,861 (ES-10), 524,288 (ES-23), 1,026,271 (ES-24), 974,634 (Abbobdanza), 572,791 (Th. intermedium), and 513,056 (Th. ponticum) SLAFs were obtained. By bioinformatics analysis, 3203 (ES-9), 4455 (ES-23), 2775 (ES-10), and 3148 (ES-24) specific sequences were selected for further sequence alignments. There were 78 out of 263 sequences from ES-24 with homology more than 90% of ES-10 (78/153). In addition, 114 out of 221 sequences from ES-23 were more than 90% homologous with ES-9 (114/177). To some degree, these results revealed the possible genomic similarity between chromosome 2St/3St of Th. intermedium and Th. ponticum.
According to the above sequence alignment results, 110 fragments from ES-23 were selected, which were regarded as 2St chromosome-specific fragments and then 73 of 3St chromosome-specific fragments from ES-24 were also selected. Subsequently, 183 pairs of primers were designed to amplify fragments from CS, Abbondanza, Zhong4, Xiaoyan784, ES-9, ES-23, ES-10, ES-24. In addition, specificity of the primers was further confirmed by analysis of Th. ponticum, Th. intermedium, tetraploid P. spicata, diploid P. spicata, Th. bessarabicum, Th. elongatum, and the wheat- Th. intermedium 1-7St addition line. A total of two 2St-chromosome-specific molecular markers, PTH-005 and PTH-013, and two 3St-chromosome-specific molecular markers, PTH-113 and PTH-135, were developed (Fig 6, Table 4).
Utility of the 3St-chromosome-specific markers in BC1F2 population
In order to validate that the stripe rust resistance gene(s) were carried by chromosome 3St, 60 BC1F2 individuals of ES-24 and HXH were further used for a genetic analysis. The evaluation of stripe rust resistance revealed that Zhong4, ES-24, and the 33 F2 individuals were highly resistant to Pst race CYR32 at the seedling stage (Fig 7a). Subsequently, 10 resistant F2 individuals as well as 10 susceptible ones were randomly selected for FISH analysis. Compared with the FISH karyotype of ES-24, chromosome 3St were actually detected in the resistant individuals (Fig 7b) and susceptible ones had undetectable FISH pattern of chromosome 3St (Fig 7b). It was indicated that the novel stripe rust resistant gene(s) originated from the chromosome 3St of Th. intermedium.
Furthermore, the specificity of newly developed 3St-chromosome-specific molecular markers was confirmed by PCR analyses of the 60 BC1F2 individuals of ES-24 and HXH (Fig 8). Combined with the result of seedling stage stripe rust resistance evaluation, it was revealed that Xiaoyan784, Zhong4, ES-9, ES-24, and the 33 BC1F2 plants conferring strong resistance to Pst race CYR32 carried 3St chromosome-specific markers. Oppositely, the other 26 BC1F2 plants without specific amplification as well as the parental line Abbondanza, and susceptible control HXH were seriously susceptible to Pst race CYR32. It was indicated that the newly developed St-chromosome-specific molecular markers could be used to trace the chromosome 3St in a common wheat background.