2022年10月10日中国电子科技大学生命科学与技术学院信息生物学中心与澳大利亚悉尼大学植物育种研究所生命与环境科学学院在Frontiers上发表了题为“Molecular and cytogenetic dissection of stripe rust resistance gene Yr83 from rye 6R and generation of resistant germplasm in wheat breeding”的研究论文。 该研究中黑麦6R衍生的条锈病抗性基因Yr83在小麦背景中通过非变性荧光原位杂交（ND-FISH）、寡核苷酸FISH染色和6R特异性PCR标记被物理定位在长臂的FL 0.87-1.00区域。 该基因对澳大利亚和中国的小麦条锈病菌株均表现出高度抗性。
1. 6R karyotype analysis of Sub6R(6D) and its derivatives in Sichuan wheat background
The researchers characterized the 6R chromosome from Triticale T-701 and Sub6R (6D) by multicolor FISH (mc-FISH) and genomic in situ hybridization (GISH). In the present study, the probe Oligo-Ku's native FISH (ND-FISH) was used instead of GISH to identify rye chromosome 6R in Sub6R (6D). A standard karyotype of 6R was generated using ND-FISH of the probes Oligo-pSc119.2 and Oligo-pSc200. The short arm of 6R carries a strong and weak pSc119.2 locus in the distal and subterminal regions, respectively, and a strong Oligo-pSc200 locus in the distal region. The 6R long arm has four Oligo-pSc119.2 and two Oligo-pSc200 signaling sites, with the Oligo-pSc200 hybridization site located between the interstitial and subterminal Oligo-pSc119.2 sites.
Using rye genomic DNA as a probe, Li et al. observed two strong hybridization bands on 6RL by GISH. Based on a comparison of GISH band patterns and FISH patterns, the two GISH bands on 6RL are physically located at the same location as the Oligo-pSc200 hybridization site. An R23 series containing 6DS.6RL translocation was selected from the BC1F4 generation of Sub6R(6D) and MY11. In addition, a new ND-FISH probe, Oligo-248, was developed, which generates specific hybridization sites in the 6RL end region of the T-701, Sub6R(6D), and R23 lines. The updated 6R ND-FISH KARYOTYPE will help to characterize the structural variants of the 6R chromosome, especially the distal end of the 6RL, in progeny or mutant lines.
Fig.1 NLD-FISH karyotype analysis of chromosome 6R in different wheat backgrounds
In order to transfer rye 6R chromosome fragments into the background of Sichuan wheat, a total of 908 sub6R(6D) and F3 generations of Sichuan wheat CM42 and MY11 were screened, and the probes Oligo-pSc119.2 and Oligo-pSc200 were used for native fluorescence in situ hybridization (ND-FISH). In the BC1F3 generation, R42 and R323 lines with a pair of 6R chromosomes were produced against the background of CM11 and MY476, respectively. In the F4 generation, telomere chromosomes 6RS and 6RL, and isochromosomes iso6RS and iso6RL were identified. In addition, 25 strains (2.7%) had different wheat-6R translocations, including T1DS.6RL, T2DS.6RL, T6DS.6RL, T6BS.6RL and T6RS.6DL, indicating a high frequency of breaks and refusions between 6R and wheat chromosomes. A homozygous T6DS.6RL translocation line R23 was developed. ND-FISH was performed using the common centromere repeat Oligo-CCS1 and rye-specific centromere repeat Oligo-pAWRC1.1 probes, and the results showed that the R23 line contained recombinant centromeres from wheat 6D and rye 6R, based on the different signal intensities using the two probes. The R476 strain showed 7-10 days earlier flowering than the parent MY11, a reduction in plant height to 80-90 cm, a significant increase in spike length, and the retention of stripe rust resistance from 6RL, suggesting that 6R from "Merced" had a positive effect on agronomic traits and rust resistance traits in the wheat background.
Fig.2 Results of sequence ND-FISH analysis
2. Identification of new 6R deletions and translocation lines
In order to further map the Yr83 gene, a total of 1,662 M3 plants from 195 M2:3 families were screened for γ-irradiated Sub6R(6D) lines, and native fluorescence in situ hybridization (ND-FISH) was performed using the probes Oligo-pSc119.2, Oligo-pTa535 and Oligo-pSc200. Approximately 4.0% of plants contain modified 6R chromosomes, including deletion, telomere, or isotelomeric 6R, and wheat-6R translocation chromosomes. Based on the standard karyotype of 6R, three types of deletions of 6RL were detected by FISH using Oligo-pSc119.2 and Oligo-pSc200. Type 6R-1 shows a deletion of the most distal Oligo-pSc119.2 locus at the end of 6RL, and the estimated break point is at FL 0.87 of 6RL. Type 6R-2 is a large deletion distal to 6RL, with a break point after the second Oligo-pSc200 site, an estimated break point of FL 0.82 and a deletion of FL 0.82-1.00. The deletion of type 6R-3 is larger than that of type 6R-2, and the break point is between the two Oligo-pSc200 sites. A T6AL.6RLdel line was developed from the hybrid progeny of the Sub6R(6D)/CS ph1b mutant Schomburgk/6R deletion and characterized by ND-FISH using Oligo-pSc200, Oligo-pTa535, and Oligo-pSc119.2. The breaking point is between the two Oligo-pSc200 sites on the 6RL, similar to FL 0.73 for the 6R-3 type.
图3 Sub6R(6D)系辐照子代小麦-黑麦6R系的ND-FISH
Sequential ND-FISH was performed using the probes Oligo-k288 or Oligo-D and Oligo-Ku, as well as Oligo-pSc119.2 and Oligo-pTa535 to detect breaks between wheat and 6R chromosomes. The probe Oligo-k288 specifically hybridizes to the A and B genome chromosomes, while Oligo-D specifically hybridizes to the D genome chromosomes. Two Robertsonian translocations involving 7A-6R and 7D-6R were observed in 0.06% of plants, while 2.13 non-Robertsonian wheat-6R translocations were detected in 12% of plants. Breaking points at FL 0.50 in 6RS and FL 0.50 and FL 0.73 in 6RL occur most frequently in non-Robertson translocations.
Table 1 Application of oligonucleotide probes in the identification of ND-FISH chromosomes
3. Confirm the breakpoint on the 6RL deletion by PCR marking
DNA of wheat lines MY11, Sub6R(6D), T6DS.6RL, R266, R76 and R367 was amplified using 190 PLUG markers, 321 CINAU markers and 190 Kustro rye 6RL specific markers. In addition, 16 6RL-specific primers were designed according to the position of the Lo7 6RL reference genome sequence. The breakpoints of R266 and T6AL.6RL22 are located in the 720.56-723.16 Mb region by marking Ku-6RL142 and Ku-6RL112. The breakpoint of R76 is located in the 784.09-786.82 Mb region by marking Ku-6RL416 and Ku-6RL912. The breaking point of R367 is located in the 806.17-807.21 Mb region by marking Ku-6RL17 and SC-6RL072. In the distal region of 6RL, the labels SC-6RL082 to SC-6RL087 fail to amplify in Sub6R (6D) but are amplified in CS-Imperial 6R. This suggests that the current 6R may have lost the 882-885 Mb region at the end of the 6RL, or perhaps the two rye varieties were highly differentiated in this region. Further investigation is needed to investigate the association between the unique hybridization of the probe Oligo-248 in ND-FISH and the apparent loss of the 6RL 882-885 Mb region. Corresponding to the reference genome of Lo7, the deletion in R367 is approximately 75 Mb (806-881 Mb) for 6RL.
Fig. 4 Physical location of the sub6R(6D) chromosome-specific marker
Table 2 6RL-specific markers for Sub6R(6D).
ND-FISH analysis was performed on the Sub6R(6D) and T6DS.6RL translocation lines R23 using the probes Oligo-pSc119.2 and Oligo-pTa535, followed by FISH mapping with a large number of oligo probes of Synt6 and Synt7. FISH mapping of Synt6 shows distinct signals across the proximal regions of 6RS and 6RL, suggesting that they have homology to the 6th group of chromosomes of wheat. Comparing the karyotype of 6RL using ND-FISH of the probes Oligo-pSc119.2 and Oligo-pTa535, it was found that tandem repeat localization of the two Oligo-pSc119.2 sites may be in the Synt7 region of 6RL of Sub6R(6D) and R23. FISH profiles hybridized using the probe Synt7 in the distal region of 6RL (approximately FL 0.82-1.00) indicate that this region has homology to wheat group 7 chromosomes.
图5 Sub6R(6D) (A, B)和R23 (C, D)的ND-FISH和Oligo-FISH图谱
4. Stripe rust resistance and physical positioning of Yr83
Wheat parents CM42 and MY11 were susceptible to Pst mixed races CYR32, CYR33 and CYR34 in the field, and the strains Sub6R(6D) and T6DS.6RL strains R23 were resistant. Homozygous 6RL deletion lines R266, R76 and R367 were susceptible at the adult stage. The results showed that the stripe rust resistance gene Yr83 of 6RL was located at FL0.87 ~ 1.00, which corresponded to the rye genome of Lo7 in the region of 806.26 ~ 881.00 Mb.
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