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Chromosome FISH karyotype of the Ns-genome chromosomes using probes pSc200 (red), pTa71A-2 (green) and Oligo-44 (red) for P. juncea and Leymus species. The arrangement of chromosomes was based on the FISH patterns similarities with P. huashanica (ZY3156) chromosomes and chromosomes morphology including arm ratio and relative length. White arrows indicate the FISH signal variations within homologous chromosomes, red lines indicate the chromosomes which can’t be discriminated from chromosomes of P. huashanica using all the three probes. Scale bar: 10 μm
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Background
Psathyrostachys huashanica Keng has long been used as a genetic resource for improving wheat cultivar because of its genes mediating the resistance to various diseases (stripe rust, leaf rust, take-all, and powdery mildew) as well as its desirable agronomic traits. However, a high-resolution fluorescence in situ hybridization (FISH) kary...
Citations
... A pair of chromosomes produced strong terminal pSc200 signals on both arms and distinct Oligo-44 signals in the middle region of both arms (Figure 2a). According to the published FISH karyotype of P. huashanica [32], this pair of chromosomes in line 18-1-5 were identified as 7Ns chromosomes. At the same time, sequential mc-FISH and GISH analyses of the same metaphase cell also demonstrated that the two abovementioned chromosomes were Ns chromosomes from P. huashanica (Figure 2b,c). ...
... The probe combination Oligo-pSc200 [70], Oligo-44 [71], and Oligo-pTa71A-2 [72], which was able to clearly distinguish all P. huashanica chromosomes from each other and all wheat chromosomes [32], was used to confirm the homoeologous group relationships of the added alien chromosomes in line 18-1-5. These oligonucleotide probes were 5 -endlabeled with 6-carboxytetramethylrhodamine or 6-carboxyfluorescein by Sangon Biotech (Chengdu, China). ...
Powdery mildew caused by Blumeria graminis f. sp. tritici is a devastating disease that reduces wheat yield and quality worldwide. The exploration and utilization of new resistance genes from wild wheat relatives is the most effective strategy against this disease. Psathyrostachys huashanica Keng f. ex P. C. Kuo (2n = 2x = 14, NsNs) is an important tertiary gene donor with multiple valuable traits for wheat genetic improvement, especially disease resistance. In this study, we developed and identified a new wheat—P. huashanica disomic addition line, 18-1-5—derived from a cross between P. huashanica and common wheat lines Chinese Spring and CSph2b. Sequential genomic and multicolor fluorescence in situ hybridization analyses revealed that 18-1-5 harbored 21 pairs of wheat chromosomes plus a pair of alien Ns chromosomes. Non-denaturing fluorescence in situ hybridization and molecular marker analyses further demonstrated that the alien chromosomes were derived from chromosome 7Ns of P. huashanica. The assessment of powdery mildew response revealed that line 18-1-5 was highly resistant at the adult stage to powdery mildew pathogens prevalent in China. The evaluation of agronomic traits indicated that 18-1-5 had a significantly reduced plant height and an increased kernel length compared with its wheat parents. Using genotyping-by-sequencing technology, we developed 118 PCR-based markers specifically for chromosome 7Ns of P. huashanica and found that 26 of these markers could be used to distinguish the genomes of P. huashanica and other wheat-related species. Line 18-1-5 can therefore serve as a promising bridging parent for wheat disease resistance breeding. These markers should be conducive for the rapid, precise detection of P. huashanica chromosomes and chromosomal segments carrying Pm resistance gene(s) during marker-assisted breeding and for the investigation of genetic differences and phylogenetic relationships among diverse Ns genomes and other closely related ones.
Psathyrostachys huashanica, which grows exclusively in Huashan, China, is an important wild relative of common wheat that has many desirable traits relevant for wheat breeding. However, the poorly characterized interspecific phylogeny and genomic variations and the relative lack of species-specific molecular markers have limited the utility of P. huashanica as a genetic resource for enhancing wheat germplasm. In this study, we sequenced the P. huashanica transcriptome, resulting in 50,337,570 clean reads that were assembled into 65,617 unigenes, of which 38,428 (58.56%) matched at least one sequence in public databases. The phylogenetic analysis of P. huashanica, Triticeae species, and Poaceae species was conducted using 68 putative orthologous gene clusters. The data revealed the distant evolutionary relationship between P. huashanica and common wheat as well as the substantial diversity between the P. huashanica genome and the wheat D genome. By comparing the transcriptomes of P. huashanica and Chinese Spring, 750,759 candidate SNPs between P. huashanica Ns genes and their common wheat orthologs were identified. Among the 90 SNPs in the exon regions with different functional annotations, 58 (64.4%) were validated as Ns genome-specific SNPs in the common wheat background by KASP genotyping assays. Marker validation analyses indicated that six specific markers can discriminate between P. huashanica and the other wheat-related species. In addition, five markers are unique to P. huashanica, P. juncea, and Leymus species, which carry the Ns genome. The Ns genome-specific markers in a wheat background were also validated regarding their specificity and stability for detecting P. huashanica chromosomes in four wheat–P. huashanica addition lines. Four and eight SNP markers were detected in wheat–P. huashanica 2Ns and 7Ns addition lines, respectively, and one marker was specific to both wheat–P. huashanica 3Ns, 4Ns, and 7Ns addition lines. These markers developed using transcriptome data may be used to elucidate the genetic relationships among Psathyrostachys, Leymus, and other closely-related species. They may also facilitate precise introgressions and the high-throughput monitoring of P. huashanica exogenous chromosomes or segments in future crop breeding programs.
