Phenotypic characterization and gene mapping of hybrid necrosis in Triticum durum-Haynaldia villosa amphiploids.

KEY MESSAGE: Phenotypical, physiological and genetic characterization was carried out on the hybrid necrosis gene from Haynaldia villosa, and the related gene Ne-V was mapped to chromosome arm 2VL. Introducing genetic variation from wild relatives into common wheat through wide crosses is a vital strategy for enriching genetic diversity and promoting wheat breeding. However, hybrid necrosis, a genetic autoimmunity syndrome, often occurs in the offspring of interspecific or intraspecific crosses, restricting both the selection of hybrid parents and the pyramiding of beneficial genes. To utilize the germplasms of Haynaldia villosa (2n = 2x = 14, VV), we conducted wide hybridization between durum wheat (2n = 4x = 28, AABB) and multiple H. villosa accessions to synthesize the amphiploids (2n = 6x = 42, AABBVV). This study revealed that 61.5% of amphiploids derived from the above crosses exhibited hybrid necrosis, with some amphiploids even dying before reaching maturity. However, the initiation time and severity of necrosis varied dramatically among the progenies, suggesting that there were multiple genetic loci or multiple alleles in the same genetic locus conferring to hybrid necrosis in H. villosa accessions. Genetic analysis was performed on the F2 and derived F2:3 populations, which were constructed between amphiploid STH59-1 with normal leaves and amphiploid STH59-2 with necrotic leaves. A semidominant hybrid necrosis-related gene, Ne-V, was mapped to an 11.8-cM genetic interval on the long arm of chromosome 2V, representing a novel genetic locus identified in Triticum-related species. In addition, the hybrid necrosis was correlated with enhanced H2O2 accumulation and cell death, and it was influenced by the temperature and light. Our findings provide a foundation for cloning the Ne-V gene and exploring its molecular mechanism.

Identification and transfer of a new Pm21 haplotype with high genetic diversity and a special molecular resistance mechanism.

KEY MESSAGE: A new functional Pm21 haplotype, Pm21(8#), was cloned from the new wheat-H. villosa translocation line T6VS(8#)·6DL, which confers the same strong resistance to powdery mildew through a different resistance mechanism. Broad-spectrum disease resistance genes are desirable in crop breeding for conferring stable, durable resistance in field production. Pm21(4#) is a gene introduced from wild Haynaldia villosa into wheat that confers broad-spectrum resistance to wheat powdery mildew and has been widely used in wheat production for approximately 30 years. The discovery and transfer of new functional haplotypes of Pm21 into wheat will expand its genetic diversity in production and avoid the breakdown of resistance conferred by a single gene on a large scale. Pm21(4#) previously found from T6VS(4#)·6AL has been cloned. In this study, a new wheat-H. villosa translocation, T6VS(8#)·6DL, was identified. A new functional Pm21 haplotype, designated Pm21(8#), was cloned and characterized. The genomic structures and the splicing patterns of Pm21(4#) and Pm21(8#) were different, and widespread sequence diversity was observed in the gene coding region and the promoter region. In the field, Pm21(8#) conferred resistance to Blumeria graminis f. sp. tritici (Bgt), similar to Pm21(4#), indicating that Pm21(8#) was also a resistance gene. However, Bgt development during the infection stage was obviously different between Pm21(4#)- and Pm21(8#)-containing materials under the microscopic observation. Pm21(4#) inhibited the formation of haustoria and the development of hyphae in the initial infection stage, while Pm21(8#) limited the growth of hyphae and inhibited the formation of conidiophores in the late infection stage. Therefore, Pm21(8#) is a new functional Pm21 haplotype that provides a new gene resource for wheat breeding.

Genome-wide identification of the NLR gene family in Haynaldia villosa by SMRT-RenSeq.

BACKGROUND: Nucleotide-binding and leucine-rich repeat (NLR) genes have attracted wide attention due to their crucial role in protecting plants from pathogens. SMRT-RenSeq, combining PacBio sequencing after resistance gene enrichment sequencing (RenSeq), is a powerful method for selectively capturing and sequencing full-length NLRs. Haynaldia villosa, a wild grass species with a proven potential for wheat improvement, confers resistance to multiple diseases. So, genome-wide identification of the NLR gene family in Haynaldia villosa by SMRT-RenSeq can facilitate disease resistance genes exploration. RESULTS: In this study, SMRT-RenSeq was performed to identify the genome-wide NLR complement of H. villosa. In total, 1320 NLRs were annotated in 1169 contigs, including 772 complete NLRs. All the complete NLRs were phylogenetically analyzed and 11 main clades with special characteristics were derived. NLRs could be captured with high efficiency when aligned with cloned R genes, and cluster expansion in some specific gene loci was observed. The physical location of NLRs to individual chromosomes in H. villosa showed a perfect homoeologous relationship with group 1, 2, 3, 5 and 6 of other Triticeae species, however, NLRs physically located on 4VL were largely in silico predicted to be located on the homoeologous group 7. Fifteen types of integrated domains (IDs) were integrated in 52 NLRs, and Kelch and B3 NLR-IDs were found to have expanded in H. villosa, while DUF948, NAM-associated and PRT_C were detected as unique integrated domains implying the new emergence of NLR-IDs after H. villosa diverged from other species. CONCLUSION: SMRT-RenSeq is a powerful tool to identify NLR genes from wild species using the baits of the evolutionary related species with reference sequences. The availability of the NLRs from H. villosa provide a valuable library for R gene mining and transfer of disease resistance into wheat.

Long-range assembly of sequences helps to unravel the genome structure and small variation of the wheat-Haynaldia villosa translocated chromosome 6VS.6AL.

Genomics studies in wild species of wheat have been limited due to the lack of references; however, new technologies and bioinformatics tools have much potential to promote genomic research. The wheat-Haynaldia villosa translocation line T6VS·6AL has been widely used as a backbone parent of wheat breeding in China. Therefore, revealing the genome structure of translocation chromosome 6VS·6AL will clarify how this chromosome formed and will help to determine how it affects agronomic traits. In this study, chromosome flow sorting, NGS sequencing and Chicago long-range linkage assembly were innovatively used to produce the assembled sequences of 6VS·6AL, and gene prediction and genome structure characterization at the molecular level were effectively performed. The analysis discovered that the short arm of 6VS·6AL was actually composed of a large distal segment of 6VS, a small proximal segment of 6AS and the centromere of 6A, while the collinear region in 6VS corresponding to 230-260 Mb of 6AS-Ta was deleted when the recombination between 6VS and 6AS occurred. In addition to the molecular mechanism of the increased grain weight and enhanced spike length produced by the translocation chromosome, it may be correlated with missing GW2-V and an evolved NRT-V cluster. Moreover, a fine physical bin map of 6VS was constructed by the high-throughput developed 6VS-specific InDel markers and a series of newly identified small fragment translocation lines involving 6VS. This study will provide essential information for mining of new alien genes carried by the 6VS·6AL translocation chromosome.

A malectin-like/leucine-rich repeat receptor protein kinase gene, RLK-V, regulates powdery mildew resistance in wheat.

Pattern recognition receptors (PRRs) can trigger plant immunity through the recognition of pathogen-associated molecular patterns. In this study, we report that a malectin-like/leucine-rich repeat receptor protein kinase gene, RLK-V, from Haynaldia villosa putatively acts as a PRR to positively regulate powdery mildew resistance caused by Blumeria graminis f. sp. tritici (Bgt) in wheat. RLK-V has two alternatively spliced transcripts corresponding to an intact RLK-V1.1 and a truncated RLK-V1.2 caused by intron retention. Expression analysis showed that both transcripts could be up-regulated by Bgt in resistant materials, whereas the functional RLK-V1.1 was expressed only after Bgt inoculation. Promoter activity assays indicated that RLK-V could respond to Bgt even in susceptible wheat. Silencing of RLK-V in Pm21-carrying resistant materials resulted in compromised resistance to Bgt. In addition, over-expression of RLK-V1.1 in Pm21-lacking susceptible Yangmai158 and SM-1 by single-cell transient expression and stable transformation in Yangmai158 could improve powdery mildew resistance. We propose that RLK-V regulates basal resistance to powdery mildew, which is also required for broad-spectrum resistance mediated by the Pm21 gene. Over-expression of RLK-V1.1 could trigger cell death in Nicotiana benthamiana, and RLK-V1.1 transgenic wheat accumulated more reactive oxygen species and displayed a stronger hypersensitive response than did the recipient, which led to enhanced Bgt resistance. However, constitutive activation of RLK-V1.1 resulted in the abnormal growth of transgenic plants.