Exploring interspecific hybridization dynamics in artificial forests of Pinus brutia and P. halepensis: Implications for sustainable afforestation.

Interspecific hybridization increases genetic diversity, which is essential for coping with changing environments. Hybrid zones, occurring naturally in overlapping habitats of closely related species, can be artificially established during afforestation. The resulting interspecific hybridization may promote sustainability in artificial forests, particularly in regions facing degradation due to climate change. Currently, there is limited evidence of hybridization during regeneration of artificial forests. Here, we studied the frequency of Pinus brutia Ten. × P. halepensis Mill. hybridization in five planted forests in Israel in three stages of forest regeneration: seeds before dispersal, emerged seedlings and recruited seedlings at the end of the dry season. We found hybrids on P. brutia, but not on P. halepensis trees due to asynchronous cone production phenology. Using 94 single-nucleotide polymorphism (SNP) markers, we found hybrids at all stages, most of which were hybrids of advanced generations. The hybrid proportions increased from 4.7 ± 2.1 to 8.2 ± 1.4 and 21.6 ± 6.4 per cent, from seeds to emerged seedlings and to recruited seedlings stages, respectively. The increased hybrid ratio implies an advantage of hybrids over P. brutia during forest regeneration. To test this hypothesis, we measured seedling growth rate and morphological traits under controlled conditions and found that the hybrid seedlings exhibited selected traits of the two parental species, which likely contributed to the fitness and survival of the hybrids during the dry season. This study highlights the potential contribution of hybrids to sustainable-planted forests and contributes to the understanding of genetic changes that occur during the regeneration of artificial forests.

Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement.

Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for the improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Aegilops longissima and Aegilops speltoides. Whole-genome comparative analysis, along with the recently sequenced Aegilops sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides genome is closest to the wheat B subgenome, and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

Virulence Survey of Puccinia striiformis in Israel Revealed Considerable Changes in the Pathogen Population During the Period 2001 to 2019.

A total of 353 urediniospore isolates of Puccinia striiformis f. sp. tritici (Pst) collected in Israel during 2001 to 2019 were analyzed. Pst pathogenicity was studied with a set of 20 differentials (17 Avocet and 3 other lines). Three periods were compared: 2001 to 2007, 2009 to 2016, and 2017 to 2019. No virulence to Yr5 or Yr15 was detected. Virulence frequencies on Yr4, Yr10, Yr24, and YrSp genes rose to the moderate level (0.28 to 0.44) in 2017 to 2019. Virulence frequencies to Yr2 and Yr9 decreased. One Pst phenotype was identified in all three periods, but its frequency drastically decreased from 0.74 in 2001 to 2016 to 0.21 in 2017 to 2019. The most probable scenario of emergence of wheat yellow rust in Israel is wind dissemination of Pst urediniospores from the Horn of Africa. Variability of the Pst population increased amid considerable evolution with two major transformations in 2009 and 2017. The first modification can be attributed to changes in wheat genetic background in Israel due to deployment of new cultivars resistant to yellow rust since 2004. The second shift in 2017 can be primarily explained by intensive deployment of wheat cultivars resistant to the stem rust race Ug99 in the 2010s in the Horn of Africa. This led to changing genetic backgrounds of the cultivated wheats in the donor region and development and long-distance spread of new Pst phenotypes to Israel. Two singular multivirulent Pst phenotypes were identified in 2019, one of them being closely related to the aggressive Warrior race. Such phenotypes may potentially defeat existing resistances.

Genome-Wide Association Study in Bread Wheat Identifies Genomic Regions Associated with Grain Yield and Quality under Contrasting Water Availability.

The objectives of this study were to identify genetic loci in the bread wheat genome that would influence yield stability and quality under water stress, and to identify accessions that can be recommended for cultivation in dry and hot regions. We performed a genome-wide association study (GWAS) using a panel of 232 wheat accessions spanning diverse ecogeographic regions. Plants were evaluated in the Israeli Northern Negev, under two environments: water-limited (D; 250 mm) and well-watered (W; 450 mm) conditions; they were genotyped with ~71,500 SNPs derived from exome capture sequencing. Of the 14 phenotypic traits evaluated, 12 had significantly lower values under D compared to W conditions, while the values for two traits were higher under D. High heritability (H2 = 0.5-0.9) was observed for grain yield, spike weight, number of grains per spike, peduncle length, and plant height. Days to heading and grain yield could be partitioned based on accession origins. GWAS identified 154 marker-trait associations (MTAs) for yield and quality-related traits, 82 under D and 72 under W, and identified potential candidate genes. We identified 24 accessions showing high and/or stable yields under D conditions that can be recommended for cultivation in regions under the threat of global climate change.

TdPm60 identified in wild emmer wheat is an ortholog of Pm60 and constitutes a strong candidate for PmG16 powdery mildew resistance.

KEY MESSAGE: We identified TdPm60 alleles from wild emmer wheat (WEW), an ortholog of Pm60 from T. urartu, which constitutes a strong candidate for PmG16 mildew resistance. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta improved the resistance to several local Bgt isolates. Wild emmer wheat (WEW), the tetraploid progenitor of durum and bread wheat, is a valuable genetic resource for resistance to powdery mildew fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). PmG16 gene, derived from WEW, confers high resistance to most tested Bgt isolates. We mapped PmG16 to a 1.4-cM interval between the flanking markers uhw386 and uhw390 on Chromosome 7AL. Based on gene annotation of WEW reference genome Zavitan_V1, 34 predicted genes were identified within the ~ 3.48-Mb target region. Six genes were annotated as associated with disease resistance, of which TRIDC7AG077150.1 was found to be highly similar to Pm60, previously cloned from Triticum urartu, and resides in the same syntenic region. The functional molecular marker (FMM) for Pm60 (M-Pm60-S1) co-segregated with PmG16, suggesting the Pm60 ortholog from WEW (designated here as TdPm60) as a strong candidate for PmG16. Sequence alignment identified only eight SNPs that differentiate between TdPm60 and TuPm60. Furthermore, TdPm60 was found to be present also in the WEW donor lines of the powdery mildew resistance genes MlIW172 and MlIW72, mapped to the same region of Chromosome 7AL as PmG16, suggesting that TdPm60 constitutes a candidate also for these genes. Furthermore, screening of additional 230 WEW accessions with Pm60 specific markers revealed 58 resistant accessions from the Southern Levant that harbored TdPm60, while none of the susceptible accessions showed the presence of this gene. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta conferred resistance against several local Bgt isolates.

Genome-Wide Mapping of Loci for Adult-Plant Resistance to Stripe Rust in Durum Wheat Svevo Using the 90K SNP Array.

Stripe rust is a foliar disease in wheat caused by Puccinia striiformis f. tritici. The best way to protect wheat from this disease is by growing resistant cultivars. Tetraploid wheat can serve as a good source of valuable genetic diversity for various traits. Here, we report the mapping of nine stripe rust resistance quantitative trait loci (QTL) effective against P. striiformis f. tritici in China and Israel. We used recombinant inbred lines (RILs) developed from a cross between the durum wheat cultivar Svevo and Triticum dicoccoides accession Zavitan. By genotyping the RIL population of 137 lines using the wheat 90K single-nucleotide polymorphism array, we mapped an adult-plant resistance locus QYrsv.swust-1BL.1, the most effective QTL, within a 0.75-centimorgan region in T. turgidum subsp. durum 'Svevo' on chromosome arm 1BL, corresponding to the region of 670.7 to 671.5 Mb on the Chinese Spring chromosome arm 1BL. Of the other eight minor-effect stripe rust QTL, seven were from Svevo and mapped on chromosomes 1A, 1B, 2B, 3A, 4A, and 5A, and one was from Zavitan and mapped on chromosome 2A. Several QTL with epistatic effects were identified as well. The markers linked to the resistance QTL can be useful in marker-assisted selection for incorporation of these resistance QTL into both durum and common wheat cultivars.

Reducing the size of an alien segment carrying leaf rust and stripe rust resistance in wheat.

BACKGROUND: Leaf and stripe rusts are two major wheat diseases, causing significant yield losses. The preferred way for protecting wheat from rust pathogens is by introgression of rust resistance traits from wheat-related wild species. To avoid genetic drag due to replacement of large wheat chromosomal segments by the alien chromatin, it is necessary to shorten the alien chromosome segment in primary recombinants. RESULTS: Here we report on shortening of an alien chromosome segment in wheat that carries leaf and stripe rust resistance from Sharon goatgrass (Aegilops sharonensis). Rust resistant wheat introgression lines were selected and the alien region was mapped using genotyping by sequencing. Single polymorphic nucleotides (SNP) were identified and used to generate diagnostic PCR markers. Shortening of the alien fragment was achieved by induced homoeologous pairing and lines with shortened alien chromosome were identified using the PCR markers. Further reduction of the segment was achieved in tertiary recombinants without losing the rust resistance. CONCLUSIONS: Alien chromatin in wheat with novel rust resistance genes was characterized by SNP markers and shortened by homoeologous recombination to avoid deleterious traits. The resulting wheat lines are resistant to highly virulent races of leaf and stripe rust pathogens and can be used as both resistant wheat in the field and source for gene transfer to other wheat lines/species.

The Israeli-Palestinian wheat landraces collection: restoration and characterization of lost genetic diversity.

BACKGROUND: For over a century, genetic diversity of wheat worldwide was eroded by continual selection for high yields and industrial demands. Wheat landraces cultivated in Israel and Palestine demonstrate high genetic diversity and a potentially wide repertoire of adaptive alleles. While most Israeli-Palestinian wheat landraces were lost in the transition to 'Green Revolution' semi-dwarf varieties, some germplasm collections made at the beginning of the 20th century survived in gene banks and private collections worldwide. However, fragmentation and poor conservation place this unique genetic resource at a high risk of genetic erosion. Herein, we describe a long-term initiative to restore, conserve, and characterize a collection of Israeli and Palestinian wheat landraces (IPLR). RESULTS: We report on (i) the IPLR construction (n = 932), (ii) the historical and agronomic context to this collection, (iii) the characterization and assessment of the IPLR's genetic diversity, and (iv) a data comparison from two distinct subcollections within IPLR: a collection made by N. Vavilov in 1926 (IPLR-VIR) and a later one (1979-1981) made by Y. Mattatia (IPLR-M). Though conducted in the same eco-geographic space, these two collections were subjected to considerably different conservation pathways. IPLR-M, which underwent only one propagation cycle, demonstrated marked genetic and phenotypic variability (within and between accessions) in comparison with IPLR-VIR, which had been regularly regenerated over ∼90 years. CONCLUSION: We postulate that long-term ex situ conservation involving human and genotype × environment selection may significantly reduce accession heterogeneity and allelic diversity. Results are further discussed in a broader context of pre-breeding and conservation. © 2019 Society of Chemical Industry.

SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene Yr26.

KEY MESSAGE: NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding. Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (> 30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.

Correction to: Landraces of snake melon, an ancient Middle Eastern crop, reveal extensive morphological and DNA diversity for potential genetic improvement.

Following publication of the original article [1], the authors reported the need for a more detailed acknowledgement of the source of the samples that were analyzed and their coordinates, which are discussed in the 'Methods' section of the article. This Correction provides an addition to the 'Methods' section, and a subsequently revised 'Acknowledgements' and 'Availability of data and materials' section.

Landraces of snake melon, an ancient Middle Eastern crop, reveal extensive morphological and DNA diversity for potential genetic improvement.

BACKGROUND: Snake melon (Cucumis melo var. flexuosus, "Faqqous") is a traditional and ancient vegetable in the Mediterranean area. A collection of landraces from 42 grower fields in Israel and Palestinian territories was grown and characterized in a "Common Garden" rain-fed experiment, at the morphological-horticultural and molecular level using seq-DArT markers. RESULTS: The different landraces ("populations") showed extensive variation in morphology and quantitative traits such as yield and femaleness, and clustered into four horticultural varieties. Yield was assessed by five harvests along the season, with middle harvests producing the highest yields. Yield correlated with early vigor, and with femaleness, but not with late vigor. At the molecular level, 2784 SNP were produced and > 90% were mapped to the melon genome. Populations were very polymorphic (46-72% of the markers biallelic in a 4 individuals sample), and observed heterozygosity was higher than the expected, suggesting gene flow among populations and extensive cross pollination among individuals in the field. Genetic distances between landraces were significantly correlated with the geographical distance between collecting sites, and with long term March precipitation average; variation in yield correlated with April temperature maxima. CONCLUSIONS: The extensive variation suggests that selection of local snake melon could result in yield improvement. Correlations between traits and climatic variables could suggest local adaptation of landraces to the diverse environment in which they evolved. This study stresses the importance of preserving this germplasm, and its potential for breeding better snake melons as an heirloom crop in our region.

Resistance of Aegilops longissima to the Rusts of Wheat.

Stem rust (caused by Puccinia graminis f. sp. tritici), leaf rust (P. triticina), and stripe rust (P. striiformis f. sp. tritici) rank among the most important diseases of wheat worldwide. The development of resistant cultivars is the preferred method of controlling rust diseases because it is environmentally benign and also cost effective. However, new virulence types often arise in pathogen populations, rendering such cultivars vulnerable to losses. The identification of new sources of resistance is key to providing long-lasting disease control against the rapidly evolving rust pathogens. Thus, the objective of this research was to evaluate the wild wheat relative Aegilops longissima for resistance to stem rust, leaf rust, and stripe rust at the seedling stage in the greenhouse. A diverse collection of 394 accessions of the species, mostly from Israel, was assembled for the study, but the total number included in any one rust evaluation ranged from 308 to 379. With respect to stem rust resistance, 18.2 and 80.8% of accessions were resistant to the widely virulent U.S. and Kenyan P. graminis f. sp. tritici races of TTTTF and TTKSK, respectively. The percentage of accessions exhibiting resistance to the U.S. P. triticina races of THBJ and BBBD was 65.9 and 52.2%, respectively. Over half (50.1%) of the Ae. longissima accessions were resistant to the U.S. P. striiformis f. sp. tritici race PSTv-37. Ten accessions (AEG-683-23, AEG-725-15, AEG-803-49, AEG-1274-20, AEG-1276-22, AEG-1471-15, AEG-1475-19, AEG-2974-0, AEG-4005-20, and AEG-8705-10) were resistant to all races of the three rust pathogens used in this study. Distinct differences in the geographic distribution of resistance and susceptibility were found in Ae. longissima accessions from Israel in response to some rust races. To P. graminis f. sp. tritici race TTKSK, populations with a very high frequency of resistance were concentrated in the central and northern part of Israel, whereas populations with a comparatively higher frequency of susceptibility were concentrated in the southern part of the country. The reverse trend was observed with respect to P. striiformis f. sp. tritici race PSTv-37. The results from this study demonstrate that Ae. longissima is a rich source of rust resistance genes for wheat improvement.

Discovery and characterization of two new stem rust resistance genes in Aegilops sharonensis.

KEY MESSAGE: We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen. Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F6 recombinant inbred line and an F2 population, two genes were identified that mapped to the short arm of chromosome 1Ssh, designated as Sr-1644-1Sh, and the long arm of chromosome 5Ssh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.

Distribution and haplotype diversity of WKS resistance genes in wild emmer wheat natural populations.

KEY MESSAGE: The wheat stripe rust resistance gene Yr36 ( WKS1 ) with a unique kinase-START domain architecture is highly conserved in wild emmer wheat natural populations. Wild emmer wheat (Triticum dicoccoides) populations have developed various resistance strategies against the stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst). The wild emmer gene, Yr36 (WKS1), which confers partial resistance to a broad spectrum of Pst races, is composed of a kinase and a START lipid-binding domain, a unique gene architecture found only in the Triticeae tribe. The analysis of 435 wild emmer accessions from a broad range of natural habitats revealed that WKS1 and its paralogue WKS2 are present only in the southern distribution range of wild emmer in the Fertile Crescent, supporting the idea that wheat domestication occurred in the northern populations. An analysis of full-length WKS1 sequence from 54 accessions identified 15 different haplotypes and very low-nucleotide diversity (π = 0.00019). The high level of WKS1 sequence conservation among wild emmer populations is in contrast to the high level of diversity previously observed in NB-LRR genes (e.g., Lr10 and Pm3). This phenomenon may reflect the different resistance mechanisms and different evolutionary pathways that shaped these genes, and may shed light on the evolution of genes that confer partial resistance to stripe rust. Only five WKS1 coding sequence haplotypes were revealed among all tested accessions, encoding four different putative WKS1 proteins (designated P0, P1, P2, and P3). Infection tests showed that P0, P1, and P3 haplotypes display a resistance response, while P2 displayed a susceptible response. These results show that the WKS1 proteins (P0, P1, and P3) can be useful to improve wheat resistance to stripe rust.

Three-dimensional modeling and diversity analysis reveals distinct AVR recognition sites and evolutionary pathways in wild and domesticated wheat Pm3 R genes.

The Pm3 gene confers resistance against wheat powdery mildew. Studies of Pm3 diversity have shown that Pm3 alleles isolated from southern populations of wild emmer wheat located in Lebanon, Jordan, Israel, and Syria are more diverse and more distant from bread wheat alleles than alleles from the northern wild wheat populations located in Turkey, Iran, and Iraq. Therefore, southern populations from Israel were studied extensively to reveal novel Pm3 alleles that are absent from the cultivated gene pool. Candidate Pm3 genes were isolated via a polymerase chain reaction cloning approach. Known and newly identified Pm3 genes were subjected to variation analysis and polymorphic amino acid residues were superimposed on a three-dimensional (3D) model of PM3. The region of highest interspecies diversity between Triticum aestivum and T. dicoccoides lies in leucine-rich repeats (LRR) 19 to 24, whereas most intraspecies diversity in T. aestivum is located in LRR 25 to 28. Interestingly, these two regions are separated by one large LRR whose propensity for flexibility facilitates the conformation of the PM3 LRR domain into two differently structured models. The combination of evolutionary and protein 3D structure analysis revealed that Pm3 genes in wild and domesticated wheat show different evolutionary histories which might have been triggered through different interactions with the powdery mildew pathogen.

Linkage disequilibrium and association analysis of stripe rust resistance in wild emmer wheat (Triticum turgidum ssp. dicoccoides) population in Israel.

KEY MESSAGE: Rapid LD decay in wild emmer population from Israel allows high-resolution association mapping. Known and putative new stripe rust resistance genes were found. Genome-wide association mapping (GWAM) is becoming an important tool for the discovery and mapping of loci underlying trait variation in crops, but in the wild relatives of crops the use of GWAM has been limited. Critical factors for the use of GWAM are the levels of linkage disequilibrium (LD) and genetic diversity in mapped populations, particularly in those of self-pollinating species. Here, we report LD estimation in a population of 128 accessions of self-pollinating wild emmer, Triticum turgidum ssp. dicoccoides, the progenitor of cultivated wheat, collected in Israel. LD decayed fast along wild emmer chromosomes and reached the background level within 1 cM. We employed GWAM for the discovery and mapping of genes for resistance to three isolates of Puccinia striiformis, the causative agent of wheat stripe rust. The wild emmer population was genotyped with the wheat iSelect assay including 8643 gene-associated SNP markers (wheat 9K Infinium) of which 2,278 were polymorphic. The significance of association between stripe rust resistance and each of the polymorphic SNP was tested using mixed linear model implemented in EMMA software. The model produced satisfactory results and uncovered four significant associations on chromosome arms 1BS, 1BL and 3AL. The locus on 1BS was located in a region known to contain stripe rust resistance genes. These results show that GWAM is an effective strategy for gene discovery and mapping in wild emmer that will accelerate the utilization of this genetic resource in wheat breeding.

Resistance of Aegilops Species from Israel to Widely Virulent African and Israeli Races of the Wheat Stem Rust Pathogen.

Widely virulent races of the stem rust pathogen (Puccinia graminis f. sp. tritici) such as those isolated from Africa (e.g., TTKSK, isolate synonym Ug99) threaten wheat production worldwide. To identify Aegilops accessions with effective resistance to such virulent stem rust races, up to 10 different species from Israel were evaluated against African races TTKSK, TTKST, and TTTSK and the Israeli race TTTTC as seedlings in the greenhouse. A wide diversity of stem rust reactions was observed across the Aegilops spp. and ranged from highly resistant (i.e., infection type 0) to highly susceptible (infection type 4). The frequency of resistance within a species to races TTTTC and TTKSK ranged from 7 and 14%, respectively, in Aegilops searsii to 98 and 100% in AE. speltoides. In all, 346 accessions were found resistant to the three African races and 138 accessions were resistant (or heterogeneous with a resistant component) to all four races. The species with broadly resistant accessions included Ae. longissima (59 accessions), Ae. peregrina (47 accessions), Ae. sharonensis (15 accessions), Ae. geniculata (9 accessions), Ae. kotschyi (5 accessions), and Ae. bicornis (3 accessions). Few geographical trends or correlations with climatic variables were observed with respect to stem rust resistance in the Aegilops spp. The exception was Ae. longissima infected with race TTTTC, where a high frequency of resistance was found in central and northern Israel and a very low frequency in southern Israel (Negev desert region). This geographical trend followed a pattern of annual precipitation in Israel, and a significant correlation was found between this variable and resistance in Ae. longissima. Although difficult, it is feasible to transfer resistance genes from Aegilops spp. into wheat through conventional wide-crossing schemes or, alternatively, a cloning and transformation approach. The broadly resistant accessions identified in this study will be valuable in these research programs.

Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

Genome-wide association mapping of rust resistance in Aegilops longissima.

The rust diseases, including leaf rust caused by Puccinia triticina (Pt), stem rust caused by P. graminis f. sp. tritici (Pgt), and stripe rust caused by P. striiformis f. sp. tritici (Pst), are major limiting factors in wheat production worldwide. Identification of novel sources of rust resistance genes is key to developing cultivars resistant to rapidly evolving pathogen populations. Aegilops longissima is a diploid wild grass native to the Levant and closely related to the modern bread wheat D subgenome. To explore resistance genes in the species, we evaluated a large panel of Ae. longissima for resistance to several races of Pt, Pgt, and Pst, and conducted a genome-wide association study (GWAS) to map rust resistance loci in the species. A panel of 404 Ae. longissima accessions, mostly collected from Israel, were screened for seedling-stage resistance to four races of Pt, four races of Pgt, and three races of Pst. Out of the 404 accessions screened, two were found that were resistant to all 11 races of the three rust pathogens screened. The percentage of all accessions screened that were resistant to a given rust pathogen race ranged from 18.5% to 99.7%. Genotyping-by-sequencing (GBS) was performed on 381 accessions of the Ae. longissima panel, wherein 125,343 single nucleotide polymorphisms (SNPs) were obtained after alignment to the Ae. longissima reference genome assembly and quality control filtering. Genetic diversity analysis revealed the presence of two distinct subpopulations, which followed a geographic pattern of a northern and a southern subpopulation. Association mapping was performed in the genotyped portion of the collection (n = 381) and in each subpopulation (n = 204 and 174) independently via a single-locus mixed-linear model, and two multi-locus models, FarmCPU, and BLINK. A large number (195) of markers were significantly associated with resistance to at least one of 10 rust pathogen races evaluated, nine of which are key candidate markers for further investigation due to their detection via multiple models and/or their association with resistance to more than one pathogen race. The novel resistance loci identified will provide additional diversity available for use in wheat breeding.