ABSTRACT
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease that can cause severe yield losses. Identification and utilization of stripe rust resistance genes are essential for effective breeding against the disease. Wild emmer accession TZ-2, originally collected from Mount Hermon, Israel, confers near-immunity resistance against several prevailing Pst races in China. A set of 200 F6:7 recombinant inbred lines (RILs) derived from a cross between susceptible durum wheat cultivar Langdon and TZ-2 was used for stripe rust evaluation. Genetic analysis indicated that the stripe rust resistance of TZ-2 to Pst race CYR34 was controlled by a single dominant gene, temporarily designated YrTZ2. Through bulked segregant analysis (BSA) and SSR mapping, YrTZ2 was located on chromosome arm 1BS and flanked by SSR markers Xwmc230 and Xgwm413 with genetic distance of 0.8 cM (distal) and 0.3 cM (proximal), respectively. By applying wheat 90K iSelect SNP genotyping assay, 11 polymorphic loci (consist of 250 SNP markers) closely linked with YrTZ2 were identified. YrTZ2 was further delimited into a 0.8 cM genetic interval between SNP marker IWB19368 and SSR marker Xgwm413, and co-segregated with SNP marker IWB28744 (attached with 28 SNP markers). Comparative genomics analyses revealed high level of collinearity between the YrTZ2 genomic region and the orthologous region of Aegilops tauschii 1DS. The genomic region between loci IWB19368 and IWB31649 harboring YrTZ2 is orthologous to a 24.5 Mb genomic region between AT1D0112 and AT1D0150, spanning 15 contigs on chromosome 1DS. The genetic and comparative maps of YrTZ2 provide framework for map-based cloning and marker-assisted selection (MAS) of YrTZ2.
INTRODUCTION
Bread wheat (Triticum aestivum L.) is one of the top three most important food crops which production affects worldwide food security. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the severest wheat diseases worldwide. Growing resistance cultivars is the most cost-effective and environmentally friendly method to control this disease. Up to date, more than 60 formally named and many other provisionally designated stripe rust resistance genes or quantitative trait loci (QTL) have been reported (McIntosh et al. 2013, 2014, 2016). Out of them, two stripe rust resistance genes, Yr36 and Yr18, have been isolated through map-based cloning strategy (Fu et al. 2009; Krattinger et al. 2009). However, stripe rust resistance genes tend to become ineffective due to the continuous evolution of Pst races and monoculture deployment of resistance cultivars in wide area (Wan et al. 2004, 2007; de Vallavieille-Pope et al. 2012). So there is a continued need for identification and utilization of diversified resistance genes from various wheat germplasm resources. Wild emmer (Triticum turgidum ssp. dicoccoides, 2n = 4x = 28, AABB) is allotetraploidy with A and B sub-genomes, which was derived from a spontaneous hybridization of two diploid wild grasses Triticum urartu (2n=14, AA) and an as yet unidentified Aegilops species related to Aegilops speltoides (2n=14, SS) (Dvorak et al. 1993; Dvorak and Zhang 1990). Wild emmer is the progenitor of cultivated tetraploid durum wheat (Triticum turgidum ssp. durum, AABB) and hexaploid bread wheat (Triticum aestivum, AABBDD) (Feldman 2001), harboring abundant genetic resources for wheat improvement, including abiotic stress tolerances (salt, drought and heat), biotic stress tolerances (powdery mildew, rusts and Fusarium head blight), grain protein quality and quantity, and micronutrient concentrations (Zn, Fe, and Mn) (Xie and Nevo 2008). Manual selection during wheat domestication resulted in an inadvertent loss of genes and quantitative trait loci (QTL) beneficial for improving wheat agronomic and economic traits. Although they could be introgressed into modern wheat cultivars through traditional long-term breeding methods, molecular breeding provides an improved strategy for wheat improvement and can greatly shorten the breeding period.
Previously, molecular markers used for genetic linkage maps are mainly comprised of restriction fragment length polymorphisms (RFLPs) (Blanco et al. 1998), amplified fragment length polymorphisms (AFLPs) (Nachit et al. 2001), simple sequence repeats (SSRs) (Somers et al. 2004; Song et al. 2005), and diversity arrays technology (DArT) (Akbari et al. 2006; Peleg et al. 2008). Recently, high-throughput genotyping technology became more and more important for genetic studies. With the advantages of abundance, usual biallelism and availability of genotyping platform, single nucleotide polymorphisms (SNPs) are increasingly applied for high-density genetic mapping, physical map construction, comparative genomics analysis, genome-wide association studies (GWAS) and genomic selection in rice (Zhao et al. 2011), maize (Ganal et al. 2011; Riedelsheimer et al. 2012), and wheat (Akhunov et al. 2009; Luo et al. 2009; Cavanagh et al. 2013; Wang et al. 2014).
Fine mapping and map-based cloning of wheat genes is tedious because of the characteristics of wheat genome: allopolyploid (AABBDD), large genome size (17 gigabase) and numerous repeat DNA (90%). The availability of draft genome sequences and International Wheat Genome Sequencing Consortium (IWGSC) survey sequences of T. aestivum cv. Chinese Spring, T. urartu accession G1812 and Ae. tauschii accession AL8/78 (Brenchley et al. 2012; Jia et al. 2013; Ling et al. 2013; IWGSC 2014) facilitates wheat gene mapping. In particular, the released high-resolution SNP genetic linkage map and accurate physical map of Ae. tauschii accession AL8/78 provides closely wheat-related target for comparative genomics analyses (Luo et al. 2013).
In present study, we report: (1) the identification and genetic mapping a near-immunity stripe rust resistance gene YrTZ2 derived from wild emmer with microsatellite markers and 90K iSelect SNP genotyping assay, and (2) comparative genomics analysis of the genomic regions of YrTZ2 with the genetic linkage map and physical map of Ae. tauschii.
MATERIALS AND METHODS
Plant Material
The wild emmer accession TZ-2 was used as the stripe rust resistant parent to make cross with a highly susceptible durum wheat cultivar Langdon. A set of 200 F6:7 recombinant inbred lines (RILs) advanced by single-seed descent approach and the parental Langdon and TZ-2 were evaluated for stripe rust resistance with the prevailing Pst race CYR34. A highly susceptible wheat variety Mingxian169 was used as the susceptible control.
Stripe rust evaluations
The parental lines Langdon and TZ-2, Langdon/TZ-2 hybrid F1, 200 F6:7 RILs and susceptible control Mingxian169 were inoculated with Pst race CYR34 at the jointing stage in Chengdu of Sichuan Province, China. At 18 - 20 days post inoculation when the susceptible control Mingxian169 had become severely infected, the infection type (IT) was recorded with a scale of 0 - 4, with 0 (immune reaction), 0; (hypersensitive reaction), 1 (highly resistant), 2 (moderately resistant), 3 (moderately susceptible) and 4 (highly susceptible), the values of 0 - 2 were rated as resistant, and those of 3 - 4 were rated as susceptible (Zhang et al. 2001). ITs were recorded again ten days later.
Genomic DNA isolation and SSR marker analysis
Genomic DNAs of the parental lines and the F6:7 RILs population were extracted from seeding leaves using the Plant Genomic DNA Kit (Tiangen Biotech, CO., Ltd, Beijing, China). DNA concentration was quantified using NanoPhotometer® P360 (Implem GmbH, Munich, Germany) and normalized to 100 ng/ul. Resistant and susceptible DNA bulks were produced by separately mixing equal amounts of DNA from ten homozygous resistant and ten homozygous susceptible F6:7 families for bulked segregant analysis (Michelmore et al. 1991). Wheat genomic SSRs (Xgwm, Xwmc, Xbarc, Xcfa, and Xcfd series, https://wheat.pw.usda.gov) were used for polymorphism surveys between the two DNA bulks, and the polymorphic SSR markers were subsequently genotyped in the RIL mapping populations.
PCR reactions were carried out in a 10 μl reaction volume with the following conditions: one denaturation cycle at 94° for 5 min, followed by 35 cycles at 94° for 45 s, 55 - 65° (depending on specific primers) for 45 s, and 72° for 1 min, followed by an extension step of 72° for 10 min. Fragment analysis of PCR products were carried out on 8% non-denaturing polyacrylamide gels (39 acrylamide: 1 bisacrylamide). After electrophoresis, the gels were silver stained and photographed.
Infinium 90K iSelect SNP Genotyping
To saturate the genomic region harboring the stripe rust resistance gene, the 200 F6:7 RILs were genotyped using wheat 90K iSelect SNP genotyping assay platform at the Genome Center of University of California, Davis according to the manufacturer’s protocol. SNP allele clustering was conducted with two population-based detection algorithms: Density Based Spatial Clustering of Applications with Noise (DBSCAN) and Ordering Points to Identify the Clustering Structure (OPTICS) using the polyploid version of GenomeStudio software as described in Wang et al. (2014). Subsequently, the cluster matrix of polymorphic SNP markers was output from the polyploid version of GenomeStudio, and the genotypes of samples assigned in TZ-2 cluster were marked ‘1’, and the genotypes of sample located in Langdon cluster were marked ‘2’, the others were marked ‘0’.
Genetic mapping of the stripe rust resistance gene
The polymorphic SNP markers, SSR markers and stripe rust resistance genotypes were used for linkage analysis with the MultiPoint mapping software as described in Peleg et al. (2008) and Luo et al. (2013). Co-segregating SNP markers were regarded as a polymorphic locus. The linkage map was constructed with the software Mapdraw V2.1 (Liu and Meng 2003).
Data availability
The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.
RESULTS
Inheritance of the stripe rust resistance gene in TZ-2
The wild emmer accession TZ-2 and durum wheat cultivar Langdon showed nearly immune and highly susceptible to stripe rust, respectively. The F1 plants are highly resistant to CYR34, indicating the dominant nature of the stripe rust resistance in TZ-2. Of the 200 F6:7 RILs derived from the cross between Langdon and TZ-2, 103 were resistant (IT 0-2) and 97 were susceptible (3-4), which fits the expected 1:1 ratio for a single gene inheritance (χ21:1= 0.18, P<0.05), indicating that a single dominant locus, provisionally designated YrTZ2, in TZ-2 is responsible for the stripe rust resistance.
Identification of microsatellite markers linked to YrTZ2
Initially, 194 SSR primer pairs distributed randomly throughout the whole genome were screened for polymorphisms between the parental lines as well as the resistant and susceptible DNA bulks. SSR markers, Xwmc406, Xwmc230, Xgwm413, Xwmc128 and Xcfd65 revealed polymorphisms between the resistant and susceptible parents as well as the bulked segregants. After testing the F6:7 segregating population, a linkage map for stripe rust disease resistance gene YrTZ2 was constructed. The gene YrTZ2 was localized into a 1.1 cM genetic interval between SSR markers Xwmc230 and Xgwm413 (Fig. 1).
Chromosome arm assignment and physical bin mapping
In order to locate the YrTZ2 in the deletion bins on chromosome 1BS, Chinese Spring homoeologous group 1 nullisomic-tetrasomics, ditelosomics and deletion lines were used to assign the chromosomal and physical bin locations of the YrTZ2-linked SSR markers. Both SSR markers Xgwm413 and Xwmc230 were detected in N1A-T1B, N1D-T1A, Dt1BS and 1BS-9, but absent in N1B-T1A, Dt1BL and 1BS-10 (Fig. 2), indicated that YrTZ2 is located on chromosome 1BS bin 0.50-0.84 (Fig. 1).
Identification of SNP markers linked to YrTZ2
The 200 F6:7 RILs were genotyped with 90K iSelect SNP genotyping assay. After clustering, 15625 SNP markers were polymorphic between the parental lines, which were subsequently used for whole genome linkage map construction utilizing the MultiPoint mapping software. Polymorphic SNP and SSR markers linked to YrTZ2 were used to construct a high-resolution linkage map of YrTZ2. Due to the limitation of population size, multiple co-segregating SNP markers were attached to one polymorphic locus with minimum missing scores and used as skeleton marker. All together, 11 polymorphic loci (consisting of 250 SNP markers), IWB33689, IWB10487, IWB54031, IWB21709, IWB19368, IWB28744, IWB31649, IWB56173, IWB57972, IWB46473 and IWB40316, were integrated into the genetic linkage map of YrTZ2 (Fig. 1). YrTZ2 was finally delimited into an 0.8 cM interval between SNP locus IWB19368 and SSR marker Xgwm413, and co-segregated with SNP locus IWB28744 (attached with 28 SNP markers) (Fig. 1).
Identification collinearity genomic region of YrTZ2 in Ae. tauschii and comparative genomics analysis
The sequences of the 250 SNP markers clustered into 11 polymorphic loci were used as queries to search the Ae. tauschii SNP marker extended sequence database to identify the orthologous gene pairs between T. dicoccoides 1BS and Ae. tauschii 1DS. Out of the 11 polymorphic loci, 7 loci, IWB54031, IWB19368, IWB28744, IWB31649, IWB56173, IWB57972 and IWB40316, identified 31 orthologous SNP marker extended sequence in Ae. tauschii. Compararive genomics analysis revealed high levels collinearity between YrTZ2 genomic region and its orthologous genomic regions in Ae. tauschii 1DS (Fig. 1; Table S1).
YrTZ2 was mapped between SNP markers IWB19368 and IWB31649, and co-segregated with IWB28744. IWB19368 and IWB31649 are corresponding to the extended sequences of markers AT1D0112 (distal) and AT1D0150 (proximal), respectively, on chromosome 1DS that were anchored to the assembled BAC contigs ctg220 and ctg2295 in the physical map of Ae. tauschii. Therefore, the genomic region between IWB19368 and IWB31649 was orthologous to a 24.5Mb genomic region containing 15 BAC contigs, ctg220, ctg4623, ctg1063, ctg5929, ctg3163, ctg699, ctg1065, ctg6879, ctg554, ctg2446, ctg393, ctg2286, ctg4912, ctg798 and ctg2295 on chromosome 1DS (Fig. 1).
DISCUSSION
Bread wheat is serving as an important global food crop all the time. Maximizing wheat production is becoming a big challenge for researchers, breeders and growers. Wild relatives of wheat harbor rich genetic resource for wheat improvement (Schneider et al. 2008; Xie and Nevo 2008). Wild emmer is the ancestor of modern cultivated wheat and mainly distributed in central-eastern (Turkey, Iran and Iraq) and western areas (Syria, Lebanon, Jordan and Israel) of the Fertile Crescent (Avni et al. 2014). Wild emmer harbors abundant beneficial traits that can be introgressed into tetraploid and hexaploid wheat in modern wheat breeding programs. However, wild emmer has not been explored thoroughly and its potential in wheat breeding programs remains to be further characterized (Xie and Nevo 2008).
Wild emmer accession TZ-2 was collected from Mount Hermon, Israel, and showed highly stripe rust resistance to many Pst races (CYR29, CYR30, CYR31, CYR32, CYR33 and CYR34) in greenhouse seedling and field adult plant stage tests. In this study, genetic analysis showed that the stripe rust resistance to CYR34 in TZ-2 is controlled by a single dominant gene YrTZ2 that was mapped between SNP locus IWB19368 and SSR marker Xgwm413 in a 0.8 cM genetic interval on chromosome 1BS Bin 0.50-0.84. Up to date, another two stripe rust resistance genes, Yr15 and YrH52, were derived from Israeli wild emmer wheat and located on chromosome 1BS. Yr15 was identified from wild emmer accession G25 and mapped on chromosome 1BS using cytogenetic analysis (McIntosh et al. 1996) and molecular markers (Sun et al. 1997; Chagué et al. 1999; Ramirez-Gonzalez et al. 2015). When studying the stripe rust resistance gene in T. dicoccoides accession Hermon 52, Peng et al. (1999, 2000) found the resistance locus YrH52 was linked to SSR marker Xgwm413 with a genetic distance of 1.3 cM (proximal). YrH52-linked polymorphic microsatellite markers analysis revealed that Yr15 (Xgwm413/UBC212a-Yr15-Nor1) is different from YrH52 (Xgwm413/UBC212a/Nor1-YrH52-Xgwm273) on 1BS (Peng et al. 2000). In current study, YrTZ2 (Xgwm413-YrTZ2-IWB19368) was located at similar portion of chromosome 1BS as that of Yr15 and YrH52. Phytopathology and allelism tests need to be conducted in the future to clarify if YrTZ2 is allelic or closely linked to Yr15 or YrH52.
In addition to Yr15, YrH52 and YrTZ2, several other stripe rust resistance genes have been identified on chromosome 1BS. Yr10 was identified from Turkish hexaploid wheat accession PI 178383 and mapped at the terminal region of chromosome 1BS (Wang et al. 2002). Yr24 was derived from T. turgidum subsp. durum accession K733 (McIntosh and Lagudah 2000). Yr26 was assumed to be from durum line γ80-1, a γ-radiated mutant (Ma et al. 2001). YrCH42 was identified from Chinese wheat cultivar Chuanmai 42 (Li et al. 2006). Evidences showed that Yr24, Yr26 and YrCH42 were the same gene (Ma et al. 2001; Li et al. 2006; McIntosh et al. 2013) and is losing resistance to the new virulent Pst race CYR34 in China (Han et al. 2012). YrAlp was derived from spring wheat cultivar Alpowa with race-specific all-stage resistance (Lin and Chen 2007). Cheng et al. (2014) identified broad-spectrum all-stage stripe rust resistance genes Yr64 and Yr65 in different bins of chromosome 1BS from durum wheat accessions PI 331260 and PI 480016, respectively. Pyramiding these genes on chromosome 1BS via marker-assisted selection would benefit the development of durable and broad-spectrum stripe rust resistance varieties in wheat breeding program. The characteristics of large genome size, hexaploid nature and numerous repetitive DNA sequences presented a formidable challenge to fine mapping and map-based cloning of wheat genes. Single nucleotide polymorphisms (SNPs) are the most abundant sequence variability in wheat genome. The nature of biallelic, cost-effective and high-throughput genotyping makes SNPs more suitable for genetic studies. The advent of wheat 90K iSelect SNP genotyping assay increased the number of gene-based markers which was applied for wheat genetic linkage map construction, genome-wide association studies and comparative genomics analysis (Cavanagh et al. 2013; Wang et al. 2014; Wu et al. 2015). In this study, YrTZ2 was initially mapped into a 1.1 cM genetic interval between SSR markers Xwmc230 and Xgwm413. To construct a high-density genetic linkage map, wheat 90K iSelect SNP genotyping assay was applied to saturate the genomic region of YrTZ2. Altogether, 250 polymorphic SNP markers clustering in 11 loci were located in the genomic region of YrTZ2. Finally, YrTZ2 was delimited within a 0.8 cM genetic interval between locus IWB19368 and marker Xgwm413, and co-segregated with locus IWB28744 (consisting of 28 attaching SNP markers) that could be served as a starting point for chromosome landing and map-based cloning as well as marker-assisted selection (MAS) of the YrTZ2 gene.
Comparative genomics analyses provided an effective way for wheat gene mapping. By applying comparative genomics analysis using genome sequences of Brachypodium, rice or sorghum, high-density genetic linkage maps of vernalization (VRN) genes (Yan et al. 2003, 2004, 2006), pairing homologous 1 (Ph1) (Griffiths et al. 2006), grain protein content-B1 (Gpc-B1) (Uauy et al. 2006), yellow rust resistance gene Yr36 (Fu et al. 2009), wax production gene W1 (Lu et al. 2015) and powdery mildew resistance gene Pm6 (Qin et al. 2011), Pm41 (Wang et al. 2014), Ml3D232 (Zhang et al. 2010), MlIW170 (Liu et al. 2012; Liang et al. 2015), MlIW172 (Ouyang et al. 2014) were constructed. The draft genome sequences of T. aestivum cv. Chinese Spring, T. urartu accession G1812 and Ae. tauschii accession AL8/78 enriched the available sequence resource and accelerated the wheat genomics research (Brenchley et al. 2012; Jia et al. 2013; Ling et al. 2013). The physical map of Ae. tauschii, anchored with 7,185 SNP marker extended sequence, provided an efficient tool for comparative genomics analyses among grass families, and marker development for fine mapping and map-based cloning of genes in wheat (Luo et al. 2013).
Comparative genomics analysis indicated highly collinearity between YrTZ2 genomic region (IWB19368-IWB31649) of 1BS and a 24.5 Mb orthologous genomic region spanning 15 BAC-contigs of Ae. tauschii 1DS. The recently finished BAC-contig sequence of Ae. tauschii and Chinese Spring IWGSC whole genome assembly Ver. 1.0 would further contribute to fine mapping, map-based cloning and marker-assisted selection (MAS) of YrTZ2.
ACKNOWLEDGEMENTS
This work was financially supported by the National Key Research and Development Program of China (2016YFD0101802).