91 Genetic Dissection of Morphological and Phenological Traits Associated with Domestication Syndrome in Durum × Wild Emmer Wheat RIL Population

Andrii Fatiukha

Institute of Evolution and the Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa

Domestication and subsequent evolution under domestication of wheat caused substantial genetic changes, which affected plant morphology, physiology and phenology. Morphological characters, such as compactness of spikes, the number of side shoots, can be mentioned as domestication related traits in cereals. We suggest to consider the angle of side shoots (Ash) as a novel trait associated with the domestication syndrome. The objective of this study is to provide a better understanding of the antagonism between natural and man-made selection of the traits under domestication in order to identify the significant changes in phenology and morphology of wheat during domestication. We used a recombinant inbred line (RIL) population derived from a cross between Triticum durum (cv. Langdon) and *Triticum dicoccoides *(acc. G18-16) for mapping of quantitative trait loci (QTL) of five morphological and three phenological traits. A total of 36 QTL effects were identified that were co-located in 21 loci. Eight of these loci showed pleiotropic effects on the studied traits (including phenology). A major QTL effect of Ash, co-located with strong phenological effect, was identified on chromosome 2BL. We found that phenological loci affected the duration of flowering and development of wheat in different manners. The duration of the reproductive stage in cereals affects the development of apical meristem and many other morphological traits, such as the number of spikelets per spike and the number of side shoots. These results shed more light on shaping of wheat plant architecture and development during its evolution under domestication.

92 Molecular Dissection of AGPase Enzyme and its Genes in Wheat and Ten Other Species

Ritu Batra

Ch. Charan Singh University

ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer with two large subunits (LS) and two small subunits (SS). It plays a critical role in starch biosynthesis. Using the well characterized Sh2 (LS: large subunit) and Bt2 *(SS: small subunit) genes of maize AGPase as references, true orthologs were identified in seven other monocots (Triticum urartu, *Aegilops tauschii, wheat, rice, barley, sorghum and Brachypodium) and three dicots (Arabidopsis, chickpea and potato). The detailed structure, function and evolution of the genes encoding the LS and the SS among monocots and dicots were studied. The results of the present study suggested that: (i) at the DNA level, the genes controlling the SS are more conserved than those controlling the LS; the variation in both is mainly due to intron number, intron length and intron phase distribution; (ii) at protein level, the SS genes are more conserved relative to those for LS; (iii) “QTCL” motif (providing thermostability to AGPase) present in SS showed evolutionary differences in AGPase belonging to wheat 7BS, T. urartu, rice and sorghum, while “LGGG” motif in LS was present in all species except T. urartu and chickpea; (iv) expression analysis revealed downregulation of both subunits under conditions of heat and drought stress. The wheat sequences identified in the present study will be utilized to design genome specific primers. These primers will be used to amplify the three copies each of AGPase LS and SS genes located on homoeologous group 1 and 7 chromosomes, respectively in a set of wheat genotypes (20 heat tolerant, 20 heat sensitive and 8 moderately heat tolerant/sensitive) to identify alleles of AGPase LS and SS genes that may impart thermotolerance.

93 NAC Transcription Factor and Laccase Gene: Key Players in Deciphering FHB Resistance Mechanism in Wheat QTL-Fhb1

Nancy Soni

McGill University

Fusarium head blight (FHB) is one of the most devastating and alarming diseases of wheat around the globe. In addition to causing a loss in wheat crop yield, it also reduces grain quality with mycotoxin contamination. Among 121 quantitative trait loci (QTLs) associated with FHB resistance, QTL-Fhb1 is considered to have major resistance effects. Wheat near isogenic lines (NILs), derived from Sumai 3 and Thatcher cross, were sequenced using Illumina HiSeq technology to capture the genes localized within the fine mapped QTL-Fhb1 region located within a 1.27cM interval. A total of 26 genes were putatively identified, of which, wheat NAC transcription factor (TaNAC), which is also known as a master regulator of plant secondary cell wall biosynthesis, was found polymorphic. Also, a laccase gene (TaLAC) which catalyzes cell wall lignification was also found polymorphic. Associated semi-comprehensive metabolomics study revealed a few important metabolites related to phenylpropanoid and flavonoid pathway with high fold change in pathogen inoculated samples. When the TaNAC or TaLACsilenced, the fungal biomass and the disease severity increased. However, no significant change in RR metabolites observed. In-silico analysis revealed secondary wall NAC binding element (SNBE) site in the promoter region of TaLAC, which suggest the regulation of laccase gene by NAC transcription factor, thus, unveiling the mechanism of FHB resistance associated with QTL-Fhb1.

94 Mapping QTL for Fusarium Head Blight Resistance in Canadian Spring Wheat AC Barrie

Dinushika Thambugala

Agriculture and Agri-Food Canada

Breeding for resistance to Fusarium head blight (FHB) in Canadian spring wheat is hampered by a poor understanding of genetics of resistance, particularly native FHB resistance. Here we dissected the genetic basis of FHB resistance in the Canadian spring wheat variety, AC Barrie which possesses an intermediate level of FHB resistance. A recombinant inbred line (RIL) population from the cross Cutler/AC Barrie and a doubled haploid (DH) population of the cross AC Barrie/Reeder were evaluated for FHB resistance in multiple field nurseries. Genotyping was performed with the Illumina Infinium 90K wheat SNP beadchip. IM and ICIM analyses identified numerous QTL controlling FHB resistance in the AC Barrie/Cutler RIL population on chromosomes 1B, 2A, 2B, 2D, 3B, 4D, 5A, and 6B and Barrie contributed most of these QTL. Major QTL for FHB resistance from AC Barrie were mapped on chromosomes 3B and 6B at the expected locations of Fhb1 and Fhb2. Plant height locus Rht-D1 was identified on 4D, and Ppd-D1 locus was mapped on chromosome 2D. An additional FHB resistance QTL from AC Barrie mapped to the same region as a QTL from Nyubai on 3BS, near the centromere (3BSc). AC Barrie has a unique haplotype at Fhb1, Fhb2, and 3BSc relative to known resistance sources such as Sumai-3, Wuhan-1, and Nyubai. The DH population of the cross AC Barrie/Reeder is also being studied and results will be presented at the meeting. This study provides insight into the genetic basis of FHB resistance in Canadian spring wheat variety AC Barrie.

95 A Major Tan Spot Race-Nonspecific Resistance Gene in Tetraploid and Hexaploid Wheat

Justin D. Faris

USDA-ARS

Tan spot, caused by the necrotrophic fungus Pyrenophora tritici-repentis *(Ptr), is a major foliar disease of both common and durum wheat. Over the past few decades, research has revealed that wheat-Ptr* interactions are based on an inverse gene-for-gene system, where pathogen-secreted necrotrophic effectors (also known as host-selective toxins) induce susceptibility when recognized by dominant sensitivity genes in the host. However, a few race-nonspecific resistance QTLs have also been reported. In 2005, Faris and Friesen reported a race-nonspecific QTL with major effects on chromosome 3B in the Brazilian hard red spring wheat line BR34, and Kariyawasam et al. (2016) reported a QTL in the same region in the soft white spring wheat cultivar ‘Penawawa’. Here, we evaluated the Langdon durum–Triticum dicoccoides accession Israel-A chromosome substitution lines (LDN-DIC) for reaction to all races. With the exception of LDN-DIC 3B being highly resistant, LDN and all the LDN-DIC lines were moderately to highly susceptible. A recombinant inbred chromosome line population derived from LDN x LDN-DIC 3B was used to map the location of a single dominant resistance gene using SSR markers. In addition, chromosome 3B linkage maps in the BR34- and Penawawa-derived mapping populations were reconstructed using the Illumina 90K SNP array and SSRs, and the disease data was reanalyzed. Comparative mapping indicated that BR34, Penawawa, and T. dicoccoidesaccession Israel-A all likely possess the same chromosome 3B tan spot resistance gene. Current progress on marker development and deployment of the gene will be presented.

96 Development of Diagnostic Markers for the Detection of Functional and Non-Functional Alleles of Yr15

Tzion Fahima

Institute of Evolution and the Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa

Stripe rust, caused by the fungus Puccinia striiformis f.s. tritici (Pst), is a destructive disease of wheat globally. Depletion of effective resistance to Pst in cultivated wheat has led to search for new resistance genes in the wild relatives of wheat. One of the most promising genes conferring broad-spectrum resistance to stripe rust is Yr15, derived from wild emmer wheat (Triticum dicoccoides) accession G25. Yr15, mapped on chromosome arm 1BS, has recently been cloned by our consortium and designated as Wheat Tandem Kinase 1 (WTK1). We found wtk1 susceptible alleles in most 274 tested durum, bread, and wild emmer wheat lines. Out of 69 tested durum and bread wheat cultivars and lines, only 33 Yr15 introgression lines contained the functional allele (Wtk1) from G25 and were resistant to Pst. The remaining 36 susceptible lines carried non-functional alleles (wtk1), which included insertions of large transposable elements that resulted in changes in reading frame. Development of reliable molecular markers can facilitate the introgression of Yr15 into new varieties via marker-assisted selection. Diagnostic markers designed based on the polymorphism between the WTK1 *alleles are preferred in order to avoid negative linkage drag. Therefore, we have designed highthroughput co-dominant KASP markers that can differentiate between the functional (Wtk1) and all known non-functional (wtk1)* alleles, and can be used in breeding programs for development of modern cultivars with high resistance to stripe rust.

97 A Novel Approach of Rapid and Targeted Gene Transfer from Wild Relatives into Crop Plants

Kanwardeep Singh

Washington State University

The focus of the USAID funded innovation lab is to develop heat tolerant wheat varieties while understanding the heat tolerance trait at molecular, genetic, physiological and biochemical level. Since wild relatives of crop plants are known for their biotic and abiotic stress tolerance, one aspect of the project is to develop a fast, accurate, targeted and efficient method of transferring value added genes such as those controlling heat stress tolerance from wild relatives into cultivated wheat. But so far targeted transfer of such genes has been difficult because of Ph1 gene imposed restriction on chromosome pairing and recombination between wheat and wild relative chromosomes. With more than 300 useful genes transferred from the wild relatives into wheat, most have not been used in breeding because the transfers were either complete chromosome/arm or large segments which often carried undesirable traits along with the useful genes. We cloned a candidate for the Ph1 gene, silencing of which resulted in a phenotype characteristic of Ph1 gene mutants. Complementation of a Ph1 gene mutant (ph1b) with the candidate gene under its native promoter restored the chromosome pairing function. In this study, we transiently silenced the gene via VIGS to induce chromosome pairing and recombination between chromosome 1BS of wheat with 1RS of rye. Out of 250 plants that were analyzed, 66 plants showed recombination between wheat and rye chromosome arm. With an average of 5, the number of rye segments in each recombinant plant ranged from 1 to 6. The size of the rye segment transferred to wheat background ranged from 2 to 100 Mb. Although recombination hot-spots were obvious, recombination events were distributed on the entire chromosome arm.

98 The Cloned Yr15 Gene (WTK1) Encodes Two Kinase-like Protein Domains, Both Required for Conferring Broad-Spectrum Resistance to Stripe Rust

Tzion Fahima

Institute of Evolution and the Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease that threatens global wheat production. The wild emmer wheat gene Yr15, *located on chromosome 1BS,* confers resistance to a broad spectrum of Pstraces. Comparative genomics, chromosome walking, BAC libraries (wild emmer and bread wheat), whole genome assemblies, EMS mutagenesis and transgenic approaches enabled us to clone Yr15 and validate its function. The Yr15 protein has a novel structure for R-genes in wheat with two kinase-like domains in tandem, designated here Wheat Tandem Kinase 1 (WTK1). We have shown that both kinase domains are essential for conferring Pst resistance. Macro- and microscopic observations of development and accumulation of fungal biomass suggest that the hypersensitive response plays a central role in the resistance mechanism, limiting the development of fungal feeding structures. Non-functional alleles of Yr15 in T. dicoccoides, T. durum and T. aestivum differ from the functional allele of DIC G25 by indels, creating truncated proteins. Therefore, we designed diagnostic markers that differentiate between functional and non-functional Yr15 alleles. Our results suggest that Yr15* has the potential to improve stripe rust resistance in a wide range of tetraploid and hexaploid wheat germplasm. The absence of the functional Yr15in tested durum and common wheat varieties highlights the value of DIC germplasm as a reservoir of resistance genes for wheat.

99 Exploring Genetic Diversity in Bread Wheat Using Nested Association Mapping

Melissa Garcia

School of Agriculture, Food and Wine, University of Adelaide

The rate of genetic gain in breeding programs can be increased by extending the amount of variation available for selection using land races and exotic germplasm. However, exotic germplasm carries a range of undesirable traits that limits their suitability for modern agriculture. Backcrossing to locally adapted varieties and pre-selection for traits is therefore required to ensure meaningful data are generated in field trials.

Multi-parental schemes such as Nested Association Mapping (NAM) populations improve the use of exotic germplasm as a resource for the discovery of novel traits and QTL/genes. NAM combines the power of linkage analysis and the precision of association mapping. When jointly analysed, NAM populations can provide higher power to detect QTL than any of the constituent biparental families separately.

We selected 75 highly diverse hexaploid spring-type wheat accessions from regions of the world that are affected by heat and drought stress. These accessions were crossed with two Australian elite varieties as founder parents, and BC1F6 populations were generated.

Twenty individuals from each of 28 NAM sub-populations were genotyped at BC1F4 using a targeted genotype by sequencing assay. These lines are being grown in a completely randomised field trial with two replications. Plots were phenotyped for NDVI, relative maturity and presence of awns. Plant height, yield, thousand grain weight and harvest index will be obtained at harvest.

Genome wide association analysis is underway. Selected populations which maximize diversity and power will be phenotyped in multiple field trials across Australia next year.

100 New Genotyping Technology, GRAS-Di, Using Next Generation Sequencer

Hiroyuki Enoki

Toyota Motor Corporation

We developed new genotyping technology, Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di). This technology consisted of sample preparation using high concentration random primer, NGS and data analysis. The sample preparation was very simple. It was not necessary to do primer design, enzyme digestion, fragmentation, size selection, adaptor ligation, and sample normalization. It was only two steps PCR for NGS library without specialized equipment. Rice BIL population was used for evaluation of genotyping by GRAS-Di (96 samples / lane of HiSeq2500). The number of reads for each amplicon was highly reproducibility, r > 0.99, with repetition. Over ten thousand SNPs were detected among the BIL population and the SNPs were distributed uniformly rice genome. The ratio of missing value was very low, 1.5%. The reproducibility of SNP was 99.9% with repetition. If there was no reference sequence, genotype data could be detected by GRAS-Di using original algorism based on amplicon analysis. Theoretically, the technology is also applicable to other creatures, including highly polyploidy creatures. We performed the applicability test for several creatures. The result shown that the technology was applicable for over fifty creatures, including wheat, soybean, tomato, potato, sugarcane, cow, pig, chicken, tuna and human. The technology could be provided over 30,000 multiplex sequencing at once. We think that GRAS-Di would be very easy and very powerful technology for genome wide genotyping in many creatures. We signed licensing agreement with Kazusa DNA Research Institute, Eurofins Genomics, and GeneBay for GRAS-Di.

101、Stacking Multiple Stem Rust Resistance Genes at a Single Locus for Durable Resistance in Wheat

Ming Luo

Commonwealth Scientific and Industrial Research Organisation

Stem rust disease caused by the fungal pathogen Puccinia graminis f.sp tritici is a significant threat to global wheat production. The most cost effective way to control this disease is by genetic resistance. However major gene resistance is often readily overcome by pathogen evolution when resistance genes are deployed singularly. Combining major resistance genes is believed to extend their durability as multiple mutations are required in the pathogen to overcome this polygenic resistance. Major resistance genes can be combined by conventional breeding however this is a labour intensive process and resistance gene combinations are difficult to maintain in segregating families. In this study, we have used cloned stem rust resistance genes Sr22, Sr35, Sr45, Sr46 and Sr50 and the multi-pathogen adult plant rust resistance gene Sr55/Lr67/Yr46/Pm46/Ltn3 to produce binary vectors containing combinations of these genes. Constructs containing either 3, 4, 5 or 6 rust resistance genes were produced and transformed into bread wheat by Agrobacterium transformation. Molecular/genetic analysis demonstrated that some transgenic wheat lines contain all the resistance genes present in the binary vector used for transformation (ie. up to six) and these genes are inherited as a single locus in progeny plants. Transgenic plants are resistant to wheat stem rust disease with resistance co-segregating with the multigene transgenic locus.

102 MAGIC Yield: Using an Eight Founder Population for the Genetic Dissection of Yield and Yield Components in UK Winter Wheat

Benedetta Saccomanno

NIAB

Multiparent advanced generation inter-cross (MAGIC) populations are a powerful mapping resource in crop genetics for the dissection of complex traits, previously hindered by relatively low genetic recombination and allelic diversity of traditional bi-parental populations. Wheat (*Triticum aestivum *L.) is a major arable crop of global importance, covering 1.6 million hectares in the UK alone (AHDB survey, 2017). Breeders and farmers must continue to improve wheat grain yield and yield stability to help meet demand from an increasing population, and to ensure food security in the face of the effects of climate change. The Magic Yield project helps address these problems by using an eight-founder MAGIC population (Mackay *et al. *2014), consisting of 1,000 lines created by inter-crossing eight elite UK winter wheat varieties over three generations, to study the genetic basis of yield and yield components. With the participation of five wheat breeding companies, we conducted field trials at five UK sites for two consecutive years, phenotyping yield and a suite of pre- and post-harvest yield components. Phenotypic data coupled with Illumina iSelect 90k SNP genotype data (Gardner *et al. *2016) allowed the detection of a total of 76 quantitative trait loci (QTL) across all year, trait and site combinations. Flanking markers for selected QTL were converted to Kompetitive Allele Specific PCR (KASP) markers to aid fine-mapping and consequent characterization of genes controlling yield. Ultimately, the resources generated will aid the selection of wheat lines with improved performance within breeding programs, for the downstream benefit to growers and end-users.

103 “Am Hidden, Feeling Hot! Do Something!” – Heat Stress on Wheat Roots

Sundaravelpandian Kalaipandian

CSIRO (Agriculture and Food)

High temperature is a major threat to plant productivity due to climate change. Often the hidden-half of the plant is more sensitive to heat stress than the above ground parts. Heat stress affects the roots by limiting water and nutrient uptake, which in turn affect shoot water demand and photosynthesis. However, the molecular mechanism of root responses to heat stress is poorly understood. Recently, we have studied the role of TaHsfC2a gene in wheat. Overexpression of TaHsfC2a-B in transgenic wheat plants increased survival rate to about 90% while only 15% of wild-type plants survived after heat treatment at 43°C. Interestingly, we observed that the shoots were drying but the roots were intact after heat treatment in the transgenic plants, which contributed to recovery of the shoots, however all parts of the wild-type plants died after heat treatment. Reactive oxygen species (ROS) was shown to have a major role in abiotic stresses including heat stress. We found that the transgenic plant roots accumulate very low hydrogen peroxide (H2O2) when compared with wild-type plant roots. To understand the molecular mechanisms underlying the heat stress and ROS in the roots, the transcriptome of TaHsfC2a transgenic and wild-type roots are being studied by using RNA-sequencing. In addition, we found that TaHsfC2a was markedly up-regulated under drought and abscisic acid treatment, and we have also identified the potential targets of this gene (TaHSP70d and TaGalSyn) and confirmed through transactivation studies. Our study will identify candidate genes to develop heat resistant varieties in wheat.

104 Leveraging the Root Angle QTLome to Enhance Climate Resilience in Wheat

Marco Maccaferri

DipSA, Department of Agricultural Science, University of Bologna

Optimisation of root system architecture (RSA) is an important objective for the sustainability of durum wheat grown under drought-stressed conditions. In the present study, linkage and association mapping (AM) for RSA evaluated at the seedling stage evidenced 20 clusters of quantitative trait loci (QTLs) for root length and number as well as 30 QTLs for root growth angle (RGA). The most divergent RGA phenotypes observed by seminal root screening were validated by root phenotyping of field-grown adult plants. QTL analysis of RSA and grain yield data indicates RGA as a valuable target to enhance grain yield and yield stability across different soil moisture regimes (Maccaferri et al. 2016). Based on their relative additive effects, allelic distribution in the AM panel and co-location with QTLs for yield, eight RGA QTLs have been prioritised in terms of breeding interest and value. These QTLs were investigated for gene content based on the chromosomal pseudomolecules of Chinese Spring T. aestivum and the TriAnnot v4.3 gene prediction and annotation pipeline and the Zavitan T. dicoccoides genome assembly (Avni et al. 2017). The chromosome regions contained 25 to 242 predicted genes (123 on average). In six RGA QTLs, from one to four gene annotations were involved in auxin pathways. The comparison between the T. aestivumand T. dicoccoides gene content indicates the high quality of the T. *dicoccoides *assembly and its usefulness to identify candidates to explore the polymorphism and the structural variation of drought-related genes present in the A and B wheat genomes.

105 A TRIM Insertion led to a Gene Resurrection Event that causes Male Sterility in Wheat

Jizeng Jia

Chinese Academy of Agricultural Sciences

The male sterile ms2 mutant has been known for 40 years1 and has become extremely important in the commercial production of wheat. However, the gene responsible for this phenotype has remained unknown. We here report the map-based-cloning of the Ms2 gene. The Ms2 *locus is remarkable in several ways that have implications in basic biology. Beyond having no functional annotation and clearly having undergone pseudogenization, we found that the *Ms2 allele in the ms2 mutant acquired a terminal-repeat retrotransposon in miniature (TRIM) element in its promoter. This TRIM element is responsible for the anther-specific Ms2 *activation that confers male sterility. The identification of *Ms2 not only unravels the genetic basis of a historically-important breeding gene, but also illustrates pseudogenization at the population level and shows that resurrection of an unfixed pseudogene in the population can contribute to genetic novelty and phenotypic plasticity.

106 Fine Mapping of D-Genome Yield QTLs in Hexaploid Wheat

Increasing crop yields is an ever more crucial endeavor as the global human population continues its near exponential growth. One strategy to meeting future food demands is identifying and incorporating yield-enhancing genes into elite crop lines. Wheat (Triticum aestivum, 2n=6x=42 AABBDD), which supplies a fifth of humans’ calories worldwide, can reap the benefits of the genetic variation from the D-genome progenitor, Aegilops tauschii *(2n=2x=14 DD), which may supply yield-boosting alleles. A nested association mapping population of the D-genome (DNAM) was created from direct hybridization of the hard white winter variety, KS05HW14, and seven *Ae. tauschii *accessions and backcrossing the F1 progeny twice to KS05HW14 to regain euploidy. BC2F4 derived lines from the DNAM population were phenotyped for grain yield in Manhattan, KS; Hays, KS; and Richville, MI in 2015 and 2016, and Marianna, AR; Champaign, IL; Brookings, SD; and Pullman, WA in 2016. A genome-wide association analysis identified QTL conferring higher grain yield. Large-effect QTL identified on chromosomes 2DS and 6DL were contributed by the recurrent parent. QTL on chromosomes 2DL and 7DS were derived from the Ae. tauschii *accessions, TA1615 and TA1718. KASP markers designed for significant SNPs on the QTL identified the following segregating regions: 25.4Mb to 29.5Mb on 2DS, 430.4Mb to 575.9Mb on 2DL, 463.5Mb to 473.3Mb on 6DL, and 517.0Kb to 12.5Mb on 7DS. These data were used to create heterozygous inbred families that will be used in QTL fine-mapping and identifying yield-enhancing genes.

107 Integrating Genomic Selection in Breeding for Resistance to Rusts and Foliar Diseases in Wheat

Fnu Philomin Juliana

CIMMYT

Genomic selection is a promising technology that could increase genetic gains for quantitative disease resistance and help eliminate susceptible lines, before costly disease screening. To evaluate the potential integration of GS as a breeding tool, we tested genomic prediction for several diseases in CIMMYT’s 1st year yield trials (YT) and 2nd year elite yield trials (EYT), from 2015-2016 and 2016-2017. While the YTs comprised about 9,000 lines, the EYTs were a subset comprising 1,092 lines. All lines were genotyped using genotyping-by-sequencing and the YTs were phenotyped for response to Ug99 stem rust (SR) race in Njoro, Kenya. The EYTs were phenotyped for SR in Njoro; yellow rust (YR) in Ludhiana, India; Fusarium head blight (FHB) in El Batan, Mexico; Septoria tritici blotch (STB) in Toluca, Mexico and spot blotch (SB) in Agua Fria, Mexico. The maximum within-nursery and across-nursery prediction accuracies were 0.74 and 0.60 for SR, 0.59 and 0.50 for YR, 0.42 and 0.21 for FHB, 0.50 and 0.18 for STB and 0.56 and 0.37 for SB, respectively. We also observed that at different selection intensities, GS could discard upto a maximum of 92% of the susceptible lines discarded by PS and select upto 61% of the resistant lines selected by PS, within nurseries. However, when selections were made across-nurseries, GS could discard 73.8-90.2% of the susceptible lines and select 24.5-61.6% of the resistant lines. While these results are promising, further efforts to improve prediction accuracies are crucial for the successful integration of GS in wheat disease resistance breeding.

108 Spatio-Temporal Asymmetry of the Meiotic Pathway in Hexaploid Bread Wheat

Kim Osman

University of Birmingham

During meiosis homologous recombination (HR) generates genetic variation and provides the physical links (crossovers - COs) necessary for accurate segregation of chromosomes. In most eukaryotes the distribution of COs along chromosomes is non-random due to the influence of multiple levels of control which ensure each chromosome pair receives at least one CO and which discourage additional COs forming in adjacent chromosomal regions. Further complexity is evident in the tendency of chiasmata (the cytological manifestation of COs) to form in favourable regions of the chromosome. In some species this has led to the restriction of COs/chiasmata to particular chromosomal locations. In hexaploid wheat and other cereals the predominantly distal location of COs creates a problem of linkage-drag in the recombinationally ‘cold’ centromere/proximal and interstitial regions where agronomically important traits cannot be readily separated from undesirable ones.

As partners in a collaborative project involving five UK research groups and two wheat-breeding companies, our aim is to understand the factors influencing CO formation in hexaploid wheat in order to manipulate the process and unlock genetic diversity for crop improvement. Building on research in Arabidopsis meiosis, we are employing molecular cytogenetic techniques to perform a detailed analysis of key stages in the recombination pathway during the progression of prophase I. Here we present data showing that early recombination events in Cadenza are spatio-temporally asynchronous, initiating in the distal chromosomal regions and later spreading throughout the chromatin. This pattern reflects the distribution of euchromatin within the nucleus as revealed by immunolocalisation of various histone modifications.

109 Accelerated Cloning and Characterization of the Wheat Adult Plant Resistance Gene Lr68

Sreya Ghosh

John Innes Centre

Adult Plant Resistance (APR) genes are broad-spectrum, partial resistance genes that can contribute to sustainable control of wheat rust diseases. However, a lack of precise molecular markers complicates their characterization and practical use in breeding programmes. At the same time, the long generation time of wheat has become a limiting factor for breeders to respond quickly to an outbreak.

As the APRs cloned so far do not belong to any common gene family, it is not possible to use general features of these identified APRs to conduct biased searches for novel APRs. This project aims to rapidly clone the recently discovered APR gene Lr68 (Leaf Rust 68) using an unbiased gene isolation technique called MutChromSeq, which combines chromosome flow-sorting and mutational genomics, and is independent of fine mapping. It also aims to combine marker-assisted selection with accelerated generation advancement (“speed breeding”) for rapid germplasm structuring and field performance evaluation. Cloning APRs allows breeders to trace genes cheaply and quickly using gene-specific markers, enabling them to build effective and durable resistance gene pyramids. It also allows us to elucidate any common mechanism of action they have, helping researchers and breeders understand better the basis of their durable resistance.

110 Functional Study of TabZIP15 in Regulation of Wheat Abiotic Stress Tolerances

Lichao Zhang

Institute of Crop Sciences

bZIP transcription factors are one of the most important transcription factor families which play important roles in response to biotic and abiotic stresses. However, few studies of the functions of bZIP transcription factors in regulation of abiotic stresses tolerance have been done in wheat.

TabZIP15 encoded a bZIP transcription factor of C subfamily, which was mapped on the wheat chromosome 7DL. TabZIP15 was induced by salt, PEG, cold stresses and exogenous ABA treatment. The protein encoded by TabZIP15 was localized in the nucleus through transient expressed in tobacco epidermal cells, and possessed transcription activation activity in yeast with an N-terminal transcriptional activation domain. Overexpression of TabZIP15 improved the drought and freeze tolerance of transgenic Arabidopsis plants. Yeast one-hybrid experiments showed that TabZIP15 transcription factor can bind to ABRE cis-acting elements. Yeast library screening experiments and luciferase complementation assay (LCI) showed that TabZIP15 can interact with enolase TaENO-b, indicating that TabZIP15 may regulate abiotic stress tolerance through glycolysis and gluconeogenesis pathway.

111 Gene Regulatory Networks Reveal Novel Genes Controlling Senescence in Wheat

Philippa Borrill

John Innes Centre

Monocarpic senescence in crops is essential to enable nutrient remobilisation from photosynthetic tissues to the grain. This process must be tightly regulated to prevent premature senescence adversely affecting yields, however few genes controlling senescence have been identified in wheat. We are using a combination of approaches to identify novel regulatory genes affecting the early processes controlling senescence. We have generated a high-resolution RNA-Seq time-course of ten time-points from anthesis until the first visible signs of flag leaf senescence. To understand the key genes driving transcriptional changes, we used a combination of gene regulatory network analyses to identify modules of co-expressed genes and hub genes regulating the transcriptional processes across this time-course. From these networks, we selected ten transcription factors as candidate genes for further characterisation. We have generated double knock-out mutants of these candidate genes using the sequenced tetraploid TILLING population. Preliminary results show that two out of five candidate genes tested to date have roles in monocarpic senescence. Further studies are in progress to characterise the effects of these novel senescence regulators on nutrient remobilisation. The availability of new genomic resources, such as high-quality genome sequences and TILLING knock-out mutants, has enabled the study of genes regulating senescence at an unprecedented resolution. These genes may represent new breeding targets to adapt senescence to the environment and to modulate grain nutrient content which is influenced by the rate of senescence.

112 Identification of a Candidate Gene for* Sr9h*-Mediated Wheat Stem Rust Resistance by MutRenSeq

Matthew N. Rouse

USDA-ARS Cereal Disease Lab

The wheat stem rust resistance gene Sr9h confers major-effect resistance to stem rust pathogen race TTKSK (Ug99) and maps to chromosome arm 2BL in the cultivar ‘Gabo 56’. Sr9h is one of seven phenotypic Sr9 *alleles and the only *Sr9 *allele effective to TTKSK. We report here the identification of a candidate *Sr9h gene, by the rapid MutRenSeq approach. A total of 1603 EMS-mutagenized M2 families were screened with race TTKSK. We identified eight TTKSK-susceptible mutants that shared greater than 99% genome identity with Gabo 56 based on the 90K SNP chip. Nonsense or missense mutations were identified in the same NB-LRR candidate gene for seven of the eight mutants. A KASP marker derived from the candidate NB-LRR gene co-segregated with TTKSK resistance in two populations but appears to be a part of an NB-LRR gene family with multiple copies and pseudo genes based on the syntenic 2BL region of Chinese Spring and wild emmer wheat. Therefore we are currently sequencing chromosome 2B from Gabo56 and CM664, a second line with Sr9h, to fully characterize the Sr9h locus. Cloning the Sr9h gene and understanding the variation and unique phenotypic diversity underlying this complex locus will greatly enhance our understanding of the molecular mechanisms of resistance and race specificity and could provide extensive knowledge for long-term projects including the development of new resistance alleles and for the deployment of durable resistance.

113 Association Mapping of Stem Rust in Minnesota Spring Wheat Lines

Cyrus Kimani Ndung'u

University of Minnesota

Stem rust caused by Puccinia graminis f. sp. tritici, especially the Ug99 (TTKSK) race, is a serious threat to wheat production around the world and can cause up to 100% yield loss in susceptible cultivars. However, there are some Minnesota cultivars that have shown resistance to stem rust, including that caused by Ug99. It is therefore important to identify the QTLs for stem rust resistance in this germplasm. Association mapping is one of the most common method used to detect QTLs and genetically characterize germplasm. Our objective was to identify QTLs for resistance to the Ug99 family of stem rust pathogen races in a collection of 384 spring wheat breeding lines from the University of Minnesota.

The germplasm was screened for stem rust both in the field and as seedlings in a greenhouse. Field screening in Kenya and Ethiopia (2016 and 2017) facilitated data collection on the germplasm response to virulent races of the Ug99 race group. The seedling screening was done at the USDA-ARS CDL BSL3 greenhouse using TTKSK and TRTTF races. The germplasm was genotyped using the wheat 90K SNP Chip. The data was then analyzed using the GAPIT package in R using the Q+K model.

Significant QTLs were detected in both field seasons in Kenya but none were detected in Ethiopia. Additionally, significant QTLs for resistance to TTKSK and TRTTF races were detected in the greenhouse. Resistance to TTKSK in the greenhouse seemed to be temperature sensitive, with different QTLs being detected at different temperatures.

114  Resource Optimization with Multi-Trait Genomic Prediction for Bread Wheat Quality

Bettina Lado

Universidad de la Republica

Multi-trait genomic prediction models are a useful strategy to predict traits that otherwise are challenging due to labor intensity, difficulty, and cost. This is particularly important in the context of resource allocation in plant breeding programs. However, is not well known the amount of phenotyping that could be replaced by including phenotypic information on correlated traits. The objective of this work was to compare the predictive ability of multi-trait models, 1) by using different training population sizes for different quality traits, and 2) by testing different proportions of lines with phenotypic information for correlated traits. A group of 495 wheat lines were genotyped using genotyping by sequencing and phenotyped for eight bread quality traits. Cross-validation was used to evaluate the predictive ability of different multi-trait models using 10 to 80% of lines as training population and 50, 75 or 100% information on correlated traits. The results showed that predictive ability for all traits did not change when using more than 30% of lines as training population and 100% of the information on correlated traits. Moreover, the predictive ability of multi-trait models decreased when information on correlated traits was reduced to 50%. Overall, our results indicate that inclusion of information on correlated traits in training and testing wheat lines is a useful approach to replace phenotyping of expensive traits, allowing to reduce costs and better allocate resources in breeding programs.

115  Evidence for the Lr46 Leaf Rust Resistance Gene in the Wheat Cultivar Carberry

Ron E. Knox

Agriculture and Agri-Food Canada, SCRDC

Stacking and deployment of pleiotropic genes for resistance to multiple fungal diseases in wheat variety development is expected to increase the durability of resistance. To achieve this gene stacking objective through molecular breeding, an understanding of which genes currently exist in adapted germplasm is necessary. The cultivar Carberry is a popular hard red spring wheat variety in Canada with good rust resistance. Pedigree, phenotype, and DNA marker evidence suggested Carberry possesses the leaf rust resistance gene Lr46. Lr46 is a slow rusting adult plant resistance gene located on chromosome 1B that provides resistance against leaf rust and other diseases such as stripe rust, stem rust, and powdery mildew. We undertook an investigation to test the hypothesis that Carberry possesses Lr46. A doubled haploid population comprising 297 lines was developed from the F1 of a cross of Carberry with the universally leaf rust susceptible cultivar Thatcher. The population was evaluated for leaf rust reaction in four field nursery environments: near Swift Current SK from 2014 to 2016, and Morden MB in 2016. The population was also assessed for stem rust response in 2014 and 2016 near Swift Current. The population was genotyped using the 90K Infinium iSelect assay and following linkage map construction with JoinMap 4.1, 5457 markers were used for quantitative trait locus (QTL) analysis using MapQTL 6. Two QTL for leaf rust resistance were identified from Carberry on chromosome 1B, one of which was coincident with a stem rust resistance QTL that mapped to the location of Lr46.

116 Starp: A User-Friendly and Broadly Applicable Technique for SNP Genotyping in Wheat and Other Crops

Yunming Long

North Dakota State University

Single nucleotide polymorphisms (SNPs) are widely distributed in the genome of every organism. Recent advances in DNA sequencing technology have accelerated the discovery of variations in DNA sequences. Multiplex chip-based technology for genome-scale SNP genotyping has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs remains a challenge task. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP), which combines all of the advantages in accuracy, throughput, simplicity, and operational costs as well as compatibility with multiple platforms. STARP assays employ two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers are used to specifically amplify target alleles and generate PEA-primer binding sites. The two PEA-primers are common for all genotyping assays to stringently target AMAS-primer amplicons with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. STARP is a broadly applicable and more user-friendly alternative to KASP. We developed numerous STARP markers associated with important agronomic genes for low cadmium accumulation and resistance to Hessian fly, Fusarium head blight, and stem rust in wheat. These STARP markers have being employed in wheat breeding. In addition, STARP technique has been successfully extended to analyze the differential expression of the homologous genes and specifically amplify target DNA fragments in highly repetitive regions. STARP will facilitate genomic research in wheat and other species with large and complex genomes.

117 Molecular Genetic Characterisation of Triple Rust Resistance in Aegilops tauschii

Naveenkumar Athiyannan

Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation, University of QueenslandCommonwealth Scientific and Industrial Research Organisation

Bread wheat (Triticum aestivum) is the third most cultivated crop worldwide, and a major caloric source for the human population. Global wheat production is under constant threat due to the constant evolution of highly virulent fungal pathogens such as Puccinia sp that cause rust disease. Losses due to rust disease are routinely minimised through the deployment of host plant-mediated genetic resistance in commercial cultivars. However, pathogens evolve virulence to overcome this resistance. Therefore continuous supply of new sources of resistance is essential for sustainable rust management. Resistance from the wild relatives of hexaploid wheat is a valuable resource as they broaden the gene pool of available resistance genes.

In this study, CPI110672, an accession of the D genome progenitor Aegilops tauschii, *was chosen for in-depth analysis as it resists the three wheat rust diseases namely leaf, stem and stripe rust. To characterise this triple rust resistance, we conducted genetic analysis using a mapping population derived from the cross between CPI110672 and a susceptible accession CPI110717. Through rust infection screening and 90K SNP marker analysis, the chromosome position and closely linked markers were identified. Physical maps for the chromosome region carrying these rust resistance genes were generated using the new reference genome sequences of hexaploid wheat Chinese Spring IWGSC Ref Seq v1.01 and the diploid Ae. tauschiiaccession, AL8/782,3. Comparative genomics of these reference sequences together with contigs assembled from the sequenced genome of CPI110672 facilitated the identification of candidate genes. Functional analysis will be conducted through transformation into the rust-susceptible wheat cultivar fielder.