Individuals with JAK1 variants are affected by syndromic features encompassing autoimmunity, atopy, colitis, and dermatitis.

Inborn errors of immunity lead to autoimmunity, inflammation, allergy, infection, and/or malignancy. Disease-causing JAK1 gain-of-function (GoF) mutations are considered exceedingly rare and have been identified in only four families. Here, we use forward and reverse genetics to identify 59 individuals harboring one of four heterozygous JAK1 variants. In vitro and ex vivo analysis of these variants revealed hyperactive baseline and cytokine-induced STAT phosphorylation and interferon-stimulated gene (ISG) levels compared with wild-type JAK1. A systematic review of electronic health records from the BioME Biobank revealed increased likelihood of clinical presentation with autoimmunity, atopy, colitis, and/or dermatitis in JAK1 variant-positive individuals. Finally, treatment of one affected patient with severe atopic dermatitis using the JAK1/JAK2-selective inhibitor, baricitinib, resulted in clinically significant improvement. These findings suggest that individually rare JAK1 GoF variants may underlie an emerging syndrome with more common presentations of autoimmune and inflammatory disease (JAACD syndrome). More broadly, individuals who present with such conditions may benefit from genetic testing for the presence of JAK1 GoF variants.

Identification of KASP markers and candidate genes for drought tolerance in wheat using 90K SNP array genotyping of near-isogenic lines targeting a 4BS quantitative trait locus.

KEY MESSAGE: This study identified a novel SNP and developed a highly efficient KASP marker for drought tolerance in wheat by genotyping NILs targeting a major QTL for drought tolerance using an SNP array and validation with commercial varieties. Common wheat (Triticum aestivum L.) is an important winter crop worldwide and a typical allopolyploid with a large and complex genome. With global warming, the environmental volatility and incidence of drought in wheat-producing areas will increase. Molecular markers for drought tolerance are urgently needed to enhance drought tolerance breeding. Here, we genotyped four near-isogenic line (NIL) pairs targeting a major QTL qDSI.4B.1 on wheat chromosome arm 4BS for drought tolerance using the 90K SNP Illumina iSelect array and discovered a single nucleotide polymorphism (SNP) (Excalibur_c100336_106) with consistent genotype-phenotype associations among all four NIL pairs and their parents. Then, we converted the SNP into a Kompetitive Allele-Specific PCR (KASP) marker, with an accuracy of 100% for the four NIL pairs and their parents and as high as 81.8% for the 44 tested wheat lines with known phenotypes collected from Australia and China. Two genes near this SNP were suggested as candidate genes for drought tolerance in wheat after checking the Chinese Spring reference genome annotation version 1.1. One gene, TraesCS4B02G085300, encodes an F-box protein reportedly related to the ABA network, a main pathway for drought tolerance, and another gene, TraesCS4B02G085400, encodes a calcineurin-like metallophos-phoesterase transmembrane protein, which participates in Ca2+-dependent phosphorylation regulatory system. Based on this work and previous research on pre-harvest sprouting, we established a quick and efficient general SQV-based approach for KASP marker development, integrating genotyping by SNP arrays (S) using NILs targeting major QTL for a specific trait (Q) and validating them with commercial varieties (V). The identified SNP and developed KASP marker could be applied to marker-assisted selection in drought breeding, and further study of the candidate genes may improve our understanding of drought tolerance in wheat.

Improved multi-trait prediction of wheat end-product quality traits by integrating NIR-predicted phenotypes.

Historically, end-product quality testing has been costly and required large flour samples; therefore, it was generally implemented in the late phases of variety development, imposing a huge cost on the breeding effort and effectiveness. High genetic correlations of end-product quality traits with higher throughput and nondestructive testing technologies, such as near-infrared (NIR), could enable early-stage testing and effective selection of these highly valuable traits in a multi-trait genomic prediction model. We studied the impact on prediction accuracy in genomic best linear unbiased prediction (GBLUP) of adding NIR-predicted secondary traits for six end-product quality traits (crumb yellowness, water absorption, texture hardness, flour yield, grain protein, flour swelling volume). Bread wheat lines (1,400-1,900) were measured across 8 years (2012-2019) for six end-product quality traits with standard laboratory assays and with NIR, which were combined to generate predicted data for approximately 27,000 lines. All lines were genotyped with the Infinium™ Wheat Barley 40K BeadChip and imputed using exome sequence data. End-product and NIR phenotypes were genetically correlated (0.5-0.83, except for flour swelling volume 0.19). Prediction accuracies of end-product traits ranged between 0.28 and 0.64 and increased by 30% through the inclusion of NIR-predicted data compared to single-trait analysis. There was a high correlation between the multi-trait prediction accuracy and genetic correlations between end-product and NIR-predicted data (0.69-0.77). Our forward prediction validation revealed a gradual increase in prediction accuracy when adding more years to the multi-trait model. Overall, we achieved genomic prediction accuracy at a level that enables selection for end-product quality traits early in the breeding cycle.

Nested association mapping-based GWAS for grain yield and related traits in wheat grown under diverse Australian environments.

KEY MESSAGE: Utilising a nested association mapping (NAM) population-based GWAS, 98 stable marker-trait associations with 127 alleles unique to the exotic parents were detected for grain yield and related traits in wheat. Grain yield, thousand-grain weight, screenings and hectolitre weight are important wheat yield traits. An understanding of their genetic basis is crucial for improving grain yield in breeding programmes. Nested association mapping (NAM) populations are useful resources for the dissection of the genetic basis of complex traits such as grain yield and related traits in wheat. Coupled with phenotypic data collected from multiple environments, NAM populations have the power to detect quantitative trait loci and their multiple alleles, providing germplasm that can be incorporated into breeding programmes. In this study, we evaluated a large-scale wheat NAM population with two recurrent parents in unbalanced trials in nine diverse Australian field environments over three years. By applying a single-stage factor analytical linear mixed model (FALMM) to the NAM multi-environment trials (MET) data and conducting a genome-wide association study (GWAS), we detected 98 stable marker-trait associations (MTAs) with their multiple alleles. 74 MTAs had 127 alleles that were derived from the exotic parents and were absent in either of the two recurrent parents. The exotic alleles had favourable effects on 46 MTAs of the 74 MTAs, for grain yield, thousand-grain weight, screenings and hectolitre weight. Two NAM RILs with consistently high yield in multiple environments were also identified, highlighting the potential of the NAM population in supporting plant breeding through provision of germplasm that can be readily incorporated into breeding programmes. The identified beneficial exotic alleles introgressed into the NAM population provide potential target alleles for the genetic improvement of wheat and further studies aimed at pinpointing the underlying genes.

Benefit of Introgression Depends on Level of Genetic Trait Variation in Cereal Breeding Programmes.

We investigated the benefit from introgression of external lines into a cereal breeding programme and strategies that accelerated introgression of the favourable alleles while minimising linkage drag using stochastic computer simulation. We simulated genomic selection for disease resistance and grain yield in two environments with a high level of genotype-by-environment interaction (G × E) for the latter trait, using genomic data of a historical barley breeding programme as the base generation. Two populations (existing and external) were created from this base population with different allele frequencies for few (N = 10) major and many (N ~ 990) minor simulated disease quantitative trait loci (QTL). The major disease QTL only existed in the external population and lines from the external population were introgressed into the existing population which had minor disease QTL with low, medium and high allele frequencies. The study revealed that the benefit of introgression depended on the level of genetic variation for the target trait in the existing cereal breeding programme. Introgression of external resources into the existing population was beneficial only when the existing population lacked variation in disease resistance or when minor disease QTL were already at medium or high frequency. When minor disease QTL were at low frequencies, no extra genetic gain was achieved from introgression. More benefit in the disease trait was obtained from the introgression if the major disease QTL had larger effect sizes, more selection emphasis was applied on disease resistance, or more external lines were introgressed. While our strategies to increase introgression of major disease QTL were generally successful, most were not able to completely avoid negative impacts on selection for grain yield with the only exception being when major introgression QTL effects were very large. Breeding programmes are advised to carefully consider the level of genetic variation in a trait available in their breeding programme before deciding to introgress germplasms.

Genomic regions controlling yield-related traits in spring wheat: a mini review and a case study for rainfed environments in Australia and China.

A genome-wide association study (GWAS) was performed in six environments to identify major or consistent alleles responsible for wheat yield traits in Australia and North China where rainfed farming system is adopted. A panel of 228 spring wheat varieties were genotyped by double digest restriction-site associated DNA genotyping-by-sequencing. A total of 223 significant marker-trait association (MTAs) and 46 candidate genes for large- or consistent-effect MTAs were identified. The results were compared with previous studies based on a mini-review of 23 GWAS analyses on wheat yield. A phenomenon seldom reported in previous studies was that MTAs responsible for the trait tended to cluster together at certain chromosome segments, and many candidate genes were in the form of gene clusters. Although linkage disequilibrium (LD) might contribute to the co-segregation of the regions, it also suggested that marker-assisted selection (MAS) or transgenic method targeting a single gene might not be as effective as MAS targeting a larger genomic region where all the genes or gene clusters underlying play important roles.

Novel Design of Imputation-Enabled SNP Arrays for Breeding and Research Applications Supporting Multi-Species Hybridization.

Array-based single nucleotide polymorphism (SNP) genotyping platforms have low genotype error and missing data rates compared to genotyping-by-sequencing technologies. However, design decisions used to create array-based SNP genotyping assays for both research and breeding applications are critical to their success. We describe a novel approach applicable to any animal or plant species for the design of cost-effective imputation-enabled SNP genotyping arrays with broad utility and demonstrate its application through the development of the Illumina Infinium Wheat Barley 40K SNP array Version 1.0. We show that the approach delivers high quality and high resolution data for wheat and barley, including when samples are jointly hybridised. The new array aims to maximally capture haplotypic diversity in globally diverse wheat and barley germplasm while minimizing ascertainment bias. Comprising mostly biallelic markers that were designed to be species-specific and single-copy, the array permits highly accurate imputation in diverse germplasm to improve the statistical power of genome-wide association studies (GWAS) and genomic selection. The SNP content captures tetraploid wheat (A- and B-genome) and Aegilops tauschii Coss. (D-genome) diversity and delineates synthetic and tetraploid wheat from other wheat, as well as tetraploid species and subgroups. The content includes SNP tagging key trait loci in wheat and barley, as well as direct connections to other genotyping platforms and legacy datasets. The utility of the array is enhanced through the web-based tool, Pretzel (https://plantinformatics.io/) which enables the content of the array to be visualized and interrogated interactively in the context of numerous genetic and genomic resources to be connected more seamlessly to research and breeding. The array is available for use by the international wheat and barley community.

Major genomic regions responsible for wheat yield and its components as revealed by meta-QTL and genotype-phenotype association analyses.

MAIN CONCLUSION: Meta-QTL (MQTL) analysis was done for yield-related traits in wheat. Candidate genes were identified within the refined MQTL and further validated by genotype-phenotype association analysis. Extensive studies have been undertaken on quantitative trait locus/loci (QTL) for wheat yield and its component traits. This study conducted a meta-analysis of 381 QTL related to wheat yield under various environments, including irrigated, drought- and/or heat-stressed conditions. Markers flanking meta-QTL (MQTL) were mapped on the wheat reference genome for their physical positions. Putative candidate genes were examined for MQTL with a physical interval of less than 20 Mbp. A total of 86 MQTL were identified as responsible for yield, of which 34 were for irrigated environments, 39 for drought-stressed environments, 36 for heat-stressed environments, and 23 for both drought- and heat-stressed environments. The high-confidence genes within the physical positions of the MQTL flanking markers were screened in the reference genome RefSeq V1.0, which identified 210 putative candidate genes. The phenotypic data for 14 contrasting genotypes with either high or low yield performance-according to the Australian National Variety Trials-were associated with their genotypic data obtained through ddRAD sequencing, which validated 18 genes or gene clusters associated with MQTL that had important roles for wheat yield. The detected and refined MQTL and candidate genes will be useful for marker-assisted selection of high yield in wheat breeding.

Accelerated Generation of Selfed Pure Line Plants for Gene Identification and Crop Breeding.

Production of pure lines is an important step in biological studies and breeding of many crop plants. The major types of pure lines for biological studies and breeding include doubled haploid (DH) lines, recombinant inbred lines (RILs), and near isogenic lines (NILs). DH lines can be produced through microspore and megaspore culture followed by chromosome doubling while RILs and NILs can be produced through introgressions or repeated selfing of hybrids. DH approach was developed as a quicker method than conventional method to produce pure lines. However, its drawbacks of genotype-dependency and only a single chance of recombination limited its wider application. A recently developed fast generation cycling system (FGCS) achieved similar times to those of DH for the production of selfed pure lines but is more versatile as it is much less genotype-dependent than DH technology and does not restrict recombination to a single event. The advantages and disadvantages of the technologies and their produced pure line populations for different purposes of biological research and breeding are discussed. The development of a concept of complete in vitro meiosis and mitosis system is also proposed. This could integrate with the recently developed technologies of single cell genomic sequencing and genome wide selection, leading to a complete laboratory based pre-breeding scheme.

Detection of two major grain yield QTL in bread wheat (Triticum aestivum L.) under heat, drought and high yield potential environments.

A large proportion of the worlds' wheat growing regions suffers water and/or heat stress at some stage during the crop growth cycle. With few exceptions, there has been no utilisation of managed environments to screen mapping populations under repeatable abiotic stress conditions, such as the facilities developed by the International Wheat and Maize Improvement Centre (CIMMYT). Through careful management of irrigation and sowing date over three consecutive seasons, repeatable heat, drought and high yield potential conditions were imposed on the RAC875/Kukri doubled haploid population to identify genetic loci for grain yield, yield components and key morpho-physiological traits under these conditions. Two of the detected quantitative trait loci (QTL) were located on chromosome 3B and had a large effect on canopy temperature and grain yield, accounting for up to 22 % of the variance for these traits. The locus on chromosome arm 3BL was detected under all three treatments but had its largest effect under the heat stress conditions, with the RAC875 allele increasing grain yield by 131 kg ha(-1) (or phenotypically, 7 % of treatment average). Only two of the eight yield QTL detected in the current study (including linkage groups 3A, 3D, 4D 5B and 7A) were previously detected in the RAC875/Kukri doubled haploid population; and there were also different yield components driving grain yield. A number of discussion points are raised to understand differences between the Mexican and southern Australian production environments and explain the lack of correlation between the datasets. The two key QTL detected on chromosome 3B in the present study are candidates for further genetic dissection and development of molecular markers.

Development of wheat-Lophopyrum elongatum recombinant lines for enhanced sodium 'exclusion' during salinity stress.

Lophopyrum elongatum (tall wheatgrass), a wild relative of wheat, can be used as a source of novel genes for improving salt tolerance of bread wheat. Sodium 'exclusion' is a major physiological mechanism for salt tolerance in a wheat-tall wheatgrass amphiploid, and a large proportion ( approximately 50%) for reduced Na(+) accumulation in the Xag leaf, as compared to wheat, was earlier shown to be contributed by genetic effects from substitution of chromosome 3E from tall wheatgrass for wheat chromosomes 3A and 3D. Homoeologous recombination between 3E and wheat chromosomes 3A and 3D was induced using the ph1b mutant, and putative recombinants were identified as having SSR markers specific for tall wheatgrass loci. As many as 14 recombinants with smaller segments of tall wheatgrass chromatin were identified and low-resolution breakpoint analysis was achieved using wheat SSR loci. Seven recombinants were identified to have leaf Na+ concentrations similar to those in 3E(3A) or 3E(3D) substitution lines, when grown in 200 mM NaCl in nutrient solution. Phenotypic analysis identified recombinants with introgressions at the distal end on the long arm of homoeologous group 3 chromosomes being responsible for Na(+) 'exclusion'. A total of 55 wheat SSR markers mapped to the long arm of homoeologous group 3 markers by genetic and deletion bin mapping were used for high resolution of wheat-tall wheatgrass chromosomal breakpoints in selected recombinants. Molecular marker analysis and genomic in situ hybridisation confirmed the 524-568 recombinant line as containing the smallest introgression of tall wheatgrass chromatin on the distal end of the long arm of wheat chromosome 3A and identified this line as suitable for developing wheat germplasm with Na(+) 'exclusion'.

Arabidopsis-rice-wheat gene orthologues for Na+ transport and transcript analysis in wheat-L. elongatum aneuploids under salt stress.

Lophopyrum elongatum is a wild relative of wheat that provides a source of novel genes for improvement of the salt tolerance of bread wheat. Improved Na(+) 'exclusion' is associated with salt tolerance in a wheat-L. elongatum amphiploid, in which a large proportion (ca. 50%) of the improved regulation of leaf Na(+) concentrations is controlled by chromosome 3E. In this study, genes that might control Na(+) accumulation, such as for transporters responsible for Na(+) entry (HKT1) and exit (SOS1) from cells, or compartmentalisation within vacuoles (NHX1, NHX5, AVP1, AVP2) in the model plant, Arabidopsis thaliana, were targeted for comparative analyses in wheat. Putative rice orthologues were identified and characterised as a means to bridge the large evolutionary distance between genomes from the model dicot and the more complex grass species. Wheat orthologues were identified through BLAST searching to identify either FL-cDNAs or ESTs and were subsequently used to design primers to amplify genomic DNA. The probable orthologous status of the wheat genes was confirmed through demonstration of similar intron-exon structure with their counterparts in Arabidopsis and rice. The majority of exons for Arabidopsis, rice and wheat orthologues of NHX1, NHX5 and SOS1 were conserved except for those at the amino and carboxy terminal ends. However, additional exons were identified in the predicted NHX1 and SOS1 genes of rice and wheat, as compared with Arabidopsis, indicating gene rearrangement events during evolution from a common ancestor. Nullisomic-tetrasomic, deletion and addition lines in wheat were used to assign gene sequences to chromosome regions in wheat and L. elongatum. Most sequences were assigned to homoeologous chromosomes, however, in some instances, such as for SOS1, genes were mapped to other unpredicted locations. Differential transcript abundance under salt stress indicated a complex pattern of expression for wheat orthologues that may regulate Na(+) accumulation in wheat lines containing chromosomes from L. elongatum. The identification of wheat orthologues to well characterized Arabidopsis genes, map locations and gene expression profiles increases our knowledge on the complex mechanisms regulating Na(+) transport in wheat and wheat-L. elongatum lines under salt stress.

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.

Application of comparative genomics to narrow-leafed lupin (Lupinus angustifolius L.) using sequence information from soybean and Arabidopsis.

The completion of genome-sequencing initiatives for model plants and EST databases for major crop species provides a large resource for gaining fundamental knowledge of complex gene interactions and the functional significance of proteins. There are increasingly numerous opportunities to transfer this information to other plant species with uncharacterized genomes and make advances in genome analysis, gene expression, and predicted protein function. In this study, we have used DNA sequences from soybean and Arabidopsis to determine the feasibility of applying comparative genomics to narrow-leafed lupin. We have used transcribed sequences from soybean and showed that a high proportion cross hybridize to lupin DNA, identifying similar genes and providing landmarks for estimating the degree of chromosomal synteny between species. To further investigate comparative relationships in this study, a detailed analysis of three lupin genes and comparison of orthologs from soybean and Arabidopsis shows that, in some cases, gene structure and expression are highly conserved and their proteins may have similar function. In other cases, genes show variation in expression profiles indicating alternative functions across species. The advantages and limitation of using soybean and Arabidopsis sequences for comparative genomics in lupins are discussed.