Capitalizing genebank core collections for rare and novel disease resistance loci to enhance barley resilience.

In the realm of agricultural sustainability, the utilization of plant genetic resources (PGRs) for enhanced disease resistance is paramount. Preservation efforts in genebanks are justified by their potential contributions to future crop improvement. To capitalize on the potential of PGRs, we focused on a barley core collection from the German ex situ genebank, and contrasted it with a European elite collection. The phenotypic assessment included 812 PGRs and 298 elites with a particular emphasis on four disease traits (Puccinia hordei, Blumeria graminis hordei, Ramularia collo-cygni, and Rhynchosporium commune). An integrated genome-wide association study, employing both Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) and a linear mixed model, was performed to unravel the genetic underpinnings of disease resistance. A total of 932 marker-trait associations were identified and assigned to 49 quantitative trait loci. The accumulation of novel and rare resistance alleles significantly bolstered the overall resistance level in PGRs. Three PGR donors with high counts of novel/rare alleles and exhibited exceptional resistance to leaf rust and powdery mildew were identified, offering promise for targeted pre-breeding goals and enhanced resilience in forthcoming varieties. Our findings underscore the critical contribution of PGRs to strengthening crop resilience and advancing sustainable agricultural practices.

Advanced domestication: harnessing the precision of gene editing in crop breeding.

Human population growth has increased the demand for food crops, animal feed, biofuel and biomaterials, all the while climate change is impacting environmental growth conditions. There is an urgent need to develop crop varieties which tolerate adverse growth conditions while requiring fewer inputs. Plant breeding is critical to global food security and, while it has benefited from modern technologies, it remains constrained by a lack of valuable genetic diversity, linkage drag, and an effective way to combine multiple favourable alleles for complex traits. CRISPR/Cas technology has transformed genome editing across biological systems and promises to transform agriculture with its high precision, ease of design, multiplexing ability and low cost. We discuss the integration of CRISPR/Cas-based gene editing into crop breeding to advance domestication and refine inbred crop varieties for various applications and growth environments. We highlight the use of CRISPR/Cas-based gene editing to fix desirable allelic variants, generate novel alleles, break deleterious genetic linkages, support pre-breeding and for introgression of favourable loci into elite lines.

Exploring diverse wheat germplasm for novel alleles in HMW-GS for bread quality improvement.

Wheat is one of the most important cereals used worldwide in the form of a range of products. Crop landraces have been an immense source of diversity for the breeders. In the present study, 517 Indian wheat landraces have been observed for the difference in bread making quality by assessing allelic behaviour of high molecular weight-glutenin subunits (HMW-GS). A total of 33 Glu-1 alleles (3 at Glu-A1, 15 at Glu-B1 and 15 at Glu-D1) were detected in wheat landraces. Allelic frequency of HMW-GS allelic band pattern null, 17 + 18, 2 + 12 (24.75%) was found to be the highest. Allelic frequency of HMW-GS allele null (68.27%) at Glu-A1, 17 + 18 (49.14%) at Glu-B1, and 2 + 12 (72.81%) at Glu-D1 was found to be the highest Five Novel alleles were identified at Glu-D1 locus, 12*, 12.1, 12.1*, 12.2 and 12.3. As Glu-D1 has highest quality contribution as compared to Glu-A1 and Glu-B1, reporting novel alleles at Glu-D1 represents that genetic variability available for selection is increased and it will provide tools for breeders to further improve dough properties and bread making quality.

2.7 million samples genotyped for HLA by next generation sequencing: lessons learned.

BACKGROUND: At the DKMS Life Science Lab, Next Generation Sequencing (NGS) has been used for ultra-high-volume high-resolution genotyping of HLA loci for the last three and a half years. Here, we report on our experiences in genotyping the HLA, CCR5, ABO, RHD and KIR genes using a direct amplicon sequencing approach on Illumina MiSeq and HiSeq 2500 instruments. RESULTS: Between January 2013 and June 2016, 2,714,110 samples largely from German, Polish and UK-based potential stem cell donors have been processed. 98.9% of all alleles for the targeted HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) were typed at high resolution or better. Initially a simple three-step workflow based on nanofluidic chips in conjunction with 4-primer amplicon tagging was used. Over time, we found that this setup results in PCR artefacts such as primer dimers and PCR-mediated recombination, which may necessitate repeat typing. Split workflows for low- and high-DNA-concentration samples helped alleviate these problems and reduced average per-locus repeat rates from 3.1 to 1.3%. Further optimisations of the workflow included the use of phosphorothioate oligos to reduce primer degradation and primer dimer formation, and employing statistical models to predict read yield from initial template DNA concentration to avoid intermediate quantification of PCR products. Finally, despite the populations typed at DKMS Life Science Lab being relatively homogenous genetically, an analysis of 1.4 million donors processed between January 2015 and May 2016 led to the discovery of 1,919 distinct novel HLA alleles. CONCLUSIONS: Amplicon-based NGS HLA genotyping workflows have become the workhorse in high-volume tissue typing of registry donors. The optimisation of workflow practices over multiple years has led to insights and solutions that improve the efficiency and robustness of short amplicon based genotyping workflows.

Identification of novel HLA alleles discovered in 2022-2023.

In this paper, we describe 10 novel HLA alleles discovered, submitted and officially named in the calendar years 2022 through the end of 2023.

Next-generation sequencing identifies two novel HLA-DPB1 alleles: HLA-DPB1*1069:01 and DPB1*1072:01.

Characterization of two novel HLA-DPB1 alleles: HLA-DPB1*1069:01, and DPB1*1072:01 containing non-synonymous nucleotide substitutions.

Identification and characterization of 122 novel HLA alleles in bone marrow donors recruited to the Ezer Mizion Bone Marrow Donor Registry.

Between January 2018 and June 2021, the Ezer Mizion recruited 223,960 donors. All donors were typed for their HLA class I and II alleles at high resolution by Next Generation Sequencing techniques. Comparison between the sequences obtained from these donors and those in the IPD-IMGT/HLA Database, revealed 122 Novel HLA alleles that were found in 133 donors. Most of the alleles, 94 (77%) were identified in the HLA class I genes (30, 35, and 29 in HLA-A, -B, and -C, respectively), and 28 (23%) were detected in the HLA class II genes (9 in HLA-DRB1 and -DQB1 and 10 in -DPB1). Most of these novel alleles, 106 (86.9%) comprised single nucleotide variation (SNV), 9 (7.4%) present multiple amino acids variation, 4 and 3 were generated because of deletions and insertions, respectively. Ten of these novel alleles were seen to be null alleles.

Identification of six novel HLA alleles, HLA-A*31:208, -B*08:306, -C*03:582, -C*04:494, -C*18:18 and -DRB1*07:133.

Characterization of six novel HLA alleles by next-generation sequencing.

Genomic characterization of HLA class I and class II genes in ethnically diverse sub-Saharan African populations: A report on novel HLA alleles.

HLA allelic variation has been well studied and documented in many parts of the world. However, African populations have been relatively under-represented in studies of HLA variation. We have characterized HLA variation from 489 individuals belonging to 13 ethnically diverse populations from rural communities from the African countries of Botswana, Cameroon, Ethiopia, and Tanzania, known to practice traditional subsistence lifestyles using next generation sequencing (Illumina) and long-reads from Oxford Nanopore Technologies. We identified 342 distinct alleles among the 11 HLA targeted genes: HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, and -DPB1, with 140 of those alleles containing novel sequences that were submitted to the IPD-IMGT/HLA database. Sixteen of the 140 alleles contained novel content within the exonic regions of the genes, while 110 alleles contained novel intronic variants. Four alleles were found to be recombinants of already described HLA alleles and 10 alleles extended the sequence content of already described alleles. All 140 alleles include complete allelic sequence from the 5' UTR to the 3' UTR that are inclusive of all exons and introns. This report characterizes the HLA allelic variation from these individuals and describes the novel allelic variation present within these specific African populations.

Characterization of 40 novel HLA class I and class II alleles identified in umbilical cord blood units.

Forty novel HLA class I and class II alleles were identified in umbilical cord blood (UCB) samples and categorized based on various types of mutations: non-synonymous, synonymous, frameshift, and premature termination codon. This study described 14 novel HLA-A alleles, 9 novel HLA-B alleles, 4 novel HLA-C alleles, 3 novel HLA-DRB1 alleles and 10 novel HLA-DQB1 alleles. Comparing the new allele sequence with the most homologous sequence, 60% of the novel alleles exhibited non-synonymous substitution, 32.5% observed with synonymous substitution, 5% displayed premature stop codon, and 2.5% presented with frameshift mutation. The majority of the new alleles contained a single nucleotide variation when compared with the most similar sequence.

Novel alleles in the era of next-generation sequencing-based HLA typing calls for standardization and policy.

Next-Generation Sequencing (NGS) has transformed clinical histocompatibility laboratories through its capacity to provide accurate, high-throughput, high-resolution typing of Human Leukocyte Antigen (HLA) genes, which is critical for transplant safety and success. As this technology becomes widely used for clinical genotyping, histocompatibility laboratories now have an increased capability to identify novel HLA alleles that previously would not be detected using traditional genotyping methods. Standard guidelines for the clinical verification and reporting of novelties in the era of NGS are greatly needed. Here, we describe the experience of a clinical histocompatibility laboratory's use of NGS for HLA genotyping and its management of novel alleles detected in an ethnically-diverse population of British Columbia, Canada. Over a period of 18 months, 3,450 clinical samples collected for the purpose of solid organ or hematopoietic stem cell transplantation were sequenced using NGS. Overall, 29 unique novel alleles were identified at a rate of ∼1.6 per month. The majority of novelties (52%) were detected in the alpha chains of class II (HLA-DQA1 and -DPA1). Novelties were found in all 11 HLA classical genes except for HLA-DRB3, -DRB4, and -DQB1. All novelties were single nucleotide polymorphisms, where more than half led to an amino acid change, and one resulted in a premature stop codon. Missense mutations were evaluated for changes in their amino acid properties to assess the potential effect on the novel HLA protein. All novelties identified were confirmed independently at another accredited HLA laboratory using a different NGS assay and platform to ensure validity in the reporting of novelties. The novel alleles were submitted to the Immuno Polymorphism Database-Immunogenetics/HLA (IPD-IMGT/HLA) for official allele name designation and inclusion in future database releases. A nationwide survey involving all Canadian HLA laboratories confirmed the common occurrence of novel allele detection but identified a wide variability in the assessment and reporting of novelties. In summary, a considerable proportion of novel alleles were identified in routine clinical testing. We propose a framework for the standardization of policies on the clinical management of novel alleles and inclusion in proficiency testing programs in the era of NGS-based HLA genotyping.

An analysis of sugary endosperm in sorghum: Characterization of mutant phenotypes depending on alleles of the corresponding starch debranching enzyme.

Sorghum is the fifth most important cereal crop. Here we performed molecular genetic analyses of the 'SUGARY FETERITA' (SUF) variety, which shows typical sugary endosperm traits (e.g., wrinkled seeds, accumulation of soluble sugars, and distorted starch). Positional mapping indicated that the corresponding gene was located on the long arm of chromosome 7. Within the candidate region of 3.4 Mb, a sorghum ortholog for maize Su1 (SbSu) encoding a starch debranching enzyme ISA1 was found. Sequencing analysis of SbSu in SUF uncovered nonsynonymous single nucleotide polymorphisms (SNPs) in the coding region, containing substitutions of highly conserved amino acids. Complementation of the rice sugary-1 (osisa1) mutant line with the SbSu gene recovered the sugary endosperm phenotype. Additionally, analyzing mutants obtained from an EMS-induced mutant panel revealed novel alleles with phenotypes showing less severe wrinkles and higher Brix scores. These results suggested that SbSu was the corresponding gene for the sugary endosperm. Expression profiles of starch synthesis genes during the grain-filling stage demonstrated that a loss-of-function of SbSu affects the expression of most starch synthesis genes and revealed the fine-tuned gene regulation in the starch synthetic pathway in sorghum. Haplotype analysis using 187 diverse accessions from a sorghum panel revealed the haplotype of SUF showing severe phenotype had not been used among the landraces and modern varieties. Thus, weak alleles (showing sweet and less severe wrinkles), such as in the abovementioned EMS-induced mutants, are more valuable for grain sorghum breeding. Our study suggests that more moderate alleles (e.g. produced by genome editing) should be beneficial for improving grain sorghum.

Novel SERPINA1 Alleles Identified through a Large Alpha-1 Antitrypsin Deficiency Screening Program and Review of Known Variants.

The SERPINA1 gene encodes the serine protease inhibitor alpha-1 antitrypsin (AAT) and is located on chromosome 14q31-32.3 in a cluster of homologous genes likely formed by exon duplication. AAT has a variety of anti-inflammatory properties. Its clinical relevance is best illustrated by the genetic disease alpha-1 antitrypsin deficiency (AATD) which is associated with an increased risk for chronic obstructive pulmonary disease (COPD) and cirrhosis. While 2 single nucleotide polymorphisms (SNPs) , S and Z, are responsible for more than 95% of all individuals with AATD, there are a number of rare variants associated with deficiency and dysfunction, as well as those associated with normal levels and function. Our laboratory has identified a number of novel AAT alleles that we report in this manuscript. We screened more than 500,000 individuals for AATD alleles through our testing program over the past 20 years. The characterization of these alleles was accomplished by DNA sequencing, measurement of AAT plasma levels and isoelectric focusing at pH 4-5. We report 22 novel AAT alleles discovered through our screening programs, such as Zlittle rock and QOchillicothe, and review the current literature of known AAT genetic variants.

Uncovering novel MHC alleles from RNA-Seq data: expanding the spectrum of MHC class I alleles in sheep.

BACKGROUND: Major histocompatibility complex (MHC) class I glycoproteins present selected peptides or antigens to CD8 + T cells that control the cytotoxic immune response. The MHC class I genes are among the most polymorphic loci in the vertebrate genome, with more than twenty thousand alleles known in humans. In sheep, only a very small number of alleles have been described to date, making the development of genotyping systems or functional studies difficult. A cost-effective way to identify new alleles could be to use already available RNA-Seq data from sheep. Current strategies for aligning RNA-Seq reads against annotated genome sequences or transcriptomes fail to detect the majority of class I alleles. Here, I combine the alignment of RNA-Seq reads against a specific reference database with de novo assembly to identify alleles. The method allows the comprehensive discovery of novel MHC class I alleles from RNA-Seq data (DinoMfRS). RESULTS: Using DinoMfRS, virtually all expressed MHC class I alleles could be determined. From 18 animals 75 MHC class I alleles were identified, of which 69 were novel. In addition, it was shown that DinoMfRS can be used to improve the annotation of MHC genes in the sheep genome sequence. CONCLUSIONS: DinoMfRS allows for the first time the annotation of unknown, more divergent MHC alleles from RNA-Seq data. Successful application to RNA-Seq data from 16 animals has approximately doubled the number of known alleles in sheep. By using existing data, alleles can now be determined very inexpensively for populations that have not been well studied. In addition, MHC expression studies or evolutionary studies, for example, can be greatly improved in this way, and the method should be applicable to a broader spectrum of other multigene families or highly polymorphic genes.

Characterization of six novel HLA alleles, HLA-a*24:565, -a*29:160, -b*07:444, -b*57:156, -c*18:15 and -DPB1*795:01:02.

Six novel HLA alleles were characterized in the Spanish population.

Novel HLA-A, HLA-B, and HLA-DRB1 alleles identified in Brazilian individuals.

Identification of 12 novel HLA class I and II alleles in Brazilian bone marrow donors.

Characterization of the novel alleles: HLA-A*01:305, HLA-B*58:120, HLA-C*04:428Q and HLA-C*05:01:56.

Four new HLA class I alleles were characterized by next-generation sequencing and haplotypes determined.

A novel HLA-DQA1*01 allele, HLA-DQA1*01:99, identified by next-generation sequencing.

DQA1*01:99 differs from DQA1*01:01 by a missense nucleotide substitution in exon 4.

Potential of Genome Editing to Capture Diversity From Australian Wild Rice Relatives.

Rice, a staple food worldwide and a model crop, could benefit from the introduction of novel genetics from wild relatives. Wild rice in the AA genome group closely related to domesticated rice is found across the tropical world. Due to their locality outside the range of domesticated rice, Australian wild rice populations are a potential source of unique traits for rice breeding. These rice species provide a diverse gene pool for improvement that could be utilized for desirable traits such as stress resistance, disease tolerance, and nutritional qualities. However, they remain poorly characterized. The CRISPR/Cas system has revolutionized gene editing and has improved our understanding of gene functions. Coupled with the increasing availability of genomic information on the species, genes in Australian wild rice could be modified through genome editing technologies to produce new domesticates. Alternatively, beneficial alleles from these rice species could be incorporated into cultivated rice to improve critical traits. Here, we summarize the beneficial traits in Australian wild rice, the available genomic information and the potential of gene editing to discover and understand the functions of novel alleles. Moreover, we discuss the potential domestication of these wild rice species for health and economic benefits to rice production globally.

Characterization of Novel CYP2D6 Alleles across Sub-Saharan African Populations.

The CYP2D6 gene has been widely studied to characterize variants and/or star alleles, which account for a significant portion of variability in drug responses observed within and between populations. However, African populations remain under-represented in these studies. The increasing availability of high coverage genomes from African populations has provided the opportunity to fill this knowledge gap. In this study, we characterized computationally predicted novel CYP2D6 star alleles in 30 African subjects for whom DNA samples were available from the Coriell Institute. CYP2D6 genotyping and resequencing was performed using a variety of commercially available and laboratory-developed tests in a collaborative effort involving three laboratories. Fourteen novel CYP2D6 alleles and multiple novel suballeles were identified. This work adds to the growing catalogue of validated African ancestry CYP2D6 allelic variation in pharmacogenomic databases, thus laying the foundation for future functional studies and improving the accuracy of CYP2D6 genotyping, phenotype prediction, and the refinement of clinical pharmacogenomic implementation guidelines in African and global settings.