Most Tibetan weedy barleys originated via recombination between Btr1 and Btr2 in domesticated barley.

Tibetan weedy barleys reside at the edges of qingke (hulless barley) fields in Tibet (Xizang). The spikes of these weedy barleys contain or lack a brittle rachis, with either two- or six-rowed spikes and either hulled or hulless grains at maturity. Although the brittle rachis trait of Tibetan weedy barleys is similar to that of wild barley (Hordeum vulgare ssp. spontaneum Thell.), these plants share genetic similarity with domesticated barley. The origin of Tibetan weedy barleys continues to be debated. Here, we show that most Tibetan weedy barleys originated from cross-pollinated hybridization of domesticated barleys, followed by hybrid self-pollination and recombination between Non-brittle rachis 1 (btr1) and 2 (btr2). We discovered the specific genetic ancestry of these weedy barleys in South Asian accessions. Tibetan weedy barleys exhibit lower genetic diversity than wild and Chinese landraces/cultivars and share a close relationship with qingke, genetically differing from typical eastern and western barley populations. We classified Tibetan weedy barleys into two groups, brittle rachis (BR) and non-brittle rachis (NBR); these traits align with the haplotypes of the btr1 and btr2 genes. Whereas wild barleys carry haplotype combinations of Btr1 and Btr2, each showing lower proportions in a population, the recombinant haplotype BTR2H8+BTR1H24 is predominant in the BR group. Haplotype block analysis based on whole-genome sequencing revealed two recombination breakpoints, which are present in 80.6% and 16.8% of BR accessions according to marker-assisted analysis. Hybridization events between wild and domesticated barley were rarely detected. These findings support the notion that Tibetan weedy barleys originated via recombination between Btr1 and Btr2 in domesticated barley.

Conjunctive Analyses of BSA-Seq and BSR-Seq to Identify Candidate Genes Controlling the Black Lemma and Pericarp Trait in Barley.

Black barley seeds are a health-beneficial diet resource because of their special chemical composition and antioxidant properties. The black lemma and pericarp (BLP) locus was mapped in a genetic interval of 0.807 Mb on chromosome 1H, but its genetic basis remains unknown. In this study, targeted metabolomics and conjunctive analyses of BSA-seq and BSR-seq were used to identify candidate genes of BLP and the precursors of black pigments. The results revealed that five candidate genes, purple acid phosphatase, 3-ketoacyl-CoA synthase 11, coiled-coil domain-containing protein 167, subtilisin-like protease, and caffeic acid-O-methyltransferase, of the BLP locus were identified in the 10.12 Mb location region on the 1H chromosome after differential expression analysis, and 17 differential metabolites, including the precursor and repeating unit of allomelanin, were accumulated in the late mike stage of black barley. Phenol nitrogen-free precursors such as catechol (protocatechuic aldehyde) or catecholic acids (caffeic, protocatechuic, and gallic acids) may promote black pigmentation. BLP can manipulate the accumulation of benzoic acid derivatives (salicylic acid, 2,4-dihydroxybenzoic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid, protocatechuic aldehyde, and syringaldehyde) through the shikimate/chorismite pathway other than the phenylalanine pathway and alter the metabolism of the phenylpropanoid-monolignol branch. Collectively, it is reasonable to infer that black pigmentation in barley is due to allomelanin biosynthesis in the lemma and pericarp, and BLP regulates melanogenesis by manipulating the biosynthesis of its precursors.

Mutation of barley HvPDIL5-1 improves resistance to yellow mosaic virus disease without growth or yield penalties.

The soil-borne yellow mosaic virus disease, which is caused by the bymoviruses barley yellow mosaic virus (BaYMV) and/or barley mild mosaic virus (BaMMV), seriously threatens winter barley production in Europe and East Asia. Both viruses are transmitted by the soil-borne plasmodiophorid Polymyxa graminis and are difficult to eliminate through chemical or physical measures in the field, making breeding for resistant cultivars the optimal strategy for disease control. The resistance locus rym1/11 was cloned encoding the host factor gene Protein Disulfide Isomerase Like 5-1 (PDIL5-1), whose loss-of-function variants confer broad-spectrum resistance to multiple strains of BaMMV/BaYMV. Most resistance-conferring variants have been identified in six-rowed barley landraces/historic cultivars, and their introgression into modern two-rowed malting cultivars is difficult because PDIL5-1 is located in a peri-centromeric region with suppressed recombination. In this study, we used CRISPR/Cas9 genome editing to modify PDIL5-1 in the BaYMV/BaMMV-susceptible elite malting barley cv. 'Golden Promise' and obtained the mutants pdil5-1-a and pdil5-1-b. PDIL5-1 in the pdil5-1-a mutant encodes a protein lacking a cysteine residue, and pdil5-1-b contains a protein-coding frameshift. Both mutants were completely resistant to BaYMV. The knockout mutant pdil5-1-b showed complete BaMMV resistance, while pdil5-1-a showed decreased viral accumulation but no disease symptoms if compared to 'Golden Promise'. Both PDIL5-1 edited lines, as well as the previously produced EMS-induced pdil5-1 mutant '10253-1-5' in the elite malting barley cv. 'Barke' background, displayed no growth or yield penalties in garden experiments or bymovirus-free field trials. Line '10253-1-5' showed improved resistance and yield performance compared to the wild-type and its sibling line when grown in infectious fields. Therefore, genome editing of the host factor gene PDIL5-1 could facilitate the breeding of barley varieties with resistance to bymoviruses.

Genome-Wide Association Mapping of Hulless Barely Phenotypes in Drought Environment.

Drought stress is one of the main factors restricting hulless barley (Hordeum vulgare L. var. nudum Hook. f.) yield. Genome-wide association study was performed using 269 lines of hulless barley to identify single-nucleotide polymorphism (SNP) markers associated with drought-resistance traits. The plants were cultured under either normal or drought conditions, and various quantitative traits including shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, leaf fresh weight, leaf saturated fresh weight, leaf dry weight, ratio of root and shoot fresh weight, ratio of root and shoot dry weight, shoot water loss rate, root water loss rate, leaf water content and leaf relative water content, and field phenotypes including main spike length, grain number per plant, grain weight per plant, thousand grain weight (TGW), main spike number, plant height, and effective spike number of plants were collected. After genotyping the plants, a total of 8,936,130 highly consistent population SNP markers were obtained with integrity > 0.5 and minor allele frequency > 0.05. Eight candidate genes potentially contributed to the hulless barley drought resistance were obtained at loci near significant SNPs. For example, EMB506, DCR, and APD2 genes for effective spike number of plants, ABCG11 gene for main spike number (MEN), CLPR2 gene for main spike length, YIP4B gene for root and shoot dry weight (RSWD), and GLYK and BTS genes for TGW. The SNPs and candidate genes identified in this study will be useful in hulless barley breeding under drought resistance.

A reference-guided TILLING by amplicon-sequencing platform supports forward and reverse genetics in barley.

Barley is a diploid species with a genome smaller than those of other members of the Triticeae tribe, making it an attractive model for genetic studies in Triticeae crops. The recent development of barley genomics has created a need for a high-throughput platform to identify genetically uniform mutants for gene function investigations. In this study, we report an ethyl methanesulfonate (EMS)-mutagenized population consisting of 8525 M3 lines in the barley landrace "Hatiexi" (HTX), which we complement with a high-quality de novo assembly of a reference genome for this genotype. The mutation rate within the population ranged from 1.51 to 4.09 mutations per megabase, depending on the treatment dosage of EMS and the mutation discrimination platform used for genotype analysis. We implemented a three-dimensional DNA pooling strategy combined with multiplexed amplicon sequencing to create a highly efficient and cost-effective TILLING (targeting induced locus lesion in genomes) platform in barley. Mutations were successfully identified from 72 mixed amplicons within a DNA pool containing 64 individual mutants and from 56 mixed amplicons within a pool containing 144 individuals. We discovered abundant allelic mutants for dozens of genes, including the barley Green Revolution contributor gene Brassinosteroid insensitive 1 (BRI1). As a proof of concept, we rapidly determined the causal gene responsible for a chlorotic mutant by following the MutMap strategy, demonstrating the value of this resource to support forward and reverse genetic studies in barley.

Genomic and Pathogenic Diversity of Barley Yellow Mosaic Virus and Barley Mild Mosaic Virus Isolates in Fields of China and Their Compatibility with Resistance Genes of Cultivated Barley.

Plant viruses transmitted by the soilborne plasmodiophorid Polymyxa graminis constantly threaten global production of cereal crops. Although the yellow mosaic virus disease of barley has been known to be present for a long time in China, the understanding of the diversity of the viral pathogens and their interactions with host resistance remains limited. In this study, we conducted a nationwide survey of P. graminis and the barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) it transmits, followed by genomic and pathogenic diversity analyses of both viruses. BaYMV and BaMMV were found exclusively in the region downstream of the Yangtze River, despite the national distribution of its transmission vector P. graminis. Analysis of the genomic variations of BaYMV and BaMMV revealed an elevated rate of nonsynonymous substitutions in the viral genome-linked protein (VPg), in which most substitutions were located in its interaction surface with the host eukaryotic translation initiation factor 4E (eIF4E). VPg sequence diversity was associated with the divergence in virus pathogenicity that was identified through multiple field trials. The majority of the resistance genes, including the widely applied rym4 and rym5 (alleles of eIF4E), as well as the combination of rym1/11 and rym5, are not sufficient to protect cultivated barley against viruses in China. Collectively, these results provide insights into virulence specificity and interaction mode with host resistance in cultivated barley, which has significant implications in breeding for the broad-spectrum resistance barley varieties.

Assessment of genetic diversity in chinese hulless barley accessions for qualitative traits

Cultivated barley (Hordeum vulgare L.) has been proven to be an economically important model plant and having large genetic diversity among the species. The effective exploitation of qualitative characters in barley can be measured by its genetic diversity and interrelationship. This study aims to determine the assessment of genetic diversity in Chinese hulless barley accessions for qualitative traits. Presently, in this study, the genetic diversity of 208 Chinese hulless barley from different Provinces of China, 111 genotypes were from the Tibet plateau, 30 Sichuan, 2 USA, 1 Canada, 12 Gansu, 51 Qinghai, 1 Yunnan was investigated; collected. Almost all the qualitative traits including crude protein, fiber, starch, neutral detergent fiber, and acid detergent fiber exhibited significantly high variability (p≤0.0001) among the cultivars. The data were analyzed using Statistics 8.1. In this study, significantly high variation was observed between starch content and neutral detergent fiber (23.64% and 11.54%). However, the highest diversity is based on the magnitude of the coefficient of variation exhibited in crude protein (13.82%), starch (12.87%), and fiber (12.17%). There was a significantly positive correlation between fiber, acid detergent fiber, and neutral detergent fiber except for starch content with crude protein and fiber that exhibited a significant negative correlation (r= -0.38*** and r= -0.92***). A large genetic diversity was observed through cluster analysis among all the 208 barley accessions, distance coefficient ranging between 0.28 and 75.86. The histogram revealed that frequency distributions of 208 different genotypes of hulless barley crop with all five different characters, crude protein, fiber, starch, neutral detergent fiber, and acid detergent fiber, showed normal distribution. It is concluded that this hulless barley study showed genetic diversity among the accessions and confirmed genetic diversity in various traits used.

Bulked segregant RNA-sequencing (BSR-seq) identified a novel rare allele of eIF4E effective against multiple isolates of BaYMV/BaMMV.

KEY MESSAGE: A novel rare allele of the barley host factor gene eIF4E for BaMMV/BaYMV infection was identified in an Iranian landrace that showed broad resistance to barley yellow mosaic virus disease, and molecular markers facilitating efficient selection were developed. The soil-borne yellow mosaic virus disease caused by different strains of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) is a major threat to winter barley (Hordeum vulgare) production in Europe and East Asia. However, the exploration of resistant germplasm or casual genes for barley breeding is rather limited in relation to the rapid diversification of viral strains. Here, we identified an Iranian barley landrace 'HOR3298,' which represented complete resistance to BaYMV and BaMMV. In contrast to rym4 and rym5, which act as the predominant source in Europe and East Asia for breeding resistant cultivars over decades and which have been overcome by several virulent isolates, this landrace showed broad-spectrum resistance to multiple isolates of BaYMV/BaMMV in the fields of Germany and China. By employment of bulked segregant RNA sequencing, test for allelism, and haplotype analysis, a recessive resistance gene in 'HOR3298' was genetically mapped coincident with the host factor eukaryotic translation initiation factor 4E (eIF4E, causal gene of rym4 and rym5). The eIF4EHOR3298 allele encoded for a novel haplotype that contained an exclusive nucleotide mutation (G565A) in the coding sequence. The easily handled markers were developed based on the exclusively rare variation, providing precise selection of this allele. Thus, this work provided a novel reliable resistance source and the feasible marker-assisted selection assays that can be used in breeding for barley yellow mosaic virus disease resistance in cultivated barley.

Cloning and expression analysis of cDNAs encoding ADP-glucose pyrophosphorylase large and small subunits from hulless barley (Hordeum vulgare L. var. nudum).

As an important plateau cereal crop, hulless barley is the principal food for the Tibetan people in China. ADP-glucose pyrophosphorylase (AGPase) is considered as the key enzyme for starch biosynthesis in plants. In this study, cDNAs encoding the small subunit (SSU I) and large subunit (LSU I) of AGPase were isolated from hulless barley. The results showed that SSU I and LSU I were 1438 and 1786 bp in length with a complete open reading frame (ORF) of 1419 and 1572 bp. The ORF-encoded polypeptides of 472 and 523 amino acids were having calculated molecular masses of 52.01 and 58.23 kDa, and the pI values were 5.59 and 6.30. In addition, phylogenetic analysis showed that SSU I and LSU I had the same phylogenetic trends with some species. Furthermore, expression levels in different growth periods and tissues of two hulless barley varieties were analyzed by quantitative reverse transcription-polymerase chain reaction. Gene expression levels of SSU I and LSU I were consistent with the total starch accumulation rate in endosperm. In conclusion, our data confirmed that SSU I and LSU I played an important role in hulless barley starch synthesis.

Association mapping for pre-harvest sprouting resistance in white winter wheat.

Association mapping identified quantitative trait loci (QTLs) and the markers linked to pre-harvest sprouting (PHS) resistance in an elite association mapping panel of white winter wheat comprising 198 genotypes. A total of 1,166 marker loci including DArT and SSR markers representing all 21 chromosomes of wheat were used in the analysis. General and mixed linear models were used to analyze PHS data collected over 4 years. Association analysis identified eight QTLs linked with 13 markers mapped on seven chromosomes. A QTL was detected on each arm of chromosome 2B and one each on chromosome arms 1BS, 2DS, 4AL, 6DL, 7BS and 7DS. All except the QTL on 7BS are located in a location similar to previous reports and, if verified, the QTL on 7BS is likely to be novel. Principal components and the kinship matrix were used to account for the presence of population structure but had only a minor effect on the results. Although, none of the QTLs was highly significant across all environments, a QTL on the long arm of chromosome 4A was detected in three different environments and also using the best linear unbiased predictions over years. Although previous reports have identified this as a major QTL, its effects were minor in our biparental mapping populations. The results of this study highlight the benefits of association mapping and the value of using elite material in association mapping for plant breeding programs.

Co-overexpression FIT with AtbHLH38 or AtbHLH39 in Arabidopsis-enhanced cadmium tolerance via increased cadmium sequestration in roots and improved iron homeostasis of shoots.

Cadmium (Cd) is toxic to plant cells. Under Cd exposure, the plant displayed leaf chlorosis, which is a typical symptom of iron (Fe) deficiency. Interactions of Cd with Fe have been reported. However, the molecular mechanisms of Cd-Fe interactions are not well understood. Here, we showed that FER-like Deficiency Induced Transcripition Factor (FIT), AtbHLH38, and AtbHLH39, three basic helix-loop-helix transcription factors involved in Fe homeostasis in plants, also play important roles in Cd tolerance. The gene expression analysis showed that the expression of FIT, AtbHLH38, and AtbHLH39 was up-regulated in the roots of plants treated with Cd. The plants overexpressing AtbHLH39 and double-overexpressing FIT/AtbHLH38 and FIT/AtbHLH39 exhibited more tolerance to Cd exposure than wild type, whereas no Cd tolerance was observed in plants overexpressing either AtbHLH38 or FIT. Further analysis revealed that co-overexpression of FIT with AtbHLH38 or AtbHLH39 constitutively activated the expression of Heavy Metal Associated3 (HMA3), Metal Tolerance Protein3 (MTP3), Iron Regulated Transporter2 (IRT2), and Iron Regulated Gene2 (IREG2), which are involved in the heavy metal detoxification in Arabidopsis (Arabidopis thaliana). Moreover, co-overexpression of FIT with AtbHLH38 or AtbHLH39 also enhanced the expression of NICOTIANAMINE SYNTHETASE1 (NAS1) and NAS2, resulting in the accumulation of nicotiananamine, a crucial chelator for Fe transportation and homeostasis. Finally, we showed that maintaining high Fe content in shoots under Cd exposure could alleviate the Cd toxicity. Our results provide new insight to understand the molecular mechanisms of Cd tolerance in plants.

[Genetic diversity analysis of the developed Qingke (hulless barley) varieties from the plateau regions of Sichuan Province in China revealed by SRAP markers].

Genetic diversity of 25 accessions of Qingke (hulless barley) varieties from the plateau regions of Sichuan Province, China, was analyzed by using SRAP (Sequence-related Amplified Polymorphism) markers. The results showed that 64 pairs of primer combinations produced 999 clear bands, of which 62 primer pairs (96.9%) amplified 225 polymorphic bands (22.5%). Three hundred and thirty three allelic phenotypes were amplified with an average of 5.20 alleles/primer pair. The genetic diversity ranged from 0 (me9/em14, me9/em15) to 0.8928 (me6/em18) with an average of 0.5126. The 25 accessions were classified into three major groups: A, B, and C by cluster analysis using UPGMA, which showed significant relationship with the origin regions of accessions. Thus, it was suggested that the Sichuan hulless barleys could be used as elite germplasms to enhance the genetic background for super-hulless barley breeding.

Genetic diversity analysis of Tibetan wild barley using SSR markers.

One hundred and six accessions of wild barley collected from Tibet, China, including 50 entries of the two-rowed wild barley Hordeum vulgare ssp. spontaneum (HS), 29 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon (HA), and 27 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon var. lagunculiforme (HL), were analyzed using 30 SSR markers selected from the seven barley linkage groups for studying genetic diversity and evolutionary relationship of the three subspecies of Tibetan wild barley to cultivated barley in China. Over the 30 genetic loci that were studied, 229 alleles were identified among the 106 accessions, of which 70 were common alleles. H. vulgare ssp. spontaneum possesses about thrice more private alleles (2.83 alleles/locus) than HS (0.93 alleles/locus), whereas almost no private alleles were detected in HL. The genetic diversity among-subspecies is much higher than that within-subspecies. Generally, the genetic diversity among the three subspecies is of the order HS > HL > HA. Phylogenetic analysis of the 106 accessions showed that all the accessions of HS and HA was clustered in their own groups, whereas the 27 accessions of HL were separated into two groups (14 entries with group HS and the rest with group HA). This indicated that HL was an intermediate form between HS and HA. Based on this study and previous works, we suggested that Chinese cultivated barley might evolve from HS via HL to HA.

Genetic diversity and geographical differentiation of cultivated six-rowed naked barley landraces from the Qinghai-Tibet plateau of China detected by SSR analysis

Cultivated six-rowed naked barley (Hordeum vulgare ssp. hexastichon var. nudum Hsü) is the oldest cultivated barley in China. We used 35 simple sequence repeat (SSR) markers selected from seven barley linkage groups to study the genetic diversity, geographical differentiation and evolutionary relationships among 65 H. vulgare ssp. hexastichon landrace accessions collected from the Qinghai-Tibet plateau of China, 25 accessions from Tibet (TB), 20 from Qinghai (QH) and 20 from Ganzi (GZ) prefecture in Sichuan province. At the 35 SSR loci we identified 248 alleles among the 65 accessions, 119 (47.98 percent) of the alleles being common alleles. We also found that the TB accessions possessed 47 private alleles, about 1.5 times more than the 31 private alleles found in the QH accessions and about 5 times more than 9 private alleles found in the GZ accessions. Generally, the TB accessions showed significantly higher genetic diversity than either the QH or GZ accessions whereas no significant difference in genetic diversity was found between the QH and GZ accessions. Partitioning analysis of genetic diversity showed that about 81 percent of the total variation was due to within-subgroup diversity and about 19 percent was clearly accounted for by geographical differentiation among the three subgroups. The distributions of alleles for most loci (71.4 percent) were significantly different among the three subgroups and geographical differentiation could be found according to the distribution of SSR alleles. Cluster analysis indicated that most of the accessions could be clustered into groups which basically coincided with their geographical distribution. These results suggest that Tibet might be a center of genetic diversity for cultivated barley, the cultivated six-rowed naked barley on the Qinghai-Tibet plateau of China may have evolved in Tibet and spread to Qinghai and then to Ganzi prefecture of Sichuan province.

[Microsatellite markers and applications in the barley genome].

Microsatellites, also called simple sequence repeats (SSR), are simple, tandemly repeated DNA sequences with a repeat length of a few base pairs,and are very ideally used as molecular markers because of their abundance, high level of polymorphism, co-dominance and ease of assay with the polymerase chain reaction (PCR) by selecting primers as the conserved DNA sequences flanking the SSRs,as well as better stability. The experiments showed that SSRs are randomly distributed throughout the barley genome,and there are 3-18 alleles at a single SSR locus,up to 37 alleles/locus. SSR markers have being widely applied in the construction of molecular genetic map, the study of genetic diversity,the identification of germplasm, gene mapping for important traits and molecular marker-assisted selection. Meanwhile,most of markers are strongly clustered around the centromeric regions of all seven linkage groups. As a result of the clustering,genome coverage with SSRs remains incomplete with an obvious lack of markers on the long arms of chromosomes 1H and 5H and short arm of chromosome 6H. Therefore,it is very potential and necessary to further develop SSR markers in barley.