The barley pan-genome reveals the hidden legacy of mutation breeding.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

HvGST plays a key role in anthocyanin accumulation in colored barley.

Blue aleurone of barley is caused by the accumulation of delphinidin-based derivatives. Although these compounds are ideal nutrients for human health, they are undesirable contaminants in malt brewing. Therefore, the ability to add and remove this trait easily would facilitate breeding barley for different purposes. Here we identified a glutathione S-transferase gene (HvGST) that was responsible for the blue aleurone trait in Tibetan qingke barley by performing a genome-wide association study and RNA-sequencing analysis. Gene variation and expression analysis indicated that HvGST also participates in the transport and accumulation of anthocyanin in purple barley. Haplotype and the geographic distribution analyses of HvGST alleles revealed two independent natural variants responsible for the emergence of white aleurone: a 203-bp deletion causing premature termination of translation in qingke barley and two key single nucleotide polymorphisms in the promoter resulting in low transcription in Western barley. This study contributes to a better understanding of mechanisms of colored barley formation, and provides a comprehensive reference for marker-assisted barley breeding.

Acyl-CoA-binding protein (ACBP) genes involvement in response to abiotic stress and exogenous hormone application in barley (Hordeum vulgare L.).

BACKGROUND: Acyl-CoA-Binding proteins (ACBPs) function as coenzyme A transporters and play important roles in regulating plant growth and development in response to abiotic stress and phytohormones, as well as in membrane repair. To date, the ACBP family has not been a comprehensively characterized in barley (Hordeum vulgare L.). RESULTS: Eight ACBP genes were identified in the barley genome and named as HvACBP1-8. The analysis of the proteins structure and promoter elements of HvACBP suggested its potential functions in plant growth, development, and stress response. These HvACBPs are expressed in specific tissues and organs following induction by abiotic stressors such as drought, salinity, UV-B exposure, temperature extremes, and exposure to exogenous phytohormones. The HvACBP7 and HvACBP8 amino acid sequences were conserved during the domestication of Tibetan Qingke barley. CONCLUSIONS: Acyl-CoA-binding proteins may play important roles in barley growth and environmental adaptation. This study provides foundation for further analyses of the biological functions of HvACBPs in the barley stress response.

Tunable and highly sensitive fluorescent thermometers from La2CaZrO6:Cr3+ with a time-resolved technology.

Non-contact optical temperature measurement can effectively avoid the disadvantages of traditional contact thermometry and thus, become a hot research topic. Herein, a fluorescence intensity ratio (FIR) thermometry using a time-resolved technique based on La2CaZrO6:Cr3+ (LCZO) is proposed, with a maximum relative sensitivity (Sr - FIR) of 2.56% K-1 at 473 K and a minimum temperature resolution of 0.099 K. Moreover, the relative sensitivity and temperature resolution can be effectively controlled by adjusting the width of the time gate based on the time-resolved technique. Our work provides, to our knowledge, new viewpoints into the development of novel optical thermometers with adjustable relative sensitivity and temperature resolution on an as-needed basis.

CoreSNP: an efficient pipeline for core marker profile selection from genome-wide SNP datasets in crops.

BACKGROUND: DNA marker profiles play a crucial role in the identification and registration of germplasm, as well as in the distinctness, uniformity, and stability (DUS) testing of new plant variety protection. However, selecting minimal marker sets from large-scale SNP dataset can be challenging to distinguish a maximum number of samples. RESULTS: Here, we developed the CoreSNP pipeline using a "divide and conquer" strategy and a "greedy" algorithm. The pipeline offers adjustable parameters to guarantee the distinction of each sample pair with at least two markers. Additionally, it allows datasets with missing loci as input. The pipeline was tested in barley, soybean, wheat, rice and maize. A few dozen of core SNPs were efficiently selected in different crops with SNP array, GBS, and WGS dataset, which can differentiate thousands of individual samples. The core SNPs were distributed across all chromosomes, exhibiting lower pairwise linkage disequilibrium (LD) and higher polymorphism information content (PIC) and minor allele frequencies (MAF). It was shown that both the genetic diversity of the population and the characteristics of the original dataset can significantly influence the number of core markers. In addition, the core SNPs capture a certain level of the original population structure. CONCLUSIONS: CoreSNP is an efficiency way of core marker sets selection based on Genome-wide SNP datasets of crops. Combined with low-density SNP chip or genotyping technologies, it can be a cost-effective way to simplify and expedite the evaluation of genetic resources and differentiate different crop varieties. This tool is expected to have great application prospects in the rapid comparison of germplasm and intellectual property protection of new varieties.

New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.).

The green revolution was based on genetic modification of the gibberellin (GA) hormone system with "dwarfing" gene mutations that reduces GA signals, conferring shorter stature, thus enabling plant adaptation to modern farming conditions. Strong GA-related mutants with shorter stature often have reduced coleoptile length, discounting yield gain due to their unsatisfactory seedling emergence under drought conditions. Here we present gibberellin (GA) 3-oxidase1 (GA3ox1) as an alternative semi-dwarfing gene in barley that combines an optimal reduction in plant height without restricting coleoptile and seedling growth. Using large-scale field trials with an extensive collection of barley accessions, we showed that a natural GA3ox1 haplotype moderately reduced plant height by 5-10 cm. We used CRISPR/Cas9 technology, generated several novel GA3ox1 mutants and validated the function of GA3ox1. We showed that altered GA3ox1 activities changed the level of active GA isoforms and consequently increased coleoptile length by an average of 8.2 mm, which could provide essential adaptation to maintain yield under climate change. We revealed that CRISPR/Cas9-induced GA3ox1 mutations increased seed dormancy to an ideal level that could benefit the malting industry. We conclude that selecting HvGA3ox1 alleles offers a new opportunity for developing barley varieties with optimal stature, longer coleoptile and additional agronomic traits.

HSP90.2 promotes CO2 assimilation rate, grain weight and yield in wheat.

Wheat fixes CO2 by photosynthesis into kernels to nourish humankind. Improving the photosynthesis rate is a major driving force in assimilating atmospheric CO2 and guaranteeing food supply for human beings. Strategies for achieving the above goal need to be improved. Here, we report the cloning and mechanism of CO2 ASSIMILATION RATE AND KERNEL-ENHANCED 1 (CAKE1) from durum wheat (Triticum turgidum L. var. durum). The cake1 mutant displayed a lower photosynthesis rate with smaller grains. Genetic studies identified CAKE1 as HSP90.2-B, encoding cytosolic molecular chaperone folding nascent preproteins. The disturbance of HSP90.2 decreased leaf photosynthesis rate, kernel weight (KW) and yield. Nevertheless, HSP90.2 over-expression increased KW. HSP90.2 recruited and was essential for the chloroplast localization of nuclear-encoded photosynthesis units, for example PsbO. Actin microfilaments docked on the chloroplast surface interacted with HSP90.2 as a subcellular track towards chloroplasts. A natural variation in the hexaploid wheat HSP90.2-B promoter increased its transcription activity, enhanced photosynthesis rate and improved KW and yield. Our study illustrated an HSP90.2-Actin complex sorting client preproteins towards chloroplasts to promote CO2 assimilation and crop production. The beneficial haplotype of Hsp90.2 is rare in modern varieties and could be an excellent molecular switch promoting photosynthesis rate to increase yield in future elite wheat varieties.

Small noncoding RNA discovery and profiling with sRNAtools based on high-throughput sequencing.

Small noncoding RNAs (sRNA/sncRNAs) are generated from different genomic loci and play important roles in biological processes, such as cell proliferation and the regulation of gene expression. Next-generation sequencing (NGS) has provided an unprecedented opportunity to discover and quantify diverse kinds of sncRNA, such as tRFs (tRNA-derived small RNA fragments), phasiRNAs (phased, secondary, small-interfering RNAs), Piwi-interacting RNA (piRNAs) and plant-specific 24-nt short interfering RNAs (siRNAs). However, currently available web-based tools do not provide approaches to comprehensively analyze all of these diverse sncRNAs. This study presents a novel integrated platform, sRNAtools (https://bioinformatics.caf.ac.cn/sRNAtools), that can be used in conjunction with high-throughput sequencing to identify and functionally annotate sncRNAs, including profiling microRNAss, piRNAs, tRNAs, small nuclear RNAs, small nucleolar RNAs and rRNAs and discovering isomiRs, tRFs, phasiRNAs and plant-specific 24-nt siRNAs for up to 21 model organisms. Different modules, including single case, batch case, group case and target case, are developed to provide users with flexible ways of studying sncRNA. In addition, sRNAtools supports different ways of uploading small RNA sequencing data in a very interactive queue system, while local versions based on the program package/Docker/virtureBox are also available. We believe that sRNAtools will greatly benefit the scientific community as an integrated tool for studying sncRNAs.

Study on the mechanism of high energy transfer efficiency of blue light excited Cr3+, Nd3+ co-doped near infrared phosphors.

Although Cr3+ as activator for Near infrared (NIR) phosphors has been widely studied, the peaks of Cr3+ emission spectra in most hosts are less than 1000 nm. Nd3+ as an activator in many hosts has a wide distribution of absorption peaks in the Ultraviolet-visible-Near infrared (UV-vis-NIR) band, especially in the 650-900 nm band for effective NIR to NIR Stokes luminescence (4F3/2→4I9/2, 4F3/2→4I11/2 transitions). Therefore, Cr3+, Nd3+ co-doping to achieve the emission in the NIR II region (1000-1700nm) is very meaningful. Here, we report La2CaZrO6(LCZO): Cr3+, Nd3+ NIR phosphors with emission spectra covering an ultra-wide range of 700-1400 nm and reveal their luminescence mechanism. The energy transfer efficiency of Cr3+ for Nd3+ can be as high as 88.4% under 471 nm blue light excitation. In the same case, the integrated intensity of the emission spectra of Cr3+, Nd3+ co-doped can reach 847% of that of Nd3+ alone and 204% of that of Cr3+ alone. Finally, the combination of commercial blue light chips and Cr3+, Nd3+ co-doped NIR phosphors shows great potential for applications in face recognition, night lighting, and angiography.

Positional cloning identified HvTUBULIN8 as the candidate gene for round lateral spikelet (RLS) in barley (Hordeum vulgare L.).

KEY MESSAGE: Map-based cloning, subcellular localization, virus-induced-gene-silencing and transcriptomic analysis reveal HvTUB8 as a candidate gene with pleiotropic effects on barley spike and leaf development via ethylene and chlorophyll metabolism. Barley lateral spikelet morphology and grain shape play key roles in grain physical quality and yield. Several genes and QTLs for these traits have been cloned or fine mapped previously. Here, we report the phenotypic and genotypic analysis of a barley mutant with round lateral spikelet (rls) from cv. Edamai 934. rls had round lateral spikelet, short but round grain, shortened awn, thick glume and dark green leaves. Histocytologic and ultrastructural analysis revealed that the difference of grain shape of rls was caused by change of cell arrangement in glume, and the dark leaf color resulted from enlarged chloroplast. HvTUBULIN8 (HvTUB8) was identified as the candidate gene for rls by combination of RNA-Seq, map-based-cloning, virus-induced-gene-silencing (VIGS) and protein subcellular location. A single G-A substitution at the third exon of HvTUB8 resulted in change of Cysteine 354 to tyrosine. Furthermore, the mutant isoform Hvtub8 could be detected in both nucleus and cytoplasm, whereas the wild-type protein was only in cytoplasm and granular organelles of wheat protoplasts. Being consistent with the rare phenotype, the "A" allele of HvTUB8 was only detected in rls, but not in a worldwide barley germplasm panel with 400 accessions. VIGS confirmed that HvTUB8 was essential to maintain spike integrity. RNA-Seq results suggested that HvTUB8 may control spike morphogenesis via ethylene homeostasis and signaling, and control leaf color through chlorophyll metabolism. Collectively, our results support HvTUB8 as a candidate gene for barley spike and leaf morphology and provide insight of a novel mechanism of it in barley development.

Positional cloning and characterization reveal the role of a miRNA precursor gene ZmLRT in the regulation of lateral root number and drought tolerance in maize.

Lateral roots play essential roles in drought tolerance in maize (Zea mays L.). However, the genetic basis for the variation in the number of lateral roots in maize remains elusive. Here, we identified a major quantitative trait locus (QTL), qLRT5-1, controlling lateral root number using a recombinant inbred population from a cross between the maize lines Zong3 (with many lateral roots) and 87-1 (with few lateral roots). Fine-mapping and functional analysis determined that the candidate gene for qLRT5-1, ZmLRT, expresses the primary transcript for the microRNA miR166a. ZmLRT was highly expressed in root tips and lateral root primordia, and knockout and overexpression of ZmLRT increased and decreased lateral root number, respectively. Compared with 87-1, the ZmLRT gene model of Zong3 lacked the second and third exons and contained a 14 bp deletion at the junction between the first exon and intron, which altered the splicing site. In addition, ZmLRT expression was significantly lower in Zong3 than in 87-1, which might be attributed to the insertions of a transposon and over large DNA fragments in the Zong3 ZmLRT promoter region. These mutations decreased the abundance of mature miR166a in Zong3, resulting in increased lateral roots at the seedling stage. Furthermore, miR166a post-transcriptionally repressed five development-related class-III homeodomain-leucine zipper genes. Moreover, knockout of ZmLRT enhanced drought tolerance of maize seedlings. Our study furthers our understanding of the genetic basis of lateral root number variation in maize and highlights ZmLRT as a target for improving drought tolerance in maize.

Inferring regulatory element landscapes and gene regulatory networks from integrated analysis in eight hulless barley varieties under abiotic stress.

BACKGROUND: The cis-regulatory element became increasingly important for resistance breeding. There were many DNA variations identified by resequencing. To investigate the links between the DNA variations and cis-regulatory element was the fundamental work. DNA variations in cis-regulatory elements caused phenotype variations in general. RESULTS: We used WGBS, ChIP-seq and RNA-seq technology to decipher the regulatory element landscape from eight hulless barley varieties under four kinds of abiotic stresses. We discovered 231,440 lowly methylated regions (LMRs) from the methylome data of eight varieties. The LMRs mainly distributed in the intergenic regions. A total of 97,909 enhancer-gene pairs were identified from the correlation analysis between methylation degree and expression level. A lot of enriched motifs were recognized from the tolerant-specific LMRs. The key transcription factors were screened out and the transcription factor regulatory network was inferred from the enhancer-gene pairs data for drought stress. The NAC transcription factor was predicted to target to TCP, bHLH, bZIP transcription factor genes. We concluded that the H3K27me3 modification regions overlapped with the LMRs more than the H3K4me3. The variation of single nucleotide polymorphism was more abundant in LMRs than the remain regions of the genome. CONCLUSIONS: Epigenetic regulation is an important mechanism for organisms to adapt to complex environments. Through the study of DNA methylation and histone modification, we found that many changes had taken place in enhancers and transcription factors in the abiotic stress of hulless barley. For example, transcription factors including NAC may play an important role. This enriched the molecular basis of highland barley stress response.

An Acyl-CoA N-Acyltransferase Regulates Meristem Phase Change and Plant Architecture in Barley.

The modification of shoot architecture and increased investment into reproductive structures is key for crop improvement and is achieved through coordinated changes in the development and determinacy of different shoot meristems. A fundamental question is how the development of different shoot meristems is genetically coordinated to optimize the balance between vegetative and reproductive organs. Here we identify the MANY NODED DWARF1 (HvMND1) gene as a major regulator of plant architecture in barley (Hordeum vulgare). The mnd1.a mutant displayed an extended vegetative program with increased phytomer, leaf, and tiller production but a reduction in the number and size of grains. The induction of vegetative structures continued even after the transition to reproductive growth, resulting in a marked increase in longevity. Using mapping by RNA sequencing, we found that the HvMND1 gene encodes an acyl-CoA N-acyltransferase that is predominately expressed in developing axillary meristems and young inflorescences. Exploration of the expression network modulated by HvMND1 revealed differential expression of the developmental microRNAs miR156 and miR172 and several key cell cycle and developmental genes. Our data suggest that HvMND1 plays a significant role in the coordinated regulation of reproductive phase transitions, thereby promoting reproductive growth and whole plant senescence in barley.

Analysis of ß-d-glucan biosynthetic genes in oat reveals glucan synthesis regulation by light.

BACKGROUND AND AIMS: Oat (Avena sativa) has human health benefits when consumed as a whole-grain food, attributed to the high content of (1,3;1,4)-ß-d-glucan (mixed-linkage glucan [MLG]), but little is known about the synthase genes and synthesis mechanism of MLG polysaccharides in this species. METHODS: The concentration of oat MLGs under different light intensities was measured by a standard enzymatic approach and further verified by immunoelectron microscopy. The effect of light intensity on MLG synthase genes was examined by RT-qPCR and western blot analyses. The pattern of expression directed by the promoter of the oat MLG synthase gene was also investigated by histochemical ß-glucuronidase (GUS) analysis. KEY RESULTS: The oat orthologues of genes implicated in the synthesis of MLG in other cereals, including cellulose synthase-like (Csl) F, H and J gene families, were defined. Transcript profiling of these genes across oat tissues indicated that AsCslF6 transcripts dominated. Under high light intensities, the expression of AsCslF6, a major isoform of the MLG synthase genes, increased to >30 % of the dark growth control. The amount of MLG in oat rose from 0.07 to 1.06 % with increased light intensity. Histochemical tests showed that the AsCslF6 gene promoter preferentially directs GUS expression under high light intensity conditions. CONCLUSIONS: Oat MLG synthesis is regulated by light. High light intensity upregulates the expression of the MLG synthase AsCslF6 gene, leading to an increase in the amount of MLG in oat leaves.

Extracellular matrix derived by human umbilical cord-deposited mesenchymal stem cells accelerates chondrocyte proliferation and differentiation potential in vitro.

The extracellular matrix (ECM) is a dynamic and intricate three-dimensional (3D) microenvironment with excellent biophysical, biomechanical, and biochemical properties that may directly or indirectly regulate cell behavior, including proliferation, adhesion, migration, and differentiation. Compared with tissue-derived ECM, cell-derived ECM potentially has more advantages, including less potential for pathogen transfer, fewer inflammatory or anti-host immune responses, and a closer resemblance to the native ECM microenvironment. Different types of cell-derived ECM, such as adipose stem cells, synovium-derived stem cells and bone marrow stromal cells, their effects on articular chondrocytes which have been researched. In this study, we aimed to develop a 3D cell culture substrate using decellularized ECM derived from human umbilical cord-derived mesenchymal stem cells (hUCMSCs), and evaluated the effects on articular chondrocytes. We evaluated the morphology and components of hUCMSC-derived ECM using physical and chemical methods. Morphological, histological, immunohistochemical, biochemical, and real-time PCR analyses demonstrated that proliferation and differentiation capacity of chondrocytes using the 3D hUCMSC-derived ECM culture substrate was superior to that using non-coated two-dimensional plastic culture plates. In conclusion, 3D decellularized ECM derived from hUCMSCs offers a tissue-specific microenvironment for in vitro culture of chondrocytes, which not only markedly promoted chondrocyte proliferation but also preserved the differentiation capacity of chondrocytes. Therefore, our findings suggest that a 3D cell-derived ECM microenvironment represents a promising prospect for autologous chondrocyte-based cartilage tissue engineering and regeneration. The hUCMSC-derived ECM as a biomaterial is used for the preparation of scaffold or hybrid scaffold products which need to further study in the future.

Pln24NT: a web resource for plant 24-nt siRNA producing loci.

ABSTRACT: In plants, 24 nucleotide small interfering RNAs (24-nt siRNAs) account for a large percentage of the total siRNA pool, and they play an important role in guiding plant-specific RNA-directed DNA methylation (RdDM), which transcriptionally silences transposon elements, transgenes, repetitive sequences and some endogenous genes. Several loci in plant genomes produce clusters of 24-nt RNAs, and these loci are receiving increasing attention from the research community. However, at present there is no bioinformatics resource dedicated to 24-nt siRNA loci and their derived 24-nt siRNAs. Thus, in this study, Pln24NT, a freely available web resource, was created to centralize 24-nt siRNA loci and 24-nt siRNA information, including fundamental locus information, expression profiles and annotation of transposon elements, from next-generation sequencing (NGS) data for 10 popular plant species. An intuitive web interface was also developed for convenient searching and browsing, and analytical tools were included to help users flexibly analyze their own siRNA NGS data. Pln24NT will help the plant research community to discover and characterize 24-nt siRNAs, and may prove useful for studying the roles of siRNA in RNA-directed DNA methylation in plants. AVAILABILITY AND IMPLEMENTATION: http://bioinformatics.caf.ac.cn/Pln24NT . CONTACT: suxh@caf.ac.cn. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human sperm acrosome function assays are predictive of fertilization rate in vitro: a retrospective cohort study and meta-analysis.

OBJECTIVE: To determine whether acrosome function scoring-including acrosomal enzyme (AE) levels and acrosome reaction (AR) results-can predict fertilization rate in vitro. METHODS: We examined the predictive value of acrosomal enzymes (AE) determined by spectrophotometry/N-α-benzoyl-DL-arginine-p-nitroanilide for fertilization rate (FR) in vitro in a retrospective cohort study of 737 infertile couples undergoing IVF therapy. Additionally, a meta-analysis was done for prospective cohort or case-control studies; the following summary measures were reported to expand upon the findings: pooled spearman correlation coefficient (Rs), standardized mean difference (SMD), sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic score (DS), diagnostic odds ratio (DOR), and area under the summary receiver operating characteristic curve (AUC). RESULTS: Lower AE levels determined by spectrophotometry with a cut-off value of <25µIU/106 spermatozoa were predictive of total fertilization failure (TFF) with moderate SEN (88.23%) and low SPE (16.50%). On meta-analysis, a total of 44 unique articles were selected, but given the multiple techniques described there was a total of 67 total datasets extracted from these 44 articles, comprising 5356 infertile couples undergoing IVF therapy. The AE levels or induced AR% was positively correlated with FR (Rs = 0.38, SMD = 0.79; Rs = 0.40, SMD = 0.86, respectively). Lower AE levels or induced AR% was predictive of lower fertilization rate with moderate accuracy (AUC = 0.78, AUC = 0.84, respectively); this was accompanied by low SEN/moderate SPE (0.57/0.85), moderate SEN/moderate SPE (0.79/0.87), respectively. For AE assay, the diagnostic performance in Asia (Rs = 0.24, SMD = 0.50) was inferior to that in North America (Rs = 0.54, SMD = 0.81) and Europe (Rs = 0.46, SMD = 0.92). Cryopreserved spermatozoa (SMD = 0.20, P = 0.204) were inferior to fresh spermatozoa (SMD = 0.89, P <  0.001). Sperm preparation yielded inferior results as compared to no preparation; spermatozoa after swim up were weak relevant (Rs = 0.27, P = 0.044); and there was no correlation for spermatozoa after a discontinuous gradient (SMD = 1.07, P >  0.05). Lower AE levels determined by fluorometry or substrate assay were used for predicting lower FR with low sensitivity and high specificity; the spectrophotometry assay had an uncertain predictive value. For induced AR assay, the diagnostic performance in the other areas was inferior to that in Africa (Rs = 0.65, SMD = 1.86). No preparation or double preparation yielded inferior results as compared to one preparation (Rs = 0.41); discontinuous gradient (Rs = 0.17, SMD = 0.47) was inferior to swim up (Rs =0.65, SMD = 1.51). Nonphysiological triggers (SMD = 0.81) did not differ from physiological triggers (SMD = 0.95) in general; ZP (Rs = 0.63) or mannose (Rs = 0.59) was superior to other physiological or nonphysiological triggers; and there was no correlation for human follicle fluid, progesterone, cyclic adenosine 3'-5'-phosphate analogue and phorbol ester-BSA-GlcNAc Neoglycoproteins with N-acetylglucosamine residues. Lower induced AR% determined by indirect immunofluorescence, direct immunofluorescence with lection, or triple stain was used for predicting lower FR, with moderate sensitivity/high specificity, moderate sensitivity/high specificity, or high sensitivity/low specificity. CONCLUSIONS: Although the correlation between acrosome function scoring and FR was significant, the assays were neither highly sensitive nor specific. Additionally, the diagnostic performance showed regional effects as well as an effect of the sperm preparation or assay method. More studies of multicenter, large-scale, careful design and synthesizing multiple sperm functional assays and oocyte quality assays are still needed in clinical settings to better predict fertilization outcome in IVF.

Identification of QTLs controlling grain protein concentration using a high-density SNP and SSR linkage map in barley (Hordeum vulgare L.).

BACKGROUND: Grain protein concentration (GPC) is a major determinant of quality in barley (Hordeum vulgare L.). Breeding barley cultivars with high GPC has practical value for feed and food properties. The aim of the present study was to identify quantitative trait loci (QTLs) for GPC that could be detected under multiple environments. RESULTS: A population of 190 recombinant inbred lines (RILs) deriving from a cross between Chinese landrace ZGMLEL with high GPC (> 20%) and Australian cultivar Schooner was used for linkage and QTL analyses. The genetic linkage map spanned 2353.48 cM in length with an average locus interval of 2.33 cM. GPC was evaluated under six environments for the RIL population and the two parental lines. In total, six environmentally stable QTLs for GPC were detected on chromosomes 2H (1), 4H (1), 6H (1), and 7H (3) and the increasing alleles were derived from ZGMLEL. Notably, the three QTLs on chromosome 7H (QGpc.ZiSc-7H.1, QGpc.ZiSc-7H.2, and QGpc.ZiSc-7H.3) that linked in coupling phase were firstly identified. Moreover, the genetic effects of stable QTLs on chromosomes 2H, 6H and 7H were validated using near isogenic lines (NILs). CONCLUSIONS: Collectively, the identified QTLs expanded our knowledge about the genetic basis of GPC in barley and could be selected to develop cultivars with high grain protein concentration.

Characterization and fine mapping of a novel barley Stage Green-Revertible Albino Gene (HvSGRA) by Bulked Segregant Analysis based on SSR assay and Specific Length Amplified Fragment Sequencing.

BACKGROUND: Leaf color variations are common in plants. Herein we describe a natural mutant of barley cultivar Edamai No.6, whs18, whose leaf color showed stable and inheritable stage-green-revertible-albino under field condition. METHODS: Bulked Segregant Analysis (BSA) based on SSR assay and Specific Length Amplified Fragment Sequencing (SLAF-seq) was used to map the candidate gene for this trait. RESULTS: We found that leaf color of whs18 was green at seedling stage, while the seventh or eighth leaf began to show etiolation, and albino leaves emerged after a short period. The newly emerged leaves began to show stripe white before jointing stage, and normal green leaves emerged gradually. The duration of whs18 with abnormal leaf color lasted for about 3 months, which had some negative impacts on yield-related-traits. Further investigations showed that the variation was associated with changes in chlorophyII content and chloroplast development. Genetic analysis revealed that the trait was controlled by a single recessive nuclear gene, and was designed as HvSGRA in this study. Based on the F2 population derived from Edamai No.9706 and whs18, we initially mapped the HvSGRA gene on the short arm of chromosome 2H using SSR and BSA. GBMS247 on 2HS showed co-segregation with HvSGRA. The genetic distance between the other marker GBM1187 and HvSGRA was 1.2 cM. Further analysis using BSA with SLAF-seq also identified this region as candidate region. Finally, HvSGRA interval was narrowed to 0.4 cM between morex_contig_160447 and morex_contig_92239, which were anchored to two adjacent FP contigs, contig_34437 and contig_46434, respectively. Furthermore, six putative genes with high-confidence in this interval were identified by POPSEQ. Further analysis showed that the substitution from C to A in the third exon of fructokinase-1-like gene generated a premature stop codon in whs18, which may lead to loss function of this gene. CONCLUSIONS: Using SSR and SLAF-seq in conjunction with BSA, we mapped HvSGRA within two adjacent FP contigs of barley. The mutation of fructokinase-1-like gene in whs18 may cause the stage green-revertible albino of barley. The current study lays foundation for hierarchical map-based cloning of HvSGRA and utilizing the gene/trait as a visualized maker in molecular breeding in future.

Rare allele of HvLox-1 associated with lipoxygenase activity in barley (Hordeum vulgare L.).

KEY MESSAGE: Identification and allele-specific marker development of a functional SNP of HvLox - 1 which associated with barley lipoxygenase activity. Improving the stability of the flavor of beer is one of the main objectives in breeding barley for malting, and lipoxygenase-1 (LOX-1) is a key enzyme controlling this trait. In this study, a modified LOX activity assay was used for null LOX-1 mutant screening. Four barley landraces with no detected level of LOX-1 activity were screened from 1,083 barley germplasm accessions from China. The genomic sequence diversity of the HvLox-1 gene of the four null LOX-1 Chinese landraces was compared with that of a further 76 accessions. A total of 104 nucleotide polymorphisms were found, which contained 83 single-nucleotide polymorphisms (SNPs), 7 multiple-nucleotide polymorphisms, and 14 insertions and deletions. Most notably, we found a rare C/G mutation (SNP-61) in the second intron which led to null LOX-1 activity through an altered splicing acceptor site. In addition, an allele-specific polymerase chain reaction marker was developed for the genotyping of SNP-61, which could be used in breeding programs for barley to be used for malting. The objective was to improve beer quality.