Genome-wide identification of bHLH transcription factors and expression analysis under drought stress in Pseudoroegneria libanotica at germination.

The basic helix-loop-helix (bHLH) transcription factor family is the second largest in plants. bHLH transcription factor is not only universally involved in plant growth and metabolism, including photomorphogenesis, light signal transduction, and secondary metabolism, but also plays an important role in plant response to stress. However, the function of bHLH TFs in Pseudoroegneria species has not been studied yet. Pseudoroegneria (Nevski) Á. Löve is a perennial genus of the Triticeae. Pseudoroegneria species are mostly distributed in arid/semi-arid areas and they show good drought tolerance. In this study, we identified 152 PlbHLH TFs in Pseudoroegneria libanotica, which could be classified into 15 groups. Collinearity analysis indicates that 122 PlbHLH genes share homology with wbHLH genes in wheat, and it has lower homology with AtbHLH genes in Arabidopsis. Based on transcriptome profiling under an experiment with three PEG concentrations (0%, 10%, and 20%), 10 up-regulated genes and 11 down-regulated PlbHLH genes were screened. Among them, PlbHLH6, PlbHLH55 and PlbHLH64 as candidate genes may be the key genes related to drought tolerance response at germination, and they have been demonstrated to respond to drought, salt, oxidative, heat, and heavy metal stress in yeast. This study lays the foundation for an in-depth study of the biological roles of PlbHLHs in Pse. libanotica, and discovered new drought-tolerance candidate genes to enhance the genetic background of Triticeae crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01433-w.

Roegneria yenchiana: A new species in the Triticeae (Poaceae) from the Hengduan Mountain region.

Roegneria yenchiana sp. nov. (Triticeae) is a new species collected from Shangri-la of Yunnan Province in China based on morphological, cytological, and molecular data. It is morphologically characterized by one spikelet per node, rectangular glums, awns flanked by two short mucros in lemmas, distinguished from other species of Roegneria. The genomic in situ hybridization results indicate that R. yenchiana is an allotetraploid, and its genomic constitution is StY. Phylogenetic analyses based on multiple loci suggested that R. yenchiana is closely related to Pseudoroegneria and Roegneria, and the Pseudoroegneria served as the maternal donors during its polyploid speciation.

A chromosome level genome assembly of Pseudoroegneria Libanotica reveals a key Kcs gene involves in the cuticular wax elongation for drought resistance.

BACKGROUND: The genus Pseudoroegneria (Nevski) Löve (Triticeae, Poaceae), whose genome symbol was designed as "St", accounts for more than 60% of perennial Triticeae species. The diploid species Psudoroegneria libanotica (2n = 14) contains the most ancient St genome, exhibited strong drought resistance, and was morphologically covered by cuticular wax on the aerial part. Therefore, the St-genome sequencing data could provide fundamental information for studies of genome evolution and reveal its mechanisms of cuticular wax and drought resistance. RESULTS: In this study, we reported the chromosome-level genome assembly for the St genome of Pse. libanotica, with a total size of 2.99 Gb. 46,369 protein-coding genes annotated and 71.62% was repeat sequences. Comparative analyses revealed that the genus Pseudoroegneria diverged during the middle and late Miocene. During this period, unique genes, gene family expansion, and contraction in Pse. libanotica were enriched in biotic and abiotic stresses, such as fatty acid biosynthesis which may greatly contribute to its drought adaption. Furthermore, we investigated genes associated with the cuticular wax formation and water deficit and found a new Kcs gene evm.TU.CTG175.54. It plays a critical role in the very long chain fatty acid (VLCFA) elongation from C18 to C26 in Pse. libanotica. The function needs more evidence to be verified. CONCLUSIONS: We sequenced and assembled the St genome in Triticeae and discovered a new KCS gene that plays a role in wax extension to cope with drought. Our study lays a foundation for the genome diversification of Triticeae species and deciphers cuticular wax formation genes involved in drought resistance.

Genome constitution and evolution of Elymus atratus (Poaceae: Triticeae) inferred from cytogenetic and phylogenetic analysis.

BACKGROUND: Elymus atratus (Nevski) Hand.-Mazz. is perennial hexaploid wheatgrass. It was assigned to the genus Elymus L. sensu stricto based on morphological characters. Its genome constitution has not been disentangled yet. OBJECTIVE: To identify the genome constitution and origin of E. atratus. METHODS: In this study, genomic in situ hybridization and fluorescence in situ hybridization, and phylogenetic analysis based on the Acc1, DMC1 and matK sequences were performed. RESULTS: Genomic in situ hybridization and fluorescence in situ hybridization results reveal that E. atratus 2n = 6x = 42 is composed of 14 St genome chromosomes, 14 H genome chromosomes, and 14 Y genome chromosomes including two H-Y type translocation chromosomes, suggesting that the genome formula of E. atratus is StStYYHH. The phylogenetic analysis based on Acc1 and DMC1 sequences not only shows that the Y genome originated in a separate diploid, but also suggests that Pseudoroegneria (St), Hordeum (H), and a diploid species with Y genome were the potential donors of E. atratus. Data from chloroplast DNA showed that the maternal donor of E. atratus contains the St genome. CONCLUSION: Elymus atratus is an allohexaploid species with StYH genome, which may have originated through the hybridization between an allotetraploid Roegneria (StY) species as the maternal donor and a diploid Hordeum (H) species as the paternal donor.

Cytogenetic and genomic characterization of a novel wheat-tetraploid Thinopyrum elongatum 1BS·1EL translocation line with stripe rust resistance.

Stripe rust, caused by Puccinia striiformis f. sp tritici (Pst), is a destructive wheat disease pathogen. Thinopyrum elongatum is a valuable germplasm including diploid, tetraploid, and decaploid with plenty of biotic and abiotic resistance. In a previous study, we generated a stripe rust resistance wheat-tetraploid Th. elongatum 1E/1D substitution line K17-841-1. To further apply the wild germplasm for wheat breeding, we selected and obtained a new homozygous wheat-tetraploid Th. elongatum translocation line T1BS·1EL using genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), oligo-FISH-Painting, and the wheat 55K single nucleotide polymorphisms (SNPs) genotyping array. The T1BS·1EL is highly resistant to stripe rust at the seedling and adult stage. Pedigree and molecular marker analyses revealed that the resistance gene was located on chromosome arm 1EL of tetraploid Th. elongatum, tentatively named Yr1EL. Besides, we developed and validated 32 Simple Sequence Repeats (SSR) markers and two kompititive allele specific PCR (KASP) assays which were specific to tetraploid Th. elongatum chromosome arm 1EL to facilitate marker-assisted selection for alien 1EL stripe rust resistance breeding. This will help us explore and locate the stripe rust resistance gene mapping on the 1E chromosome and deploy it in the wheat breeding program.

Development, identification, and utilization of wheat-tetraploid Thinopyrum elongatum 4EL translocation lines resistant to stripe rust.

KEY MESSAGE: The new stripe rust resistance gene Yr4EL in tetraploid Th. elongatum was identified and transferred into common wheat via 4EL translocation lines. Tetraploid Thinopyrum elongatum is a valuable genetic resource for improving the resistance of wheat to diseases such as stripe rust, powdery mildew, and Fusarium head blight. We previously reported that chromosome 4E of the 4E (4D) substitution line carries all-stage stripe rust resistance genes. To optimize the utility of these genes in wheat breeding programs, we developed translocation lines by inducing chromosomal structural changes through 60Co-γ irradiation and developing monosomic substitution lines. In total, 53 plants with different 4E chromosomal structural changes were identified. Three homozygous translocation lines (T4DS·4EL, T5AL·4EL, and T3BL·4EL) and an addition translocation line (T5DS·4EL) were confirmed by the genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), FISH-painting, and wheat 55 K SNP array analyses. These four translocation lines, which contained chromosome arm 4EL, exhibited high stripe rust resistance. Thus, a resistance gene (tentatively named Yr4EL) was localized to the chromosome arm 4EL of tetraploid Th. elongatum. For the application of marker-assisted selection (MAS), 32 simple sequence repeat (SSR) markers were developed, showing specific amplification on the chromosome arm 4EL and co-segregation with Yr4EL. Furthermore, the 4DS·4EL line could be selected as a good pre-breeding line that better agronomic traits than other translocation lines. We transferred Yr4EL into three wheat cultivars SM482, CM42, and SM51, and their progenies were all resistant to stripe rust, which can be used in future wheat resistance breeding programs.

Integrated Transcriptome Analysis of miRNAs and mRNAs in the Skeletal Muscle of Wuranke Sheep.

MicroRNAs (miRNAs) are regarded as important regulators in skeletal muscle development. To reveal the regulatory roles of miRNAs and their target mRNAs underlying the skeletal muscle development of Wuranke sheep, we investigated the miRNA and mRNA expression profiles in the biceps femoris of these sheep at the fetal (3 months of gestation) and 3- and 15-month-old postnatal stages. Consequently, a total of 1195 miRNAs and 24,959 genes were identified. Furthermore, 474, 461, and 54 differentially expressed miRNAs (DEMs) and 6783, 7407, and 78 differentially expressed genes (DEGs) were detected among three comparative groups. Functional analysis demonstrated that the target mRNAs of the DEMs were enriched in multiple pathways related to muscle development. Moreover, the interactions among several predicted miRNA-mRNA pairs (oar-miR-133-HDAC1, oar-miR-1185-5p-MYH1/HADHA/OXCT1, and PC-5p-3703_578-INSR/ACTG1) that potentially affect skeletal muscle development were verified using dual-luciferase reporter assays. In this study, we identified the miRNA and mRNA differences in the skeletal muscle of Wuranke sheep at different developmental stages and revealed that a series of candidate miRNA-mRNA pairs may act as modulators of muscle development. These results will contribute to future studies on the function of miRNAs and their target mRNAs during skeletal muscle development in Wuranke sheep.

Chromosome-specific painting in Thinopyrum species using bulked oligonucleotides.

KEY MESSAGE: Chromosome-specific painting probes were developed to identify the individual chromosomes from 1 to 7E in Thinopyrum species and detect alien genetic material of the E genome in a wheat background. The E genome of Thinopyrum is closely related to the ABD genome of wheat (Triticum aestivum L.) and harbors genes conferring beneficial traits to wheat, including high yield, disease resistance, and unique end-use quality. Species of Thinopyrum vary from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70), and chromosome structural variation and differentiation have arisen during polyploidization. To investigate the variation and evolution of the E genome, we developed a complete set of E genome-specific painting probes for identification of the individual chromosomes 1E to 7E based on the genome sequences of Th. elongatum (Host) D. R. Dewey and wheat. By using these new probes in oligonucleotide-based chromosome painting, we showed that Th. bessarabicum (PI 531711, EbEb) has a close genetic relationship with diploid Th. elongatum (EeEe), with five chromosomes (1E, 2E, 3E, 6E, and 7E) maintaining complete synteny in the two species except for a reciprocal translocation between 4 and 5Eb. All 14 pairs of chromosomes of tetraploid Th. elongatum have maintained complete synteny with those of diploid Th. elongatum (Thy14), but the two sets of E genomes have diverged. This study also demonstrated that the E genome-specific painting probes are useful for rapid and effective detection of the alien genetic material of E genome in wheat-Thinopyrum derived lines.

Development and Characterization of a Novel Wheat-Tetraploid Thinopyrum elongatum 6E (6D) Disomic Substitution Line with Stripe Rust Resistance at the Adult Stage.

Stripe rust, which is caused by Puccinia striiformis f. sp. tritici, is one of the most devastating foliar diseases of common wheat worldwide. Breeding new wheat varieties with durable resistance is the most effective way of controlling the disease. Tetraploid Thinopyrum elongatum (2n = 4x = 28, EEEE) carries a variety of genes conferring resistance to multiple diseases, including stripe rust, Fusarium head blight, and powdery mildew, which makes it a valuable tertiary genetic resource for enhancing wheat cultivar improvement. Here, a novel wheat-tetraploid Th. elongatum 6E (6D) disomic substitution line (K17-1065-4) was characterized using genomic in situ hybridization and fluorescence in situ hybridization chromosome painting analyses. The evaluation of disease responses revealed that K17-1065-4 is highly resistant to stripe rust at the adult stage. By analyzing the whole-genome sequence of diploid Th. elongatum, we detected 3382 specific SSR sequences on chromosome 6E. Sixty SSR markers were developed, and thirty-three of them can accurately trace chromosome 6E of tetraploid Th. elongatum, which were linked to the disease resistance gene(s) in the wheat genetic background. The molecular marker analysis indicated that 10 markers may be used to distinguish Th. elongatum from other wheat-related species. Thus, K17-1065-4 carrying the stripe rust resistance gene(s) is a novel germplasm useful for breeding disease-resistant wheat cultivars. The molecular markers developed in this study may facilitate the mapping of the stripe rust resistance gene on chromosome 6E of tetraploid Th. elongatum.

Genome-Wide Association Study of Asian and European Common Wheat Accessions for Yield-Related Traits and Stripe Rust Resistance.

Identifying novel loci of yield-related traits and resistance to stripe rust (caused by Puccinia striiformis f. sp. tritici) in wheat will help in breeding wheat that can meet projected demands in diverse environmental and agricultural practices. We performed a genome-wide association study with 24,767 single nucleotide polymorphisms (SNPs) in 180 wheat accessions that originated in 16 Asian or European countries between latitudes 30°N and 45°N. We detected seven accessions with desirable yield-related traits and 42 accessions that showed stable, high degrees of stripe rust resistance in multienvironment field assessments. A marker-trait association analysis of yield-related traits detected 18 quantitative trait loci (QTLs) in at least two test environments and two QTLs related to stripe rust resistance in at least three test environments. Five of these QTLs were identified as potentially novel QTLs by comparing their physical locations with those of known QTLs in the Chinese Spring (CS) reference genome RefSeq v1.1 published by the International Wheat Genome Sequencing Consortium; two were for spike length, one was for grain number per spike, one was for spike number, and one was for stripe rust resistance at the adult plant stage. We also identified 14 candidate genes associated with the five novel QTLs. These QTLs and candidate genes will provide breeders with new germplasm and can be used to conduct marker-assisted selection in breeding wheat with improved yield and stripe rust resistance.

RNA-seq analysis revealed considerable genetic diversity and enabled the development of specific KASP markers for Psathyrostachys huashanica.

Psathyrostachys huashanica, which grows exclusively in Huashan, China, is an important wild relative of common wheat that has many desirable traits relevant for wheat breeding. However, the poorly characterized interspecific phylogeny and genomic variations and the relative lack of species-specific molecular markers have limited the utility of P. huashanica as a genetic resource for enhancing wheat germplasm. In this study, we sequenced the P. huashanica transcriptome, resulting in 50,337,570 clean reads that were assembled into 65,617 unigenes, of which 38,428 (58.56%) matched at least one sequence in public databases. The phylogenetic analysis of P. huashanica, Triticeae species, and Poaceae species was conducted using 68 putative orthologous gene clusters. The data revealed the distant evolutionary relationship between P. huashanica and common wheat as well as the substantial diversity between the P. huashanica genome and the wheat D genome. By comparing the transcriptomes of P. huashanica and Chinese Spring, 750,759 candidate SNPs between P. huashanica Ns genes and their common wheat orthologs were identified. Among the 90 SNPs in the exon regions with different functional annotations, 58 (64.4%) were validated as Ns genome-specific SNPs in the common wheat background by KASP genotyping assays. Marker validation analyses indicated that six specific markers can discriminate between P. huashanica and the other wheat-related species. In addition, five markers are unique to P. huashanica, P. juncea, and Leymus species, which carry the Ns genome. The Ns genome-specific markers in a wheat background were also validated regarding their specificity and stability for detecting P. huashanica chromosomes in four wheat-P. huashanica addition lines. Four and eight SNP markers were detected in wheat-P. huashanica 2Ns and 7Ns addition lines, respectively, and one marker was specific to both wheat-P. huashanica 3Ns, 4Ns, and 7Ns addition lines. These markers developed using transcriptome data may be used to elucidate the genetic relationships among Psathyrostachys, Leymus, and other closely-related species. They may also facilitate precise introgressions and the high-throughput monitoring of P. huashanica exogenous chromosomes or segments in future crop breeding programs.

Determination of selected glucocorticoids in healthy foods by ultra-performance convergence chromatography-triple quadrupole mass spectrometry.

The presence of glucocorticoids in healthy foods has recently become a topic of concern because of their side effects. In this study, we developed a method based on ultra-performance convergence chromatography-triple quadrupole mass spectrometry (UPC2-MS/MS) to detect 63 glucocorticoids in healthy foods. The analysis conditions were optimized, and the method was validated. We further compared the results of this method with those of the RPLC-MS/MS method. Glucocorticoids were separated on an Acquity Torus 2-picolylamine column (100 mm × 3.0 mm, 1.7 µm) and detected via MS/MS. CO2 and methanol (containing 0.1% formic acid) were used as mobile phases. The method demonstrated good linear relationships between 1 and 200 µg·L-1 (R2 ≥ 0.996). The limits of detection in different types of samples were 0.3-1.5 µg·kg-1 (S/N = 3). The average recoveries (n = 9) and RSDs in different types of samples were 76.6-118.2% and 1.1-13.1%, respectively. The matrix effect, calculated as the ratio between calibration curves built in matrix and pure solvent, was less than 0.21 for both a fish oil and a protein powder. This method exhibited better selectivity and resolution than RPLC-MS/MS method. Lastly, it could realize the baseline separation of 31 isomers of 13 groups, including four groups of eight epimers. This study provides new technical support for assessing the risk of exposure to glucocorticoids in healthy foods.

Manganese and copper additions differently reduced cadmium uptake and accumulation in dwarf Polish wheat (Triticum polonicum L.).

This study investigated the effects of manganese (Mn) and copper (Cu) on dwarf Polish wheat under cadmium (Cd) stress by evaluating plant growth, Cd uptake, translocation, accumulation, subcellular distribution, and chemical forms, and the expression of genes participating in cell wall synthesis, metal chelation, and metal transport. Compared with the control, Mn deficiency and Cu deficiency increased Cd uptake and accumulation in roots, and Cd levels in root cell wall and soluble fractions, but inhibited Cd translocation to shoots. Mn addition reduced Cd uptake and accumulation in roots, and Cd level in root soluble fraction. Cu addition did not affect Cd uptake and accumulation in roots, while it caused a decrease and an increase of Cd levels in root cell wall and soluble fractions, respectively. The main Cd chemical forms (water-soluble Cd, pectates and protein integrated Cd, and undissolved Cd phosphate) in roots were differently changed. Furthermore, all treatments distinctly regulated several core genes that control the main component of root cell walls. Several Cd absorber (COPT, HIPP, NRAMP, and IRT) and exporter genes (ABCB, ABCG, ZIP, CAX, OPT, and YSL) were differently regulated to mediate Cd uptake, translocation, and accumulation. Overall, Mn and Cu differently influenced Cd uptake and accumulation; Mn addition is an effective treatment for reducing Cd accumulation in wheat.

FeCl3 and Fe2(SO4)3 differentially reduce Cd uptake and accumulation in Polish wheat (Triticum polonicum L.) seedlings by exporting Cd from roots and limiting Cd binding in the root cell walls.

Wheat grown in cadmium (Cd)-contaminated soils easily accumulates more Cd in edible parts than the Chinese safety limit (0.1 mg/kg). FeCl3 and Fe2(SO4)3 have been used to extract Cd from Cd-contaminated soils. Thus, we hypothesized that FeCl3 and Fe2(SO4)3, used as iron (Fe) fertilizers, can reduce Cd uptake and accumulation in wheat. Here, a hydroponic experiment was performed with three FeCl3 and Fe2(SO4)3 concentrations under 80 µM CdCl2 stress on dwarf Polish wheat (Triticum polonicum L., 2n = 4x = 28, AABB) seedlings. Compared with Fe deficiency, FeCl3 and Fe2(SO4)3 additions competitively reduced Cd concentrations. The reductions were not associated with changes in dry weight and root morphological parameters. FeCl3 and Fe2(SO4)3 additions reduced Cd concentrations in the following order from smallest to largest reduction: 25 µM Fe2(SO4)3 < 200 µM FeCl3 < 50 µM FeCl3 < 100 µM Fe2(SO4)3. Investigation of subcellular distributions showed that the four Fe fertilizers differentially reduced Cd binding in the root cell walls and enhanced root sucrose and trehalose. Cd chemical form analysis revealed that Fe fertilizer addition also differentially reduced root FE, FW, and FNaCl. Transcriptomic analysis revealed that addition of FeCl3 and Fe2(SO4)3 differentially up-regulated several genes that hydrolyze cell wall polysaccharides and metal transporter genes for Cd uptake (IRT1 and CAX19) and export (ZIP1, ABCG11, ABCG14, ABCG28, ABCG37, ABCG44, and ABCG48) reducing Cd uptake and accumulation. Our results demonstrated that FeCl3 and Fe2(SO4)3 can reduce Cd accumulation in wheat, and 50 µM FeCl3 is the most effective treatment.

Pseudorogneria libanotica Intraspecific Genetic Polymorphism Revealed by Fluorescence In Situ Hybridization with Newly Identified Tandem Repeats and Wheat Single-Copy Gene Probes.

The genus Pseudoroegneria (Nevski) Löve (Triticeae, Poaceae) with its genome abbreviated 'St' accounts for more than 60% of perennial Triticeae species. The diploid species Psudoroegneria libanotica (2n = 14) contains the most ancient St genome. Therefore, investigating its chromosomes could provide some fundamental information required for subsequent studies of St genome evolution. Here, 24 wheat cDNA probes covering seven chromosome groups were mapped in P. libanotica to distinguish homoelogous chromosomes, and newly identified tandem repeats were performed to differentiate seven chromosome pairs. Using these probes, we investigated intraspecific population chromosomal polymorphism of P. libanotica. We found that (i) a duplicated fragment of the 5St long arm was inserted into the short arm of 2St; (ii) asymmetrical fluorescence in situ hybridization (FISH) hybridization signals among 2St, 5St, and 7St homologous chromosome pairs; and (iii) intraspecific population of polymorphism in P. libanotica. These observations established the integrated molecular karyotype of P. libanotica. Moreover, we suggested heterozygosity due to outcrossing habit and adaptation to the local climate of P. libanotica. Specifically, the generated STlib_96 and STlib_98 repeats showed no cross-hybridization signals with wheat chromosomes, suggesting that they are valuable for identifying alien chromosomes or introgressed fragments of wild relatives in wheat.

MicroRNA and circular RNA profiling in the deposited fat tissue of Sunite sheep.

As the most typical deposited fat, tail fat is an important energy reservoir for sheep adapted to harsh environments and plays an important role as a raw material in daily life. However, the regulatory mechanisms of microRNA (miRNA) and circular RNA (circRNA) in tail fat development remain unclear. In this study, we characterized the miRNA and circRNA expression profiles in the tail fat of sheep at the ages of 6, 18, and 30 months. We identified 219 differentially expressed (DE) miRNAs (including 12 novel miRNAs), which exhibited a major tendency to be downregulated, and 198 DE circRNAs, which exhibited a tendency to be upregulated. Target gene prediction analysis was performed for the DE miRNAs. Functional analysis revealed that their target genes were mainly involved in cellular interactions, while the host genes of DE circRNAs were implicated in lipid and fatty acid metabolism. Subsequently, we established a competing endogenous RNA (ceRNA) network based on the negative regulatory relationship between miRNAs and target genes. The network revealed that upregulated miRNAs play a leading role in the development of tail fat. Finally, the ceRNA relationship network with oar-miR-27a_R-1 and oar-miR-29a as the core was validated, suggesting possible involvement of these interactions in tail fat development. In summary, DE miRNAs were negatively correlated with DE circRNAs during sheep tail fat development. The multiple ceRNA regulatory network dominated by upregulated DE miRNAs may play a key role in this developmental process.

Effect of nitrogen fertilizer on seed yield and quality of Kengyilia melanthera (Triticeae, Poaceae).

Widely distributed in the alpine sandy grassland in east Qinghai-Tibet Plateau (QTP), Kengyilia melanthera is considered as an ideal pioneer grass for the restoration of degraded and desertification grassland in the region. Under the special ecological and climatic conditions in the northwest Sichuan plateau located in east QTP, it is of great significance to optimize the amount of nitrogen fertilizer for the seed production of this species. The impact of nitrogen (N) fertilizer application on seed yield and quality of K. melanthera 'Aba', the only domesticated variety in the Kengyilia genus of Poaceae, was investigated based on two-year field experiments in the northwestern Sichuan plateau. The results showed that with the increase of N fertilizer application, the number of tillers, number of fertile tillers, 1,000-seed weight and seed yield of this species increased likewise. The optimum N fertilizer rate deduced in the present study was 180 kg·hm-2, where the number of fertile tillers 1,000-seed weight and seed yield reached the peak values. Interestingly, the standard germination rate, germination energy, accelerated aging germination rate, dehydrogenase and acid phosphatase activity of seeds were not affected by the increasing the input of N fertilizer. The comprehensive evaluation of membership function showed that the optimal N fertilizer treatment was 180 kg·hm-2 both for 2016 and 2017. This study provided a certain practical suggestion for the improvement of seed production of K. melanthera in the northwest Sichuan plateau.

Cytogenetic and Molecular Marker Analyses of a Novel Wheat-Psathyrostachys huashanica 7Ns Disomic Addition Line with Powdery Mildew Resistance.

Powdery mildew caused by Blumeria graminis f. sp. tritici is a devastating disease that reduces wheat yield and quality worldwide. The exploration and utilization of new resistance genes from wild wheat relatives is the most effective strategy against this disease. Psathyrostachys huashanica Keng f. ex P. C. Kuo (2n = 2x = 14, NsNs) is an important tertiary gene donor with multiple valuable traits for wheat genetic improvement, especially disease resistance. In this study, we developed and identified a new wheat-P. huashanica disomic addition line, 18-1-5-derived from a cross between P. huashanica and common wheat lines Chinese Spring and CSph2b. Sequential genomic and multicolor fluorescence in situ hybridization analyses revealed that 18-1-5 harbored 21 pairs of wheat chromosomes plus a pair of alien Ns chromosomes. Non-denaturing fluorescence in situ hybridization and molecular marker analyses further demonstrated that the alien chromosomes were derived from chromosome 7Ns of P. huashanica. The assessment of powdery mildew response revealed that line 18-1-5 was highly resistant at the adult stage to powdery mildew pathogens prevalent in China. The evaluation of agronomic traits indicated that 18-1-5 had a significantly reduced plant height and an increased kernel length compared with its wheat parents. Using genotyping-by-sequencing technology, we developed 118 PCR-based markers specifically for chromosome 7Ns of P. huashanica and found that 26 of these markers could be used to distinguish the genomes of P. huashanica and other wheat-related species. Line 18-1-5 can therefore serve as a promising bridging parent for wheat disease resistance breeding. These markers should be conducive for the rapid, precise detection of P. huashanica chromosomes and chromosomal segments carrying Pm resistance gene(s) during marker-assisted breeding and for the investigation of genetic differences and phylogenetic relationships among diverse Ns genomes and other closely related ones.

Comparative physiological and root transcriptome analysis of two annual ryegrass cultivars under drought stress.

Annual ryegrass is a widely cultivated forage grass with rapid growth and high productivity. However, drought is one of the abiotic stresses affecting ryegrass growth and quality. In this study, we compared the physiological and transcriptome responses of Chuansi No.1 (drought-tolerant, DT) and Double Barrel (drought-sensitive, DS) under drought stress simulated by PEG-6000 for 7 days. The results showed that Chuansi No. 1 had stronger physiological and biochemical parameters such as root properties, water content, osmotic adjustment ability and antioxidant ability. In addition, RNA-seq was used to elucidate the molecular mechanism of root drought resistance. We identified 8588 differentially expressed genes related to drought tolerance in root, which were mainly enriched in oxidation-reduction process, carbohydrate metabolic process, apoplast, arginine and proline metabolism, and phenylpropanoid biosynthesis pathways. The expression levels of DEGs were consistent with physiological changes of ryegrass under drought stress. We found that genes related to sucrose and starch synthesis, root development, osmotic adjustment, ABA signal regulation and specifically up-regulated transcription factors such as WRKY41, WRKY51, ERF7, ERF109, ERF110, NAC43, NAC68, bHLH162 and bHLH148 in Chuansi No. 1 may be the reason for its higher drought tolerance. This study revealed the underlying physiological and molecular mechanisms of root response to drought stress in ryegrass and provided some new candidate genes for breeding rye drought tolerant varieties.

Fine mapping of the reduced height gene Rht22 in tetraploid wheat landrace Jianyangailanmai (Triticum turgidum L.).

KEY MESSAGE: Rht22 was fine mapped in the interval of 0.53-1.48 Mb on 7AS, which reduces cell number of internode to cause semi-dwarfism in Jianyangailanmai. As a valuable germplasm resource for wheat genetic improvement, tetraploid wheat has several reduced height (Rht) and enhanced harvest index genes. Rht22, discovered in Jianyangailanmai (JAM, Triticum turgidum L., 2n = 4x = 28, AABB), significantly increases the spikelet number per spike, but its accurate chromosomal position is still unknown. In this study, a high-density genetic map was constructed using specific-length amplified fragment sequencing in an F7 RIL_DJ population, which was derived from a cross between dwarf Polish wheat (T. polonicum L., 2n = 4x = 28, AABB) and JAM. Two plant height loci, Qph.sicau-4B and Qph.sicau-7A, were mapped on chromosomes 4BS and 7AS, respectively. Qph.sicau-7A was mapped to the 0.33-4.46 Mb interval on 7AS and likely represents the candidate region of Rht22. Fine mapping confirmed and narrowed Rht22 on chromosome arm 7AS between Xbag295.s53 and Xb295.191 in three different populations. The physical region ranged from 0.53 to 1.48 Mb and included 18 candidate genes. Transcriptome analysis of two pairs of near-isogenic lines revealed that 135 differentially expressed genes (DEGs) were associated with semi-dwarfism. Of these, the expression of 83 annotated DEGs involved in hormones synthesis and signal transduction, cell wall composition, DNA replication, microtubule and phragmoplast arrays was significantly down-regulated in the semi-dwarf line. Therefore, Rht22 causes semi-dwarfism in JAM by disrupting these cellular processes, which impairs cell proliferation and reduces internode cell number.