Multi-omic analysis reveals the effects of interspecific hybridization on the synthesis of seed reserve polymers in a Triticum turgidum ssp. durum × Aegilops sharonensis amphidiploid.

BACKGROUND: Wheat grain endosperm is mainly composed of proteins and starch. The contents and the overall composition of seed storage proteins (SSP) markedly affect the processing quality of wheat flour. Polyploidization results in duplicated chromosomes, and the genomes are often unstable and may result in a large number of gene losses and gene rearrangements. However, the instability of the genome itself, as well as the large number of duplicated genes generated during polyploidy, is an important driving force for genetic innovation. In this study, we compared the differences in starch and SSP, and analyzed the transcriptome and metabolome among Aegilops sharonensis (R7), durum wheat (Z636) and amphidiploid (Z636×R7) to reveal the effects of polyploidization on the synthesis of seed reserve polymers. RESULTS: The total starch and amylose content of Z636×R7 was significantly higher than R7 and lower than Z636. The gliadin and glutenin contents of Z636×R7 were higher than those in Z636 and R7. Through transcriptome analysis, there were 21,037, 2197, 15,090 differentially expressed genes (DEGs) in the three comparison groups of R7 vs Z636, Z636 vs Z636×R7, and Z636×R7 vs R7, respectively, which were mainly enriched in carbon metabolism and amino acid biosynthesis pathways. Transcriptome data and qRT-PCR were combined to analyze the expression levels of genes related to storage polymers. It was found that the expression levels of some starch synthase genes, namely AGP-L, AGP-S and GBSSI in Z636×R7 were higher than in R7 and among the 17 DEGs related to storage proteins, the expression levels of 14 genes in R7 were lower than those in Z636 and Z636×R7. According to the classification analysis of all differential metabolites, most belonged to carboxylic acids and derivatives, and fatty acyls were enriched in the biosynthesis of unsaturated fatty acids, niacin and nicotinamide metabolism, one-carbon pool by folate, etc. CONCLUSION: After allopolyploidization, the expression of genes related to starch synthesis was down-regulated in Z636×R7, and the process of starch synthesis was inhibited, resulting in delayed starch accumulation and prolongation of the seed development process. Therefore, at the same development time point, the starch accumulation of Z636×R7 lagged behind that of Z636. In this study, the expression of the GSe2 gene in Z636×R7 was higher than that of the two parents, which was beneficial to protein synthesis, and increased the protein content. These results eventually led to changes in the synthesis of seed reserve polymers. The current study provided a basis for a greater in-depth understanding of the mechanism of wheat allopolyploid formation and its stable preservation, and also promoted the effective exploitation of high-value alleles.

Integration of transcriptomics, metabolomics, and hormone analysis revealed the formation of lesion spots inhibited by GA and CTK was related to cell death and disease resistance in bread wheat (Triticum aestivum L.).

BACKGROUND: Wheat is one of the important grain crops in the world. The formation of lesion spots related to cell death is involved in disease resistance, whereas the regulatory pathway of lesion spot production and resistance mechanism to pathogens in wheat is largely unknown. RESULTS: In this study, a pair of NILs (NIL-Lm5W and NIL-Lm5M) was constructed from the BC1F4 population by the wheat lesion mimic mutant MC21 and its wild genotype Chuannong 16. The formation of lesion spots in NIL-Lm5M significantly increased its resistance to stripe rust, and NIL-Lm5M showed superiour agronomic traits than NIL-Lm5W under stripe rust infection.Whereafter, the NILs were subjected to transcriptomic (stage N: no spots; stage S, only a few spots; and stage M, numerous spots), metabolomic (stage N and S), and hormone analysis (stage S), with samples taken from normal plants in the field. Transcriptomic analysis showed that the differentially expressed genes were enriched in plant-pathogen interaction, and defense-related genes were significantly upregulated following the formation of lesion spots. Metabolomic analysis showed that the differentially accumulated metabolites were enriched in energy metabolism, including amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Correlation network diagrams of transcriptomic and metabolomic showed that they were both enriched in energy metabolism. Additionally, the contents of gibberellin A7, cis-Zeatin, and abscisic acid were decreased in leaves upon lesion spot formation, whereas the lesion spots in NIL-Lm5M leaves were restrained by spaying GA and cytokinin (CTK, trans-zeatin) in the field. CONCLUSION: The formation of lesion spots can result in cell death and enhance strip rust resistance by protein degradation pathway and defense-related genes overexpression in wheat. Besides, the formation of lesion spots was significantly affected by GA and CTK. Altogether, these results may contribute to the understanding of lesion spot formation in wheat and laid a foundation for regulating the resistance mechanism to stripe rust.

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.

Mapping and characterization of rust resistance genes Lr53 and Yr35 introgressed from Aegilops species.

KEY MESSAGE: The rust resistance genes Lr53 and Yr35 were introgressed into bread wheat from Aegilops longissima or Aegilops sharonensis or their S-genome containing species and mapped to the telomeric region of chromosome arm 6BS. Wheat leaf and stripe rusts are damaging fungal diseases of wheat worldwide. Breeding for resistance is a sustainable approach to control these two foliar diseases. In this study, we used SNP analysis, sequence comparisons, and cytogenetic assays to determine that the chromosomal segment carrying Lr53 and Yr35 was originated from Ae.longissima or Ae. sharonensis or their derived species. In seedling tests, Lr53 conferred strong resistance against all five Chinese Pt races tested, and Yr35 showed effectiveness against Pst race CYR34 but susceptibility to race CYR32. Using a large population (3892 recombinant gametes) derived from plants homozygous for the ph1b mutation obtained from the cross 98M71 × CSph1b, both Lr53 and Yr35 were successfully mapped to a 6.03-Mb telomeric region of chromosome arm 6BS in the Chinese Spring reference genome v1.1. Co-segregation between Lr53 and Yr35 was observed within this large mapping population. Within the candidate region, several nucleotide-binding leucine-rich repeat genes and protein kinases were identified as candidate genes. Marker pku6B3127 was completely linked to both genes and accurately predicted the absence or presence of alien segment harboring Lr53 and Yr35 in 87 tetraploid and 149 hexaploid wheat genotypes tested. We developed a line with a smaller alien segment (< 6.03 Mb) to reduce any potential linkage drag and demonstrated that it conferred resistance levels similar to those of the original donor parent 98M71. The newly developed introgression line and closely linked PCR markers will accelerate the deployment of Lr53 and Yr35 in wheat breeding programs.

Identification of candidate genes for adult plant stripe rust resistance transferred from Aegilops ventricosa 2NvS into wheat via fine mapping and transcriptome analysis.

KEY MESSAGE: An adult plant gene for resistance to stripe rust was narrowed down to the proximal one-third of the 2NvS segment translocated from Aegilops ventricosa to wheat chromosome arm 2AS, and based on the gene expression analysis, two candidate genes were identified showing a stronger response at the adult plant stage compared to the seedling stage. The 2NvS translocation from Aegilops ventricosa, known for its resistance to various diseases, has been pivotal in global wheat breeding for more than three decades. Here, we identified an adult plant resistance (APR) gene in the 2NvS segment in wheat line K13-868. Through fine mapping in a segregating near-isogenic line (NIL) derived population of 6389 plants, the candidate region for the APR gene was narrowed down to between 19.36 Mb and 33 Mb in the Jagger reference genome. Transcriptome analysis in NILs strongly suggested that this APR gene conferred resistance to stripe rust by triggering plant innate immune responses. Based on the gene expression analysis, two disease resistance-associated genes within the candidate region, TraesJAG2A03G00588940 and TraesJAG2A03G00590140, exhibited a stronger response to Puccinia striiformis f. sp. tritici (Pst) infection at the adult plant stage than at the seedling stage, indicating that they could be potential candidates for the resistance gene. Additionally, we developed a co-dominant InDel marker, InDel_31.05, for detecting this APR gene. Applying this marker showed that over one-half of the wheat varieties approved in 2021 and 2022 in Sichuan province, China, carry this gene. Agronomic trait evaluation of NILs indicated that the 2NvS segment effectively mitigated the negative effects of stripe rust on yield without affecting other important agronomic traits. This study provided valuable insights for cloning and breeding through the utilization of the APR gene present in the 2NvS segment.

A co-located QTL for seven spike architecture-related traits shows promising breeding use potential in common wheat (Triticum aestivum L.).

KEY MESSAGE: A co-located novel QTL for TFS, FPs, FMs, FFS, FFPs, KWS, and KWPs with potential of improving wheat yield was identified and validated. Spike-related traits, including fertile florets per spike (FFS), kernel weight per spike (KWS), total florets per spike (TFS), florets per spikelet (FPs), florets in the middle spikelet (FMs), fertile florets per spikelet (FFPs), and kernel weight per spikelet (KWPs), are key traits in improving wheat yield. In the present study, quantitative trait loci (QTL) for these traits evaluated under various environments were detected in a recombinant inbred line population (msf/Chuannong 16) mainly genotyped using the 16 K SNP array. Ultimately, we identified 60 QTL, but only QFFS.sau-MC-1A for FFS was a major and stably expressed QTL. It was located on chromosome arm 1AS, where loci for TFS, FPs, FMs, FFS, FFPs, KWS, and KWPs were also simultaneously co-mapped. The effect of QFFS.sau-MC-1A was further validated in three independent segregating populations using a Kompetitive Allele-Specific PCR marker. For the co-located QTL, QFFS.sau-MC-1A, the presence of a positive allele from msf was associate with increases for all traits: + 12.29% TFS, + 10.15% FPs, + 13.97% FMs, + 17.12% FFS, + 14.75% FFPs, + 22.17% KWS, and + 19.42% KWPs. Furthermore, pleiotropy analysis showed that the positive allele at QFFS.sau-MC-1A simultaneously increased the spike length, spikelet number per spike, and thousand-kernel weight. QFFS.sau-MC-1A represents a novel QTL for marker-assisted selection with the potential for improving wheat yield. Four genes, TraesCS1A03G0012700, TraesCS1A03G0015700, TraesCS1A03G0016000, and TraesCS1A03G0016300, which may affect spike development, were predicted in the physical interval harboring QFFS.sau-MC-1A. Our results will help in further fine mapping QFFS.sau-MC-1A and be useful for improving wheat yield.

Recombinant human growth hormone promotes wound angiogenesis in burned mice through the ERK signaling pathway.

Burns are the most severe type of trauma, and the resulting ischemia and hypoxia damage can promote the dysfunction and even failure of tissues and organs throughout the body, endangering patients' life safety. Recombinant human growth hormone (rhGH) has the functions of promoting protein synthesis to reverse negative nitrogen balance, accelerating wound healing, and improving immune function, which is widely used in the treatment of burns. However, the exact mechanism and pathway of rhGH's action is not yet fully understood. In this study, we observed the wound repair effect of recombinant human growth hormone (rhGH) on burned mice and further analyzed the mechanism of action, which can provide more comprehensive reference opinions for clinical practice. First, by establishing a burn mouse model and and intervening with different doses of rhGH, we found that the wound healing capacity of mice was significantly enhanced and the inflammatory and oxidative stress responses were obviously alleviated, confirming the excellent promotion of wound repair and anti-inflammatory and antioxidant effects of rhGH. Subsequently, we found that the expression of p-ERK1/2/ERK1/2, EGF, TGF-ß, and VEGF proteins was elevated in the traumatic tissues of mice after rhGH intervention, suggesting that the pathway of action of rhGH might be related to the activation of ERK pathway to promote the regeneration of traumatic capillaries.

Comparative analysis of Fusarium crown rot resistance in synthetic hexaploid wheats and their parental genotypes.

BACKGROUND: Fusarium crown rot (FCR) is a chronic disease of cereals worldwide. Compared with tetraploid wheat, hexaploid wheat is more resistant to FCR infection. The underlying reasons for the differences are still not clear. In this study, we compared FCR responses of 10 synthetic hexaploid wheats (SHWs) and their tetraploid and diploid parents. We then performed transcriptome analysis to uncover the molecular mechanism of FCR on these SHWs and their parents. RESULTS: We observed higher levels of FCR resistance in the SHWs compared with their tetraploid parents. The transcriptome analysis suggested that multiple defense pathways responsive to FCR infection were upregulated in the SHWs. Notably, phenylalanine ammonia lyase (PAL) genes, involved in lignin and salicylic acid (SA) biosynthesis, exhibited a higher level of expression to FCR infection in the SHWs. Physiological and biochemical analysis validated that PAL activity and SA and lignin contents of the stem bases were higher in SHWs than in their tetraploid parents. CONCLUSION: Overall, these findings imply that improved FCR resistance in SHWs compared with their tetraploid parents is probably related to higher levels of response on PAL-mediated lignin and SA biosynthesis pathways.

A promising QTL QSns.sau-MC-3D.1 likely superior to WAPO1 for the number of spikelets per spike of wheat shows no adverse effects on yield-related traits.

KEY MESSAGE: A likely new locus QSns.sau-MC-3D.1 associated with SNS showing no negative effect on yield-related traits compared to WAPO1 was identified and validated in various genetic populations under multiple environments. The number of spikelets per spike (SNS) is one of the crucial factors determining wheat yield. Thus, improving our understanding of the genes that regulate SNS could help develop wheat varieties with higher yield. In this study, a recombinant inbred line (RIL) population (MC) containing 198 lines derived from a cross between msf and Chuannong 16 (CN16) was used to construct a genetic linkage map using the GenoBaits Wheat 16 K Panel. The genetic map contained 5,991 polymorphic SNP markers spanning 2,813.25 cM. A total of twelve QTL for SNS were detected, and two of them, i.e., QSns.sau-MC-3D.1 and QSns.sau-MC-7A, were stably expressed. QSns.sau-MC-3D.1 had high LOD values ranging from 4.99 to 11.06 and explained 9.71-16.75% of the phenotypic variation. Comparison of QSns.sau-MC-3D.1 with previously reported SNS QTL suggested that it is likely a novel one, and two kompetitive allele-specific PCR (KASP) markers were further developed. The positive effect of QSns.sau-MC-3D.1 was also validated in three biparental populations and a diverse panel containing 388 Chinese wheat accessions. Genetic analysis indicated that WHEAT ORTHOLOG OFAPO1 (WAPO1) was a candidate gene for QSns.sau-MC-7A. Pyramiding of QSns.sau-MC-3D.1 and WAP01 had a great additive effect increasing SNS by 7.10%. Correlation analysis suggested that QSns.sau-MC-3D.1 was likely independent of effective tiller number, plant height, spike length, anthesis date, and thousand kernel weight. However, the H2 haplotype of WAPO1 may affect effective tiller number and plant height. These results indicated that utilization of QSns.sau-MC-3D.1 should be given priority for wheat breeding. Geographical distribution analysis showed that the positive allele of QSns.nsau-MC-3D.1 was dominant in most wheat-producing regions of China, and it has been positively selected among modern cultivars released in China since the 1940s. Gene prediction, qRT-PCR analysis, and sequence alignment suggested that TraesCS3D03G0216800 may be the candidate gene of QSns.nsau-MC-3D.1. Taken together, these results enrich our understanding of the genetic basis of wheat SNS and will be useful for fine mapping and cloning of the gene underlying QSns.sau-MC-3D.1.

Major and stably expressed QTL for traits related to the mature wheat embryo independent of kernel size.

KEY MESSAGE: Two major and stably expressed QTL for traits related to mature wheat embryo independent of kernel size were identified and validated in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. As the juvenile of a highly differentiated plant, mature wheat (Triticum aestivum L.) embryos are highly significant to agricultural production. To understand the genetic basis of traits related to wheat embryo size, the embryo of mature kernels in a recombination inbred line that contained 126 lines from four environments was measured. The genetic loci of embryo size, including embryo length (EL), embryo width (EW), embryo area (EA), embryo length/kernel length (EL/KL), embryo width/kernel width (EW/KW), and EL/EW, were identified based on a genetic linkage map constructed based on PCR markers and the Wheat 55 K single nucleotide polymorphism (SNP) array. A total of 50 quantitative trait loci (QTL) for traits related to wheat embryo size were detected. Among them, QEL.sicau-2SY-4A for EL and QEW.sicau-2SY-7B for EW were major and stably expressed and were genetically independent of KL and KW, respectively. Their effects were further verified in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. Further analysis showed that TraesCS4A02G343300 and TraesCS7B02G006800 could be candidate genes for QEL.sicau-2SY-4A and QEW.sicau-2SY-7B, respectively. In addition, significant positive correlations between EL and kernel-related traits and the 1,000-grain weight were detected. Collectively, this study broadens our understanding of the genetic basis of wheat embryo size and will be helpful for the further fine-mapping of interesting loci in the future.

Identification of a suppressor for the wheat stripe rust resistance gene Yr81 in Chinese wheat landrace Dahongpao.

KEY MESSAGE: Combined with BSE-Seq analysis and multiple genetic populations, three genes involved in stripe rust resistance were identified in Chinese wheat landrace Dahongpao, including a novel suppressor on 2BS. Dahongpao (DHP), a landrace of hexaploid wheat in China, exhibits a high degree of stripe rust resistance in the field for many years. In this study, bulked segregant analysis coupled with exome capture sequencing (BSE-Seq) was used to identify genes encoding stripe rust resistance in multiple genetic populations from the cross between DHP and a susceptible hexaploid Australian cultivar, Avocet S (AvS). The most effective QTL in DHP was Yr18, explaining up to 53.08% of phenotypic variance in the F2:3 families. To identify additional genes, secondary mapping populations SP1 and SP2 were produced by crossing AvS with two resistant lines derived from F2:3 families lacking Yr18. An all-stage resistance gene, Yr.DHP-6AS, was identified via BSE-Seq analysis of SP1. Combined the recombinant plants from both SP1 and SP2, Yr.DHP-6AS was located between KP6A_1.66 and KP6A_8.18, corresponding to the same region as Yr81. In addition, secondary mapping populations SP3 and SP4 were developed by selfing a segregating line from F2:3 families lacking Yr18. A novel suppressor gene on chromosome 2BS was identified from DHP for effectively suppressing the resistance of Yr.DHP-6AS in the SP3 and SP4. As a result, the wheat lines carrying both Yr18 and Yr.DHP-6AS show higher level of stripe rust resistance than DHP, providing an effective and simple combination for developing new wheat cultivars with ASR and APR genes. Further, the newly developed KASP markers, KP6A_1.99 and KP6A_5.22, will facilitate the application of Yr.DHP-6AS in wheat breeding via marker-assisted selection.

A major QTL simultaneously increases the number of spikelets per spike and thousand-kernel weight in a wheat line.

KEY MESSAGE: A novel and stably expressed QTL QSNS.sicau-SSY-7A for spikelet number per spike in wheat without negative effects on thousand-kernel weight was identified and validated in different genetic backgrounds. Spikelet number per spike (SNS) is an important determinant of yield in wheat. In the present study, we combined bulked segregant analysis (BSA) and the wheat 660 K single-nucleotide polymorphism (SNP) array to rapidly identify genomic regions associated with SNS from a recombinant inbred line (RIL) population derived from a cross between the wheat lines S849-8 and SY95-71. A genetic map was constructed using Kompetitive Allele Specific PCR markers in the SNP-enriched region on the long arm of chromosome 7A. A major and stably expressed QTL, QSNS.sicau-SSY-7A, was detected in multiple environments. It was located in a 1.6 cM interval on chromosome arm 7AL flanked by the markers AX-109983514 and AX-109820548. This QTL explained 6.86-15.72% of the phenotypic variance, with LOD values ranging from 3.66 to 8.66. Several genes associated with plant growth and development were identified in the interval where QSNS.sicau-SSY-7A was located on the 'Chinese Spring' wheat and wild emmer reference genomes. Furthermore, the effects of QSNS.sicau-SSY-7A and WHEAT ORTHOLOG OFAPO1(WAPO1) on SNS were analyzed. Interestingly, QSNS.sicau-SSY-7A significantly increased SNS without negative effects on thousand-kernel weight, anthesis date and plant height, demonstrating its great potential for breeding aimed at improving grain yield. Taken together, these results indicate that QSNS.sicau-SSY-7A is a promising locus for yield improvement, and its linkage markers are helpful for fine mapping and molecular breeding.

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.

Aegilops sharonensis HMW-GSs with unusually large molecular weight improves bread-making quality in wheat-Ae. sharonensis introgression lines.

BACKGROUND: Eighteen wheat (Triticum aestivum-Aegilops sharonensis) introgression lines were generated in the previous study. These lines possessed four types of high molecular weight glutenin subunit (HMW-GS) combinations consisting of one glutenin from Ae. sharonensis (Glu-1Ssh ) plus one or more HMW-GSs from common wheat (Glu-A1, Glu-B1, or Glu-D1). RESULTS: In this study, we conducted quality tests to explore the effects of 1Ssh x2.3 and 1Ssh y2.9 on the processing quality of 18 wheat-Aegilops sharonensis introgression lines. Our data showed that the 1Ssh x2.3 and 1Ssh y2.9 subunits had a positive effect on gluten and baking quality. The bread volume of all these lines was higher than that of the parental wheat line LM3. In these lines, the HMW-GS content and the HMW/LMW ratio of 66-36-11 were higher than those of LM3, and the 66-36-11 line exhibited greatly improved quality parameters in comparison with the parent LM3. CONCLUSION: These results demonstrated that the 1Ssh x2.3 and 1Ssh y2.9 subunits from Ae. sharonensis contributed immensely to gluten strength and bread-baking quality, and proved a positive relationship between the HMW-GS sizes and their effects on dough strength in vivo. The materials developed could be used by breeding programs aiming to increase bread-making quality. © 2022 Society of Chemical Industry.

Identification and validation of a major QTL for kernel length in bread wheat based on two F3 biparental populations.

BACKGROUND: High yield and quality are essential goals of wheat (Triticum aestivum L.) breeding. Kernel length (KL), as a main component of kernel size, can indirectly change kernel weight and then affects yield. Identification and utilization of excellent loci in wheat genetic resources is of great significance for cultivating high yield and quality wheat. Genetic identification of loci for KL has been performed mainly through genome-wide association study in natural populations or QTL mapping based on genetic linkage map in high generation populations. RESULTS: In this study, an F3 biparental population derived from the cross between an EMS mutant BLS1 selected from an EMS-induced wheat genotype LJ2135 (derived from the hybrid progeny of a spelt wheat (T. spelta L.) and a common wheat) mutant bank and a local breeding line 99E18 was used to rapidly identify loci controlling KL based on Bulked Segregant Analysis (BSA) and the wheat 660 K single-nucleotide polymorphism (SNP) array. The highest ratio of polymorphic SNPs was located on chromosome 4A. Linkage map analysis showed that 33 Kompetitive Allele Specific PCR markers were linked to the QTL for KL (Qkl.sicau-BLE18-4A) identified in three environments as well as the best linear unbiased prediction (BLUP) dataset. This QTL explained 10.87-19.30% of the phenotypic variation. Its effect was successfully confirmed in another F3 population with the two flanking markers KASP-AX-111536305 and KASP-AX-110174441. Compared with previous studies and given that the of BLS1 has the genetic background of spelt wheat, the major QTL was likely a new one. A few of predicted genes related to regulation of kernel development were identified in the interval of the detected QTL. CONCLUSION: A major, novel and stable QTL (Qkl.sicau-BLE18-4A) for KL was identified and verified in two F3 biparental populations across three environments. Significant relationships among KL, kernel width (KW) and thousand kernel weight (TKW) were identified. Four predicted genes related to kernel growth regulation were detected in the interval of Qkl.sicau-BLE18-4A. Furthermore, this study laid foundation on subsequent fine mapping work and provided a possibility for breeding of elite wheat varieties.

Development and application of specific FISH probes for karyotyping Psathyrostachys huashanica chromosomes.

BACKGROUND: Psathyrostachys huashanica Keng has long been used as a genetic resource for improving wheat cultivar because of its genes mediating the resistance to various diseases (stripe rust, leaf rust, take-all, and powdery mildew) as well as its desirable agronomic traits. However, a high-resolution fluorescence in situ hybridization (FISH) karyotype of P. huashanica remains unavailable. RESULTS: To develop chromosome-specific FISH markers for P. huashanica, repetitive sequences, including pSc119.2, pTa535, pTa713, pAs1, (AAC)5, (CTT)12, pSc200, pTa71A-2, and Oligo-44 were used for a FISH analysis. The results indicated that the combination of pSc200, pTa71A-2 and Oligo-44 probes can clearly identify all Ns genomic chromosomes in the two P. huashanica germplasms. The homoeologous relationships between individual P. huashanica chromosomes and common wheat chromosomes were clarified by FISH painting. Marker validation analyses revealed that the combination of pSc200, pTa71A-2, and Oligo-44 for a FISH analysis can distinguish the P. huashanica Ns-genome chromosomes from wheat chromosomes, as well as all chromosomes (except 4Ns) from the chromosomes of diploid wheat relatives carrying St, E, V, I, P and R genomes. Additionally, the probes were applicable for discriminating between the P. huashanica Ns-genome chromosomes in all homologous groups and the corresponding chromosomes in Psathyrostachys juncea and most Leymus species containing the Ns genome. Furthermore, six wheat-P. huashanica chromosome addition lines (i.e., 2Ns, 3Ns, 4Ns, 7Ns chromosomes and chromosomal segments) were characterized using the newly developed FISH markers. Thus, these probes can rapidly and precisely detect P. huashanica alien chromosomes in the wheat background. CONCLUSIONS: The FISH karyotype established in this study lays a solid foundation for the efficient identification of P. huashanica chromosomes in wheat genetic improvement programs.

The PGS1 basic helix-loop-helix protein regulates Fl3 to impact seed growth and grain yield in cereals.

Plant transcription factors (TFs), such as basic helix-loop-helix (bHLH) and AT-rich zinc-binding proteins (PLATZ), play critical roles in regulating the expression of developmental genes in cereals. We identified the bHLH protein TaPGS1 (T. aestivum Positive Regulator of Grain Size 1) specifically expressed in the seeds at 5-20 days post-anthesis in wheat. TaPGS1 was ectopically overexpressed (OE) in wheat and rice, leading to increased grain weight (up to 13.81% in wheat and 18.55% in rice lines) and grain size. Carbohydrate and total protein levels also increased. Scanning electron microscopy results indicated that the starch granules in the endosperm of TaPGS1 OE wheat and rice lines were smaller and tightly embedded in a proteinaceous matrix. Furthermore, TaPGS1 was bound directly to the E-box motif at the promoter of the PLATZ TF genes TaFl3 and OsFl3 and positively regulated their expression in wheat and rice. In rice, the OsFl3 CRISPR/Cas9 knockout lines showed reduced average thousand-grain weight, grain width, and grain length in rice. Our results reveal that TaPGS1 functions as a valuable trait-associated gene for improving cereal grain yield.

Quick mapping and characterization of a co-located kernel length and thousand-kernel weight-related QTL in wheat.

KEY MESSAGE: A co-located KL and TKW-related QTL with no negative effect on PH and AD was rapidly identified using BSA and wheat 660 K SNP array. Its effect was validated in a panel of 218 wheat accessions. Kernel length (KL) and thousand-kernel weight (TKW) of wheat (Triticum aestivum L.) contribute significantly to kernel yield. In the present study, a recombinant inbred line (RIL) population derived from the cross between the wheat line S849-8 with larger kernels and more spikelets per spike and the line SY95-71 was developed. Further, of both the bulked segregant analysis (BSA) and the wheat 660 K single nucleotide polymorphism (SNP) array were used to rapidly identify genomic regions for kernel-related traits from this RIL population. Kompetitive Allele Specific PCR markers were further developed in the SNP-enriched region on the 2D chromosome to construct a genetic map. Both QKL.sicau-SSY-2D for KL and QTKW.sicau-SSY-2D for TKW were identified at multiple environments on chromosome arm 2DL. These two QTLs explained 9.68-23.02% and 6.73-18.32% of the phenotypic variation, respectively. The effects of this co-located QTL were successfully verified in a natural population consisting of 218 Sichuan wheat accessions. Interestingly, the major QTL was significantly and positively correlated with spike length, but did not negatively affect spikelet number per spike (SNS), plant height, or anthesis date. These results indicated that it is possible to synchronously improve kernel weight and SNS by using this QTL. Additionally, several genes associated with kernel development and filling rate were predicted and sequenced in the QTL-containing physical intervals of reference genomes of 'Chinese spring' and Aegilops tauschii. Collectively, these results provide a QTL with great breeding potential and its linked markers which should be helpful for fine mapping and molecular breeding.

Characterization and fine mapping of a lesion mimic mutant (Lm5) with enhanced stripe rust and powdery mildew resistance in bread wheat (Triticum aestivum L.).

KEY MESSAGE: A novel light intensity-dependent lesion mimic mutant with enhanced disease resistance was physiologically, biochemically, and genetically characterized, and the causative gene was fine mapped to a 1.28 Mbp interval containing 17 high-confidence genes. Lesion mimic mutants are ideal for studying disease resistance and programmed cell death photosynthesis in plants to improve crop yield. In this study, a novel light intensity-dependent lesion mimic mutant (MC21) was obtained from the wheat variety Chuannong16 (CN16) by ethyl methane sulfonate treatment. The mutant initially developed tiny lesion spots on the basal part of the leaves, which then gradually proceeded down to leaf sheaths, stems, shells, and awns at the flowering stage. The major agronomic traits were significantly altered in the mutant compared to that in the wild-type CN16. Furthermore, the mutant exhibited a lesion phenotype with degenerated chloroplast structure, decreased chlorophyll content, increased level of reactive oxygen species, and increased resistance to stripe rust and powdery mildew. Genetic analysis indicated that the lesion phenotype was controlled by a novel single semi-dominant nuclear gene. The target gene was mapped on chromosome arm 2AL located between Kompetitive Allele Specific PCR (KASP) markers, KASP-4211 and KASP-5353, and tentatively termed as lesion mimic 5 (Lm5). The fine mapping suggested that Lm5 was located in a 1.28 Mbp interval between markers KASP-5825 and KASP-9366; 17 high-confidence candidate genes were included in this genomic region. This study provides an important foundational step for further cloning of Lm5 using a map-based approach.

Development and Identification of a Novel Wheat-Thinopyrum scirpeum 4E (4D) Chromosomal Substitution Line with Stripe Rust and Powdery Mildew Resistance.

Stripe rust caused by Puccinia striiformis f. sp. tritici and powdery mildew caused by Blumeria graminis f. sp. tritici are devastating diseases of wheat worldwide. Exploration of new disease-resistant genes from cultivated wheat and wild relatives are the most effective means of reducing the amounts of fungicides applied to combat these diseases. Thinopyrum scirpeum (2n = 4x = 28, EEEE) is an important promising reservoir of useful genes, including stripe rust and powdery mildew resistance, and may be useful for increasing wheat disease resistance. Here, we characterize a novel wheat-Th. scirpeum disomic substitution line, K16-730-3, and chromosome-specific markers were developed that can be used to trace the Th. scirpeum chromosome or chromosome segments transferred into wheat. Genomic in situ hybridization and fluorescence in situ hybridization analyses indicated that K16-730-3 is a new 4E (4D) chromosomal substitution line. Evaluation of seedling and adult disease responses revealed that K16-730-3 is resistant to stripe rust and powdery mildew. In addition, no obvious difference in grain yield was observed between K16-730-3 and its wheat parents. Genotyping-by-sequencing analyses indicated that 74 PCR-based markers can accurately trace chromosome 4E, which were linked to the disease resistance genes in the wheat background. Further marker validation analyses revealed that 13 specific markers can distinguish between the E-genome chromosomes of Th. scirpeum and the chromosomes of other wheat-related species. The new substitution line K16-730-3 carrying the stripe rust and powdery mildew resistance genes will be useful as novel germplasm in breeding for disease resistance. The markers developed in this study can be used in marker-assisted selection for increasing disease resistance in wheat.