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Abstract Photosystem I (PSI) from Fittonia albivenis, an Acanthaceae ornamental plant, is notable among green plants for its red-shifted emission spectrum. Here, we solved the structure of a PSI–light harvesting complex I (LHCI) supercomplex from F. albivenis at 2.46-Å resolution using cryo-electron microscopy. The supercomplex contains a core complex of 14 subunits and an LHCI belt with four antenna subunits (Lhca1–4) similar to previously reported angiosperm PSI–LHCI structures; however, Lhca3 differs in three regions surrounding a dimer of low-energy chlorophylls (Chls) termed red Chls, which absorb far-red beyond visible light. The unique amino acid sequences within these regions are exclusively shared by plants with strongly red-shifted fluorescence emission, suggesting candidate structural elements for regulating the energy state of red Chls. These results provide a structural basis for unraveling the mechanisms of light harvest and transfer in PSI–LHCI of under canopy plants and for designing Lhc to harness longer-wavelength light in the far-red spectral range.

Some haplotypes of the sucrose synthase gene TaSus1 are associated with thousand-grain weight (TGW) in wheat (Triticum aestivum L.). However, no mutations have been identified within the gene to test this association. The effects of TaSus1 on grain number per spike (GNS) also are largely unknown. Our previous genome-wide association study identified TaSus-A1 as a candidate gene controlling fertile spikelet number per spike (FSN). In the present study, we generated two independent mutants for the three TaSus1 homoeologs by CRISPR/Cas9-mediated genome editing. The triple mutants displayed lower FSN, GNS, grain number per spikelet (GNST), and TGW than wild-type plants. In 306 hexaploid wheat accessions, two single-nucleotide polymorphisms in TaSus-A1 contributed differently to GNS. Introgression of the two alleles into a wheat genetic background confirmed their effects. The alleles differed in geographical distribution among the accessions.

Abstract Background Large-scale genotype–phenotype association studies of crop germplasm are important for identifying alleles associated with favorable traits. The limited number of single-nucleotide polymorphisms (SNPs) in most wheat genome-wide association studies (GWASs) restricts their power to detect marker-trait associations. Additionally, only a few genes regulating grain number per spikelet have been reported due to sensitivity of this trait to variable environments. Results We perform a large-scale GWAS using approximately 40 million filtered SNPs for 27 spike morphology traits. We detect 132,086 significant marker-trait associations and the associated SNP markers are located within 590 associated peaks. We detect additional and stronger peaks by dividing spike morphology into sub-traits relative to GWAS results of spike morphology traits. We propose that the genetic dissection of spike morphology is a powerful strategy to detect signals for grain yield traits in wheat. The GWAS results reveal that TaSPL17 positively controls grain size and number by regulating spikelet and floret meristem development, which in turn leads to enhanced grain yield per plant. The haplotypes at TaSPL17 indicate geographical differentiation, domestication effects, and breeding selection. Conclusion Our study provides valuable resources for genetic improvement of spike morphology and a fast-forward genetic solution for candidate gene detection and cloning in wheat.

Abstract Background Plant architecture associated with increased grain yield and adaptation to the local environments is selected during wheat (Triticum aestivum) breeding. The internode length of individual stems and tiller length of individual plants are important for the determination of plant architecture. However, few studies have explored the genetic basis of these traits. Results Here, we conduct a genome-wide association study (GWAS) to dissect the genetic basis of geographical differentiation of these traits in 306 worldwide wheat accessions including both landraces and traditional varieties. We determine the changes of haplotypes for the associated genomic regions in frequency in 831 wheat accessions that are either introduced from other countries or developed in China from last two decades. We identify 83 loci that are associated with one trait, while the remaining 247 loci are pleiotropic. We also find 163 associated loci are under strong selective sweep. GWAS results demonstrate independent regulation of internode length of individual stems and consistent regulation of tiller length of individual plants. This makes it possible to obtain ideal haplotype combinations of the length of four internodes. We also find that the geographical distribution of the haplotypes explains the observed differences in internode length among the worldwide wheat accessions. Conclusion This study provides insights into the genetic basis of plant architecture. It will facilitate gene functional analysis and molecular design of plant architecture for breeding.

Abstract Background Founder parents play extremely important roles in wheat breeding. Studies into the genetic basis of founder parents and the transmission rules of favorable alleles are of great significance in improving agronomically important traits in wheat. Results Here, a total of 366 founder parents, widely grown cultivars, and derivatives of four representative founder parents were genotyped based on efficient kompetitive allele-specific PCR (KASP) markers in 87 agronomically important genes controlling yield, quality, adaptability, and stress resistance. Genetic composition analysis of founder parents and widely grown cultivars showed a consistently high frequency of favorable alleles for yield-related genes. This analysis further showed that other alleles favorable for resistance, strong gluten, dwarf size, and early heading date were also subject to selective pressure over time. By comparing the transmission of alleles from four representative founder parents to their derivatives during different breeding periods, it was found that the genetic composition of the representative founder parents was optimized as breeding progressed over time, with the number and types of favorable alleles carried gradually increasing and becoming enriched. There are still a large number of favorable alleles in wheat founder parents that have not been fully utilized in breeding selection. Eighty-seven agronomically important genes were used to construct an enrichment map that shows favorable alleles of four founder parents, providing an important theoretical foundation for future identification of candidate wheat founder parents. Conclusions These results reveal the genetic basis of founder parents and allele transmission for 87 agronomically important genes and shed light on breeding strategies for the next generation of elite founder parents in wheat.

Benzylisoquinoline alkaloids in lotus (Nelumbo nucifera) seed plumules and leaves exhibit significant tissue specificity for their pharmacological effects and potential nutritional properties. Herein, 46 benzylisoquinoline alkaloids were identified via UPLC-QTOF-HRMS, of which 9 were annotated as glycosylated monobenzylisoquinoline alkaloids concentrated in the seed plumules. The spatial distribution of targeted benzylisoquinoline alkaloids in leaves, seed plumules, and milky sap was determined via MALDI–MSI. Furthermore, 37 Nelumbo cultivars were investigated using targeted metabolomics to provide insights into functional tea development. While aporphine alkaloids comprised the main compounds present in lotus leaves, bisbenzylisoquinoline alkaloids were the main compounds in lotus plumules, where glycosylation primarily occurs. These findings can help understand the distribution of benzylisoquinoline alkaloids in lotus tissue and the directional breeding of varieties enriched with specific chemical functional groups for nutritional and pharmacological applications.

Bisbenzylisoquinoline and aporphine alkaloids are the two main pharmacological compounds in the ancient sacred lotus (Nelumbo nucifera). The biosynthesis of bisbenzylisoquinoline and aporphine alkaloids has attracted extensive attention because bisbenzylisoquinoline alkaloids have been reported as potential therapeutic agents for COVID-19. Our study showed that NnCYP80A can catalyze C-O coupling in both (R)-N-methylcoclaurine and (S)-N-methylcoclaurine to produce bisbenzylisoquinoline alkaloids with three different linkages. In addition, NnCYP80G catalyzed C-C coupling in aporphine alkaloids with extensive substrate selectivity, specifically using (R)-N-methylcoclaurine, (S)-N-methylcoclaurine, coclaurine and reticuline as substrates, but the synthesis of C-ring alkaloids without hydroxyl groups in the lotus remains to be elucidated. The key residues of NnCYP80G were also studied using the 3D structure of the protein predicted using Alphafold 2, and six key amino acids (G39, G69, A211, P288, R425 and C427) were identified. The R425A mutation significantly decreased the catalysis of (R)-N-methylcoclaurine and coclaurine inactivation, which might play important role in the biosynthesis of alkaloids with new configurations.

RING finger E3 ligases play an important role in regulating plant growth and development by mediating substrate degradation. In this study, we identified TaGW2L, encoding a Grain width and weight2 (GW2)-like RING finger E3 ligase, as a novel positive regulator of heading date in wheat (Triticum aestivum L.). TaGW2L exhibited high amino acid sequence similarities with TaGW2 homoeologs, particularly in the conserved RING finger domain. Expression analysis indicated that TaGW2L was constitutively expressed in various wheat tissues. TaGW2L showed transactivation activity in yeast and could interact with the ubiquitin-conjugating enzymes E2s. An in vitro ubiquitination assay verified that TaGW2L possessed a similar E3 ligase activity to TaGW2. Overexpression of the TaGW2L-7A homoeolog in wheat led to a significantly earlier heading date under both natural conditions and long-day conditions. Transcriptome analysis revealed that multiple known genes positively regulating wheat heading were significantly upregulated in the TaGW2L-7A-overexpression plants compared with the wild-type control. Together, our findings shed light on the role of TaGW2L in wheat heading date and provide potential applications of TaGW2L for the adaptation improvement of crops.

Plant architecture is a crucial influencing factor of wheat yield and adaptation. In this study, we cloned and characterized TaSPL14, a homologous gene of the rice ideal plant architecture gene OsSPL14 in wheat. TaSPL14 homoeologs (TaSPL14-7A, TaSPL14-7B and TaSPL14-7D) exhibited similar expression patterns, and they were all preferentially expressed in stems at the elongation stage and in young spikes. Moreover, the expression level of TaSPL14-7A was higher than that of TaSPL14-7B and TaSPL14-7D. Overexpression of TaSPL14-7A in wheat resulted in significant changes in plant architecture and yield traits, including decreased tiller number and increased kernel size and weight. Three TaSPL14-7A haplotypes were identified in Chinese wheat core collection, and haplotype-based association analysis showed that TaSPL14-7A-Hap1/2 were significantly correlated with fewer tillers, larger kernels and higher kernel weights in modern cultivars. The haplotype effect resulted from a difference in TaSPL14-7A expression levels among genotypes, with TaSPL14-7A-Hap1/2 leading to higher expression levels than TaSPL14-7A-Hap3. As favorable haplotypes, TaSPL14-7A-Hap1/2 underwent positive selection during global wheat breeding over the last century. Together, the findings of our study provide insight into the function and genetic effects of TaSPL14 and provide a useful molecular marker for wheat breeding.

Improving far-red light utilization could be an approach to increasing crop production under suboptimal conditions. In land plants, only a small part of far-red light can be used for photosynthesis, which is captured by the antenna proteins LHCAs of photosystem I (PSI) through the chlorophyll (Chl) pair a603 and a609. However, it is unknown how the energy level of Chls a603–a609 is fine-tuned by the local protein environment in vivo. In this study, we investigated how changing the amino acid ligand for Chl a603 in LHCA4, the most red-shifted LHCA in Arabidopsis thaliana, or one amino acid near Chl a609, affected the energy level of the resulting PSI-LHCI complexes in situ and in vitro. Substitutions of the Chl a603 ligand N99 caused a blue shift in fluorescence emission, whereas the E146Q substitution near Chl a609 expanded the emission range to the red. Purified PSI-LHCI complexes with N99 substitutions exhibited the same fluorescence emission maxima as their respective transgenic lines, while the extent of red shift in purified PSI-LHCI with the E146Q substitution was weaker than in the corresponding transgenic lines. We propose that substituting amino acids surrounding red Chls can tune their energy level higher or lower in vivo, while shifting the absorption spectrum more to the red could prove more difficult than shifting to the blue end of the spectrum. Here, we report the first in vivo exploration of changing the local protein environment on the energy level of the red Chls, providing new clues for engineering red/blue-shifted crops.

Developing effective and durable host plant resistance is crucial for controlling powdery mildew, a devastating disease caused by Blumeria graminis f. sp. tritici (Bgt). In the present study, we dissected the genetic basis of the adult plant resistance to powdery mildew using a recombinant inbred line (RIL) composed of 176 F9 RILs population derived from a cross between PuBing 3228 (P3228) and susceptible cultivar Gao 8901. P3228 exhibits stable adult-plant resistance to powdery mildew in the field over consecutive years. We identified two QTLs on chromosomes 7DS (QPm.cas-7D) and 1AL (QPm.cas-1A) contributed by P3228, and one QTL on 3DS (QPm.cas-3D) contributed by Gao 8901, which could explain 65.44%, 3.45%, and 2.18% of the phenotypic variances, respectively. By analyzing the annotated genes in the 1.168 Mb physical interval of the major QTL QPm.cas-7D, we locked a previously cloned adult-plant resistance gene Pm38 that was most probably the candidate gene of QPm.cas-7D. Sequence alignment analysis revealed that the candidate gene of QPm.cas-7D in P3228 was identical to the reported Pm38 sequence. Two haplotypes QPm-7D-R and QPm-7D-S were identified in the whole Pm38 genomic regions between P3228 and Gao 8901. To apply QPm.cas-7D in wheat breeding, we developed a kompetitive allele-specific PCR (KASP) marker Kasp5249 that is closely linked with these haplotypes. It is worth mentioning that the QPm-7D-R haplotype significantly decreased TKW and underwent negative selection for higher yields in China wheat breeding. In this study, we identified a major QTL QPm.cas-7D and revealed the relationship between its resistance and yield, which could be beneficial for further applications in wheat disease resistance and high-yield breeding.

Sacred lotus (Nelumbo nucifera) is a large economic crop, which is also cultivated as a horticultural crop. This study performed a systematic qualitative and quantitative determination of five anthocyanins and 18 non-anthocyanin flavonoids from the petals of 207 lotus cultivars. Among the compounds identified in this study, quercetin 3-O-pentose-glucuronide, quercetin 7-O-glucoside, laricitrin 3-O-hexose, and laricitrin 3-O-glucuronide were discovered for the first time in sacred lotus. The relationships between these pigments and petals colors were also evaluated. A decrease in the total content of anthocyanins and increase in the content of myricetin 3-O-glucuronide resulted in a lighter flower color. Furthermore, petals were yellow when the content of quercetin 3-O-neohesperidoside and myricetin 3-O-glucuronide were increased, whereas petals were red when the total anthocyanin content was high and the quercetin 3-O-sambubioside content was low. These investigations contribute to the understanding of mechanisms that underlie the development of flower color and provide a solid theoretical basis for the further study of sacred lotus.

NAC proteins constitute one of the largest plant-specific transcription factor (TF) families and play significant roles in plant growth and development. In the present study, three TaNAC020 homoeologous genes located on chromosomes 7A, 7B, and 7D were isolated from wheat (Triticum aestivum L.). TaNAC020s were predominantly expressed in developing grains. The developed transgenic rice lines for TaNAC020-B showed higher starch density and lower amylose contents than those of the wild type (WT). Sequence polymorphism studies showed seven and eight SNPs in TaNAC020-A/B, making three and two haplotypes, respectively. No sequence polymorphism was identified in TaNAC020-D. Association analysis revealed that HAP-2 of TaNAC020-A and TaNAC020-B was the favored haplotype for higher thousand kernel weight and length. Geographic distribution and allelic frequency showed that our favored haplotype experienced strong selection in China, and likewise, diversity increased in TaNAC020s during wheat polyploidization. The results obtained in this study demonstrate that TaNAC020s positively influence starch synthesis and accumulation and are one of the key regulators of the kernel (seed) size and kernel number and have the potential for utilization in wheat breeding to improve grain yield. Molecular markers developed in this study stand instrumental in marker-assisted selection for genetic improvement and germplasm enhancement in wheat.

Under climate change, drought is one of the most limiting factors that influences wheat (Triticum aestivum L.) production. Exploring stress-related genes is vital for wheat breeding. To identify genes related to the drought tolerance response, two common wheat cultivars, Zhengmai 366 (ZM366) and Chuanmai 42 (CM42), were selected based on their obvious difference in root length under 15% PEG-6000 treatment. The root length of the ZM366 cultivar was significantly longer than that of CM42. Stress-related genes were identified by RNA-seq in samples treated with 15% PEG-6000 for 7 days. In total, 11,083 differentially expressed genes (DEGs) and numerous single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were identified. GO enrichment analysis revealed that the upregulated genes were mainly related to the response to water, acidic chemicals, oxygen-containing compounds, inorganic substances, and abiotic stimuli. Among the DEGs, the expression levels of 16 genes in ZM366 were higher than those in CM42 after the 15% PEG-6000 treatment based on RT-qPCR. Furthermore, EMS-induced mutants in Kronos (T. turgidum L.) of 4 representative DEGs possessed longer roots than the WT after the 15% PEG-6000 treatment. Altogether, the drought stress genes identified in this study represent useful gene resources for wheat breeding.

Common wheat (Triticum aestivum L.) is one of the most important crops because it provides about 20% of the total calories for humans. T. aestivum is an excellent modern species for studying concerted evolution of sub-genomes in polyploid species, because of its large chromosome size and three well-known genome donors. Establishment of common wheat genome reference sequence and development of high-density SNP chips provide an excellent foundation to answer questions of wheat evolution and breeding at the genomic level. By genotyping more than 600 accessions of common wheat and their diploid and tetraploid ancestors using a Wheat660K SNP array, we found dramatic genome changes due to tetraploidization and hexaploidization, in contrast to weaker influences of domestication and breeding on them. Further, since common wheat was introduced in China in 1500 BCE, Chinese landraces formed two subgroups (T. aestivum-L1 and T. aestivum-L2) with considerably diverse geographic distributions and agronomic traits. T. aestivum-L2, mainly distributed in central and east China is found to have more but smaller oval grains with early maturity characteristics. We found that variation and selection in intergenic regions of the A and B sub-genomes dominated this differentiation, in which chromosomes 7A and 3B took the leading roles due to the existence of putative genes related to defense responses and environmental adaption in the highly differentiated regions. Large haplotype blocks were detected on 3B (232.6–398.3 Mb) and 7A (211.7–272.9 Mb) in the landraces, forming two distinct haplotypes, respectively. We discovered that artificial crosses in breeding promoted recombination in the whole genome, however, this recombination and differentiation was highly asymmetric among the three sub-genomes in homoeologous regions. In addition, we found that the wide use of European and northern American cultivars in breeding at early era, led dramatic changes in Chinese wheat genome, whereas, the recent breeding functioned to optimize it. This study will provide the insight for reconsideration of wheat evolution and breeding, and a new strategy for parent selection in breeding.

Abstract Background The overall genetic distribution and divergence of cloned genes among bread wheat varieties that have occurred during the breeding process over the past few decades in Ningxia Province, China, are poorly understood. Here, we report the genetic diversities of 44 important genes related to grain yield, quality, adaptation and resistance in 121 Ningxia and 86 introduced wheat cultivars and advanced lines. Results The population structure indicated characteristics of genetic components of Ningxia wheat, including landraces of particular genetic resources, introduced varieties with rich genetic diversities and modern cultivars in different periods. Analysis of allele frequencies showed that the dwarfing alleles Rht-B1b at Rht-B1 and Rht-D1b at Rht-D1, 1BL/1RS translocation, Hap-1 at GW2-6B and Hap-H at Sus2-2B are very frequently present in modern Ningxia cultivars and in introduced varieties from other regions but absent in landraces. This indicates that the introduced wheat germplasm with numerous beneficial genes is vital for broadening the genetic diversity of Ningxia wheat varieties. Large population differentiation between modern cultivars and landraces has occurred in adaptation genes. Founder parents carry excellent allele combinations of important genes, with a higher number of favorable alleles than modern cultivars. Gene flow analysis showed that six founder parents have greatly contributed to breeding improvement in Ningxia Province, particularly Zhou 8425B, for yield-related genes. Conclusions Varieties introduced from other regions with rich genetic diversity and landraces with well-adapted genetic resources have been applied to improve modern cultivars. Founder parents, particularly Zhou 8425B, for yield-related genes have contributed greatly to wheat breeding improvement in Ningxia Province. These findings will greatly benefit bread wheat breeding in Ningxia Province as well as other areas with similar ecological environments.

Abstract Background China has diverse wheat varieties that adapt to very different environments divided into ten agro-ecological zones. A better understanding of genomic differences and patterns of selection among agro-ecological zones could provide useful information in selection of specific adaptive traits in breeding. Results We genotyped 438 wheat accessions from ten zones with kompetitive allele specific PCR (KASP) markers specific to 47 cloned genes for grain yield, quality, adaptation and stress resistance. Phylogenetic trees and principle component analysis revealed clear differences in winter and spring growth habits. Nucleotide diversity (π) and π ratio (π CL/π MCC) suggested that genetic diversity had increased during breeding, and that Chinese landraces (CL) from Zones I-V contributed little to modern Chinese cultivars (MCC). π ratio and Fst identified 24 KASP markers with 53 strong selection signals specific to Zones I (9 signals), II (12), III (5), IV (5), V (6), and VI (6). Genes with clear genetic differentiation and strong response to selection in at least three zones were leaf rust resistance gene Lr34 (I, II, III and IV), photoperiod sensitivity gene Ppd-D1 (I, II, III, IV and V), vernalization gene Vrn-B1 (V, VII, VIII and X), quality-related gene Glu-B1 (I, II and III) and yield-related genes Sus1-7B (I, II, III, IV and IX), Sus2-2A (I, II, III., IV and VI) and GW2-6B (II, V and VI). Conclusions This study examined selection of multiple genes in each zone, traced the distribution of important genetic variations and provided useful information for ecological genomics and enlightening future breeding goals for different agro-ecological zones.

The “edible rhizome” variant of Nelumbo nucifera with various cultivars has a long history of use as a food in East Asia. In this study, 48 target metabolites were untargeted and identified in 212 rhizome cultivars (tropical and temperate types) using ultraperformance liquid chromatography-electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry; among these, 32 compounds were newly reported in the rhizome. Combined with the browning phenotype of 212 lotus rhizomes, (epi) catechin, norarmepavine, and N-feruloyl-3-methoxytyramine were used as predominant chemical markers to separate different degrees of lotus rhizome browning. p-Coumaroyltyramine and N-trans-feruloyltyramine were selected as predominant chemical markers to investigate the differential expression between tropical and temperate lotus using principal component analysis and orthogonal partial least squares discriminant analysis. Shared and unique structure plots were used to compare the outcomes of the ecotype and browning OPLS model, showing that variation in tropical lotus rhizome browning is not obvious; this will be of great importance for genetic improvement by providing a hereditary basis.

Grain Weight 8 (GW8) in rice is a SQUAMOSA Promoter-Binding Protein-Like (SPL) family transcription factor with multiple biological functions. In this study, three GW8 homoeologs were cloned from homoeologous group 7 chromosomes of wheat. Subcellular localization and trans-activation activity assays suggested that TaGW8 is a transcriptional activator. TaGW8 genes were preferentially expressed in young spikes and developing grains. Ectopic expressions of TaGW8 in Arabidopsis and rice resulted in enhanced vegetative growth, earlier flowering and larger seeds. TaGW8-7A was the most highly variable of the three homoeologs with four haplotypes (Hap-1/2/3/4). TaGW8-7B had two haplotypes (Hap-L/H). TaGW8-7A-Hap-2 was associated with high thousand-grain weight (TGW) and large kernel length and showed higher transcriptional levels and binding activity than the other haplotypes. The high frequency of TaGW8-7A-Hap-2 in Chinese wheat populations suggested that it had been extensively selected in breeding. This haplotype showed a high potential for exploitation in global wheat breeding because its frequency was low in exotic germplasm. TaGW8-7B-Hap-H produced abundant transcripts and was associated with early heading and maturity, less tiller number and high TGW. This haplotype might be suitable for multiple cropping systems due to short wheat season. In this study we identified sub-functionalization among TaGW8 homoeologs and obtained functional molecular markers that can be used in breeding for high grain yield. Keywords: Grain yield, Haplotype association, Marker assisted breeding, Thousand grain weight, Triticum aestivum

GATA transcription factors (TFs) are type IV zinc-finger proteins that have roles in plant development and growth. The 27 GATA TFs identified in the Brachypodium distachyon genome in this study were unevenly distributed across all five chromosomes and classified into four subgroups. Phylogenesis-related GATAs shared similar gene structures and conserved motifs. Expression profiles showed that all BdGATA genes were expressed in leaves and most were induced by PEG treatment. BdGATA13 was predominantly expressed in leaf tissue and phylogenetically close to OsSNFL1, AtGNC, and AtGNL. Its protein was detected in the nucleus by subcellular localization analysis. Overexpression of BdGATA13 in transgenic Arabidopsis resulted in darker green leaves, later flowering, and more importantly, enhanced drought tolerance compared to the wild type. BdGATA13 also promoted primary root development under GA treatment. These results lay a foundation for better understanding the function of GATA genes in B. distachyon and other plants.

Abscisic acid (ABA), one of phytohormones, plays an important regulatory role in plant growth and development. ABA receptor PYL4 (pyrabactin resistance 1-like 4) was previously detected to be involved in plant response to a variety of stresses. TaPYL4 overexpression could enhance wheat (Triticum aestivum) drought resistance. In order to further investigate TaPYL4’s role in regulating development of other major agronomic traits in wheat, genes of TaPYL4-2A, TaPYL4-2B, and TaPYL4-2D were cloned from wheat, respectively. Polymorphism analysis on TaPYL4 sequences revealed that encoding regions of the three genes were highly conserved, without any SNP (single nucleotide polymorphism) presence. However, nine SNPs and four SNPs were identified in the promoter regions of TaPYL4-2A and TaPYL4-2B, respectively. Functional molecular markers were developed based on these polymorphisms, which were then used to scan a natural population of 323 common wheat accessions for correlation analysis between genotype and the target phenotypic traits. Both TaPYL4-2A and TaPYL4-2B markers were significantly correlated with plant growth-related traits under multiple environments (well-watered, drought and heat stress treatments). The additive effects of TaPYL4-2A and TaPYL4-2B were verified by the combinational haplotype (Hap-AB1∼Hap-AB4) effects determined from field data. Cis-acting elements were analyzed in the promoters of TaPYL4-2A and TaPYL4-2B, showing that a TGA-element bound by ARFs (auxin response factors) existed only in Hap-2A-1 of TaPYL4-2A. Gene expression assays indicated that TaPYL4-2A was constitutively expressed in various tissues, with higher expression in Hap-2A-1 genotypes than in Hap-2A-2 materials. Notably, TaARF4 could act as TaPYL4-2A transcription activator in Hap-2A-1 materials, but not in Hap-2A-2 genotypes. Analysis of geographic distribution and temporal frequency of haplotypes indicated that Hap-AB1 was positively selected in wheat breeding in China. Therefore, TaPYL4-2A and TaPYL4-2B could be a valuable target gene in wheat genetic improvement to develop the ideal plant architecture.

Abstract Background Premature senescence of flag leaf severely affects wheat yield and quality. Chlorophyll (Chl) degradation is the most obvious symptom during leaf senescence and catalyzed by a series of enzymes. Pheophytin pheophorbide hydrolase (Pheophytinase, PPH) gene encodes a Chl degradation hydrolase. Results In this study, the coding, genomic and promoter sequences of wheat TaPPH-A gene were cloned. The corresponding lengths were 1467 bp, 4479 bp and 3666 bp, respectively. Sequence structure analysis showed that TaPPH-A contained five exons and four introns. After the multiple sequences alignment of TaPPH-A genome from 36 accessions in a wheat diversity panel, four SNPs and one 2-bp InDel were observed, which formed two haplotypes, TaPPH-7A-1 and TaPPH-7A-2. Based on the SNP at 1299 bp (A/G), a molecular marker TaPPH-7A-dCAPS was developed to distinguish allelic variation (A/G). Using the molecular markers, 13 SSR, and 116 SNP markers, a linkage map of chromosome 7A were integrated. TaPPH-A was mapped on the chromosome region flanked by Xwmc9 (0.94 cM) and AX-95634545 (1.04 cM) on 7A in a DH population. Association analysis between TaPPH-7A allelic variation and agronomic traits found that TaPPH-7A was associated with TGW in 11 of 12 environments and Chl content at grain-filling stage under drought stress using Population 1 consisted of 323 accessions. The accessions possessed TaPPH-7A-1 (A) had higher TGW and Chl content than those possessed TaPPH-7A-2 (G), thus TaPPH-7A-1 (A) was a favorable allelic variation. By analyzing the frequency of favorable allelic variation TaPPH-7A-1 (A) in Population 2 with 157 landraces and Population 3 with 348 modern cultivars, we found it increased from pre-1950 (0) to 1960s (54.5%), then maintained a relatively stable level about 56% from 1960s to 1990s. Conclusion These results suggested the favorable allelic variation TaPPH-7A-1 (A) should be valuable in enhancing grain yield by improving the source (chlorophyll content) and sink (the developing grain) simultaneously. Furthermore, the newly developed molecular marker TaPPH-7A-dCAPS could be integrated into a breeding kit of screening high TGW wheat for marker-assisted selection.

Abstract Background Wheat breeding over the last 100 years has increased productivity by adapting genotypes to local conditions, but the genomic changes and selection signals that caused phenotypic change during breeding are essentially unknown. Studying and understanding human selection of multiple important genes controlling key phenotypic traits will promote wheat molecular breeding. Results A total of 1152 diverse global wheat materials were genotyped based on KASP markers from 47 genes controlling grain yield, grain quality, adaptation, and stress resistance. Significant phenotypic variations between landraces and modern cultivars were found in 11 adaptive and yield-related traits. Thirty-six improvement-selective favorable alleles, including 22 positive prolonged and 14 negative selection alleles, were identified through comparing frequency spectra. Sus1-7A-Hap-H, Sus1-7B-Hap-T, Sus2-2A-Hap-A, TGW6-A1a, Cwi-4A-Hap-C, vrn-A1, PHS1-PHS+ and Lr34+ were subjected to strong selection, and overwhelmingly strong selection had occurred before improvement selection at Psy-A1b, Psy-B1a or b, Psy-D1a and Cwi-5D-Hap-C. However, Rht-B1b, Rht-D1b and 1BL.1RS were rare or absent in Chinese landraces but present in modern Chinese cultivars and introduced accessions. Importantly, Lr68+, Fhb1+, Wx-B1b and Yr15+, currently existing at a low frequency, should be regarded as further major improvement targets in global wheat breeding. Gene flow analysis showed that introduced cultivars especially from the former USSR and Italy contributed to enriched genetic variation in modern Chinese cultivars. Conclusions This work objectively reports human selection on favorable alleles of multiple crucial genes in Asia, Europe, North America and CIMMYT, and traces the distribution of important genes in global wheat for molecular breeding.

Diversity surveys of germplasm are important for gaining insight into the genomic basis for crop improvement; especially InDels, which are poorly understood in hexaploid common wheat. Here, we describe a map of 89,923 InDels from exome sequencing of 262 accessions of a Chinese wheat mini-core collection. Population structure analysis, principal component analysis and selective sweep analysis between landraces and cultivars were performed. Further genome-wide association study (GWAS) identified five QTL (Quantitative Trait Loci) that were associated with spike length, two of them, on chromosomes 2B and 6A, were detected in 10 phenotypic data sets. Assisted with RNA-seq data, we identified 14 and 21 genes, respectively that expressed in spike and rachis within the two QTL regions that can be further investigated for candidate genes discovery. Moreover, InDels were found to be associated with awn length on chromosomes 5A, 6B and 4A, which overlapped with previously reported genetic loci B1 (Tipped 1), B2 (Tipped 2) and Hd (Hooded). One of the genes TaAGL6 that was previously shown to affect floral organ development was found at the B2 locus to affect awn length development. Our study shows that trait-associated InDels may contribute to wheat improvement and may be valuable molecular markers for future wheat breeding

Kernel weight is a key determinant of yield in wheat (Triticum aestivum L.). Starch consists of amylose and amylopectin and is the major constituent of mature grain. Therefore, starch metabolism in the endosperm during grain filling can influence kernel weight. In this study, we sequenced 87 genes involved in starch metabolism from 300 wheat accessions and detected 8,141 polymorphic sites. We also characterized yield-related traits across different years in these accessions. Although the starch contents fluctuated, thousand kernel weight (TKW) showed little variation. Polymorphisms in six genes were significantly associated with TKW. These genes were located on chromosomes 2A, 2B, 4A, and 7A; none were associated with starch content or amylose content. Variations of 15 genes on chromosomes 1A and 7A formed haplotype blocks in 26 accessions. Notably, accessions with higher TKWs had more of the favorable haplotypes. We thus conclude that these haplotypes contribute additive effects to TKW.

Abstract Background Yield improvement is an ever-important objective of wheat breeding. Studying and understanding the phenotypes and genotypes of yield-related traits has potential for genetic improvement of crops. Results The genotypes of 215 wheat cultivars including 11 founder parents and 106 derivatives were analyzed by the 9 K wheat SNP iSelect assay. A total of 4138 polymorphic single nucleotide polymorphism (SNP) loci were detected on 21 chromosomes, of which 3792 were mapped to single chromosome locations. All genotypes were phenotyped for six yield-related traits including plant height (PH), spike length (SL), spikelet number per spike (SNPS), kernel number per spike (KNPS), kernel weight per spike (KWPS), and thousand kernel weight (TKW) in six irrigated environments. Genome-wide association analysis detected 117 significant associations of 76 SNPs on 15 chromosomes with phenotypic explanation rates (R 2 ) ranging from 2.03 to 12.76%. In comparing allelic variation between founder parents and their derivatives (106) and other cultivars (98) using the 76 associated SNPs, we found that the region 116.0–133.2 cM on chromosome 5A in founder parents and derivatives carried alleles positively influencing kernel weight per spike (KWPS), rarely found in other cultivars. Conclusion The identified favorable alleles could mark important chromosome regions in derivatives that were inherited from founder parents. Our results unravel the genetic of yield in founder genotypes, and provide tools for marker-assisted selection for yield improvement.

Kernel number per spike (KNPS) in wheat is a key factor that limits yield improvement. In this study, we genotyped a set of 264 cultivars, and a RIL population derived from the cross Yangmai 13/C615 using the 90 K wheat iSelect SNP array. We detected 62 significantly associated signals for KNPS at 47 single nucleotide polymorphism (SNP) loci through genome-wide association analysis of data obtained from multiple environments. These loci were on 19 chromosomes, and the phenotypic variation attributable to each one ranged from 1.53 to 39.52%. Twelve (25.53%) of the loci were also significantly associated with KNPS in the RIL population grown in multiple environments. For example, BS00022896_51-2ATT, BobWhite_c10539_201-2DAA, Excalibur_c73633_120-3BGG, and Kukri_c35508_426-7DTT were significantly associated with KNPS in all environments. Our findings demonstrate the effective integration of association mapping and linkage analysis for KNPS, and underpin KNPS as a target trait for marker-assisted selection and genetic fine mapping.

The group C-bZIP transcription factors (TFs) are involved in diverse biological processes, such as the regulation of seed storage protein (SSP) production and the responses to pathogen challenge and abiotic stress. However, our knowledge of the abiotic functions of group C-bZIP genes in wheat remains limited. Here, we present the function of a novel TabZIP14-B gene in wheat. This gene belongs to the group C-bZIP TFs and contains six exons and five introns; three haplotypes were identified among accessions of tetraploid and hexaploid wheat. A subcellular localization analysis indicated that TabZIP14-B was targeted to the nucleus of tobacco epidermal cells. A transactivation assay demonstrated that TabZIP14-B showed transcriptional activation ability and was capable of binding the abscisic acid (ABA) responsive element (ABRE) in yeast. RT-qPCR revealed that TabZIP14-B was expressed in the roots, stems, leaves, and young spikes and was up-regulated by exogenous ABA, salt, low-temperature, and polyethylene glycol (PEG) stress treatments. Furthermore, Arabidopsis plants overexpressing TabZIP14-B exhibited enhanced tolerance to salt, freezing stresses and ABA sensitivity. Overexpression of TabZIP14-B resulted in increased expression of the AtRD29A, AtCOR47, AtRD20, AtGSTF6, and AtRAB18 genes and changes in several physiological characteristics. These results suggest that TabZIP14-B could function as a positive regulator in mediating the abiotic stress response.

AP2/EREBPs play significant roles in plant growth and development. The novel, pleiotropic TaPARG (PLANT ARCHITECTURE-RELATED GENE), a member of the AP2/EREBP transcription factor gene family, and its flanking sequences were isolated in wheat (Triticum aestivum L.). Two TaPARG genes were identified and named as TaPARG-2A and TaPARG-2D. Their amino acid sequences were highly similar especially in the functional domains. TaPARG-2A on chromosome 2A was flanked by markers Xwmc63 and Xgwm372. TaPARG-2D was mapped to chromosome 2D. Subcellular localization revealed that TaPARG-2D was localized in the nucleus. The results of tissue expression pattern, overexpression in rice, association analysis and distinct population verification jointly revealed that TaPARG functions during the entire growth cycle of wheat. Its functions include regulation of plant architecture-related and yield-related traits. Association analysis, geographic distribution and allelic frequencies suggested that favored haplotypes Hap-2A-2 and Hap-2A-3 were selected in Chinese wheat breeding programs. Both favored haplotypes might be caused by a single amino acid substitution (His/Tyr). These results suggest that TaPARG is a regulatory factor in plant growth and development, and that the favored alleles might be useful for improving plant architecture and grain yield of wheat.

Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development.

Common wheat is one of the most important crops in China, which is the largest producer in the world. A set of 230 cultivars was used to identify yield-related loci by association mapping. This set was tested for seven yield-related traits, viz. plant height (PH), spike length (SL), spikelet number per spike (SNPS), kernel number per spike (KNPS), thousand-kernel weight (TKW), kernel weight per spike (KWPS), and sterile spikelet number (SSN) per plant in four environments. A total of 106 simple sequence repeat (SSR) markers distributed on all 21 chromosomes were used to screen the set. Twenty-one and 19 of them were associated with KNPS and TKW, respectively. Association mapping detected 73 significant associations across 50 SSRs, and the phenotypic variation explained (R2) by the associations ranged from 1.54 to 23.93%. The associated loci were distributed on all chromosomes except 4A, 7A, and 7D. Significant and potentially new alleles were present on 8 chromosomes, namely 1A, 1D, 2A, 2D, 3D, 4B, 5B, and 6B. Further analysis showed that genetic effects of associated loci were greatly influenced by association panels, and the R2 of crucial loci were lower in modern cultivars than in the mini core collection, probably caused by strong selection in wheat breeding. In order to confirm the results of association analysis, yield-related favorable alleles Xgwm135-1A138, Xgwm337-1D186, Xgwm102-2D144, and Xgwm132-6B128 were evaluated in a double haploid (DH) population derived from Hanxuan10 xLumai14.These favorable alleles that were validated in various populations might be valuable in breeding for high-yield.

To better understand the transcriptional regulation of high molecular weight glutenin subunit (HMW-GS) expression, we isolated four Glu-1Bx promoters from six wheat cultivars exhibiting diverse protein expression levels. The activities of the diverse Glu-1Bx promoters were tested and compared with β-glucuronidase (GUS) reporter fusions. Although all the full-length Glu-1Bx promoters showed endosperm-specific activities, the strongest GUS activity was observed with the 1Bx7OE promoter in both transient expression assays and stable transgenic rice lines. A 43 bp insertion in the 1Bx7OE promoter, which is absent in the 1Bx7 promoter, led to enhanced expression. Analysis of promoter deletion constructs confirmed that a 185 bp MITE (miniature inverted-repeat transposable element) in the 1Bx14 promoter had a weak positive effect on Glu-1Bx expression, and a 54 bp deletion in the 1Bx13 promoter reduced endosperm-specific activity. To investigate the effect of the 43 bp insertion in the 1Bx7OE promoter, a functional marker was developed to screen 505 Chinese varieties and 160 European varieties, and only 1Bx7-type varieties harboring the 43 bp insertion in their promoters showed similar overexpression patterns. Hence, the 1Bx7OE promoter should be important tool in crop genetic engineering as well as in molecular assisted breeding.

Chinese wheat mini core collection (262 accessions) was genotyped at 531 microsatellite loci representing a mean marker density of 5.1 cM. One-thousand-kernel weights (TKW) of lines were measured in five trials (three environments in four growing seasons). Structure analysis based on 42 unlinked SSR loci indicated that the materials formed two sub-populations, viz., landraces and modern varieties. A large difference in TKW (7.08 g, P

A previous study provided an in-depth understanding of molecular population genetics of European and Asian wheat gene pools using a sequenced 3.1-Mb contig (ctg954) on chromosome 3BS. This region is believed to carry the Fhb1 gene for response to Fusarium head blight. In this study, 266 wheat accessions were evaluated in three environments for Type II FHB response based on the single floret inoculation method. Hierarchical clustering (UPGMA) based on a Manhattan dissimilarity matrix divided the accessions into eight groups according to five FHB-related traits which have a high correlation between them; Group VIII comprised six accessions with FHB response levels similar to variety Sumai 3. Based on the compressed mixed linear model (MLM), association analysis between five FHB-related traits and 42 molecular markers along the 3.1-Mb region revealed 12 significant association signals at a threshold of P0.1 and P0.05 within each HapB at r(2)>0.1 and P

Two hundred and fifty bread wheat lines, mainly Chinese mini core accessions, were assayed for polymorphism and linkage disequilibrium (LD) based on 512 whole-genome microsatellite loci representing a mean marker density of 5.1 cM. A total of 6,724 alleles ranging from 1 to 49 per locus were identified in all collections. The mean PIC value was 0.650, ranging from 0 to 0.965. Population structure and principal coordinate analysis revealed that landraces and modern varieties were two relatively independent genetic sub-groups. Landraces had a higher allelic diversity than modern varieties with respect to both genomes and chromosomes in terms of total number of alleles and allelic richness. 3,833 (57.0%) and 2,788 (41.5%) rare alleles with frequencies of 0.05 (P