Identification of structural variations related to drought tolerance in wheat (Triticum aestivum L.).

KEY MESSAGE: Structural variations are common in plant genomes, affecting meiotic recombination and distorted segregation in wheat. And presence/absence variations can significantly affect drought tolerance in wheat. Drought is a major abiotic stress limiting wheat production. Common wheat has a complex genome with three sub-genomes, which host large numbers of structural variations (SVs). SVs play critical roles in understanding the genetic contributions of plant domestication and phenotypic plasticity, but little is known about their genomic characteristics and their effects on drought tolerance. In the present study, high-resolution karyotypes of 180 doubled haploids (DHs) were developed. Signal polymorphisms between the parents involved with 8 presence-absence variations (PAVs) of tandem repeats (TR) distributed on the 7 (2A, 4A, 5A, 7A, 3B, 7B, and 2D) of 21 chromosomes. Among them, PAV on chromosome 2D showed distorted segregation, others transmit normal conforming to a 1:1 segregation ration in the population; and a PAVs recombination occurred on chromosome 2A. Association analysis of PAV and phenotypic traits under different water regimes, we found PAVs on chromosomes 4A, 5A, and 7B showed negative effect on grain length (GL) and grain width (GW); PAV.7A had opposite effect on grain thickness (GT) and spike length (SL), with the effect on traits differing under different water regimes. PAVs on linkage group 2A, 4A, 7A, 2D, and 7B associated with the drought tolerance coefficients (DTCs), and significant negative effect on drought resistance values (D values) were detected in PAV.7B. Additionally, quantitative trait loci (QTL) associated with phenotypic traits using the 90 K SNP array showed QTL for DTCs and grain-related traits in chromosomes 4A, and 5A, 3B were co-localized in differential regions of PAVs. These PAVs can cause the differentiation of the target region of SNP and could be used for genetic improvement of agronomic traits under drought stress via marker-assisted selection (MAS) breeding.

A simple and robust HPLC method to analyze lutein in wheat.

A method for the determination of lutein, the main component of carotenoid in wheat by high performance liquid chromatography (HPLC) was established. The samples were extracted with acetone and methanol (acetone : methanol 7 : 3, v / v, 0.1 % BHT w / v), and fully dissolved in methanol / acetonitrile / n-hexane (7 : 2 : 1, v / v / v) mixed solution. Poroshell 120 EC-C18 column (4.6 mm× 150 mm, 4µm) was used as the separation column, acetonitrile,methanol and n-hexane solution were used as mobile phase with gradient elution, flow rate was 1.0 mL·min-1, column temperature was 25 ℃, injection volume was 50 µL, and detection wave length was 450 nm. The mass concentration of lutein had a good linear relationships with the chromatographic peak area in the range of 0.5-5.0 µg·mL-1, the correlation coefficients were all not less than 0.999 8, and the detection limits was 12 ng·mL-1. The recoveries rate of standard addition in sampling were 95.50%-97.33%, and the relative standard deviations of determination results were 1.20%-1.66%(n=5). The method is sensitive, precise, rapid, accurate and efficient. The advantages of this study can be summarized in the next bullet points:•Can be used for the determination of lutein in wheat.•The detection time is short.•Limit of detection (LOD) and limit of quantification (LOQ) for lutein was 12 ng·mL-1 and 42 ng·mL-1.

Folate (vitamin B9) content analysis in bread wheat (Triticum aestivum L.).

Vitamin B9, particularly folic acid, is an essential molecule for human health. Wheat flour is one of the major sources of calorie intake by humans. The selection of folate-rich genotypes in wheat breeding can enhance the natural folate value in the daily diet. This study used a precise, high-performance liquid chromatography (HPLC) assay to analyze folate content in a 262-accession Chinese wheat mini-core collection (MCC) grown under three environments. Four folate derivatives in grains including tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-CH3-THF), 5-formyltetrahydrofolate (5-CHO-THF), and 5,10-methenyltetrahydrofolate (5,10-CH+THF) were considered. An association analysis of water regimes, accession types, released years, geographical origin, and agronomic traits with folate content was conducted for the first time. There was a large amount of variation in folate content in the analyzed accessions, with genotype identified as the main influencing factor. Total folate content was significantly correlated with the content of the four MCC derivatives under the three environments. 5-CH3-THF and 5-CHO-THF were the most abundant among the four folate derivatives and were positively correlated with high folate content. The 12 accessions with the highest folate content showed an average of more than 80 µg/100 g. The analysis demonstrated that this Chinese wheat had not undergone extensive selection for folate content during breeding, which is unrelated to the geographical origin, accession types, winter/spring types, and grain colors of wheat. The content of THF, 5-CH3-THF, and 5,10-CH+THF was significantly negatively correlated with grain width, grain thickness, and thousand kernel weight. A relatively weak negative relationship manifested between folate contents and flowering date, whereas no significant correlation with tiller number, grain number per spike, maturity date, height, and spike length was detected. The investigation benefits wheat breeders for folate enhancement.

Genome-wide association study reveals structural chromosome variations with phenotypic effects in wheat (Triticum aestivum L.).

Structural chromosome variations (SCVs) are large-scale genomic variations that can be detected by fluorescence in situ hybridization (FISH). SCVs have played important roles in the genome evolution of wheat (Triticum aestivum L.), but little is known about their genetic effects. In this study, a total of 543 wheat accessions from the Chinese wheat mini-core collection and the Shanxi Province wheat collection were used for chromosome analysis using oligonucleotide probe multiplex FISH. A total of 139 SCVs including translocations, pericentric inversions, presence/absence variations (PAVs), and copy number variations (CNVs) in heterochromatin were identified at 230 loci. The distribution frequency of SCVs varied between ecological regions and between landraces and modern cultivars. Structural analysis using SCVs as markers clearly divided the landraces and modern cultivars into different groups. There are very clear instances illustrating alien introgression and wide application of foreign germplasms improved the chromosome diversity of Chinese modern wheat cultivars. A genome-wide association study (GWAS) identified 29 SCVs associated with 12 phenotypic traits, and five (RT4AS•4AL-1DS/1DL•1DS-4AL, Mg2A-3, Mr3B-10, Mr7B-13, and Mr4A-7) of them were further validated using a doubled haploid population and advanced sib-lines, implying the potential value of these SCVs. Importantly, the number of favored SCVs that were associated with agronomic trait improvement was significantly higher in modern cultivars compared to landraces, indicating positive selection in wheat breeding. This study demonstrates the significant effects of SCVs during wheat breeding and provides an efficient method of mining favored SCVs in wheat and other crops.

Genome-wide association study of coleoptile length with Shanxi wheat.

In arid and semi-arid regions, coleoptile length is a vital agronomic trait for wheat breeding. The coleoptile length determines the maximum depth that seeds can be sown, and it is critical for establishment of the crop. Therefore, identifying loci associated with coleoptile length in wheat is essential. In the present study, 282 accessions from Shanxi Province representing wheat breeding for the Loess Plateau were grown under three experimental conditions to study coleoptile length. The results of phenotypic variation indicated that drought stress and light stress could lead to shortening of coleoptile length. Under drought stress the growth rate of environmentally sensitive cultivars decreased more than insensitive cultivars. The broad-sense heritability (H 2) of BLUP (best linear unbiased prediction) under various conditions showed G × E interaction for coleoptile length but was mainly influenced by heredity. Correlation analysis showed that correlation between plant height-related traits and coleoptile length was significant in modern cultivars whereas it was not significant in landraces. A total of 45 significant marker-trait associations (MTAs) for coleoptile length in the three conditions were identified using the 3VmrMLM (3 Variance-component multi-locus random-SNP-effect Mixed Linear Model) and MLM (mixed linear model). In total, nine stable genetic loci were identified via 3VmrMLM under the three conditions, explaining 2.94-7.79% of phenotypic variation. Five loci on chromosome 2B, 3A, 3B, and 5B have not been reported previously. Six loci had additive effects toward increasing coleoptile length, three of which are novel. Molecular markers for the loci with additive effects on coleoptile length can be used to breed cultivars with long coleoptiles.

Identification of genetic loci for flag-leaf-related traits in wheat (Triticum aestivum L.) and their effects on grain yield.

Flag-leaf-related traits including length (FLL), width (FLW), area (FLA), thickness (FLT), and volume (FLV) of flag leaves are the most important determinants of plant architecture and yield in wheat. Understanding the genetic basis of these traits could accelerate the breeding of high yield wheat varieties. In this study, we constructed a doubled haploid (DH) population and analyzed flag-leaf-related traits in five experimental locations/years using the wheat 90K single-nucleotide polymorphism array. It's worth noting that a novel method was used to measure FLT and FLV easily. Leaf thickness at two-thirds of the leaf length from tip to collar represented the average leaf thickness as measured with freehand sections and was used to calculate the leaf volume. In addition, flag-leaf-related traits showed positive correlations with yield related traits under two different water regimes. A total of 79 quantitative trait loci (QTL) controlling the five traits were detected among all chromosomes except 4D and 5A, explaining 3.09-14.52% of the phenotypic variation. Among them, 15 stable QTL were identified in more than three environments, including two major QTL for FLT, six for FLW, three for FLA, two for FLT and two for FLV. DH lines with positive alleles at both QTL regions had an average FLL (9.90%), FLW (32.87%), FLT (6.62%), FLA (18.47%), and FLV (20.87%) greater than lines with contrasting alleles. QFLT-2B, QFLV-2A, and QFLV-7D were co-located with yield-related traits. The 15 QTL were validated by tightly linked kompetitive allele specific PCR (KASP) markers in a recombinant inbred line (RIL) population derived from a different cross. QFLL-4A, QFLW-4B, QFLA-5D.1, QFLA-7A, QFLA-7D.1, QFLT-2B, QFLT-6A, QFLV-2A, and QFLV-7D are likely novel loci. These results provide a better understanding of the genetic basis underlying flag-leaf-related traits. Also, target regions for fine mapping and marker-assisted selection were identified and these will be valuable for breeding high yielding bread wheat.

Genetic dissection of lutein content in common wheat via association and linkage mapping.

KEY MESSAGE: Genetic architecture controlling grain lutein content of common wheat was investigated through an integration of genome-wide association study (GWAS) and linkage analysis. Putative candidate genes involved in carotenoid metabolism and regulation were identified, which provide a basis for gene cloning and development of nutrient-enriched wheat varieties through molecular breeding. Lutein, known as 'the eye vitamin', is an important component of wheat nutritional and end-use quality. However, the genetic manipulation of grain lutein content (LUC) in common wheat has not previously been well studied. Here, quantitative trait loci (QTL) associated with the LUC measured by high performance liquid chromatography (HPLC) were first identified by integrating a genome-wide association study (GWAS) and linkage mapping. A Chinese wheat mini-core collection (MCC) of 262 accessions and a doubled haploid (DH) population derived from Jinchun 7 and L1219 were genotyped using the 90K SNP array. A total of 124 significant marker-trait associations (MTAs) on all 21 wheat chromosomes except for 1A, 4D, and 5B that formed 58 QTL were detected. Among them, six stable QTL were identified on chromosomes 2AL, 2DS, 3BL, 3DL, 7AL, and 7BS. Meanwhile, three of the ten QTL identified in the DH population, QLuc.5A.1 and QLuc.5A.2 on chromosome 5AL and QLuc.6A.2 on 6AS, were stable and independently explained 5.58-10.86% of the phenotypic variation. The QLuc.6A.2 region colocalized with two MTAs identified by GWAS. Moreover, 71 carotenoid metabolism-related candidate genes were identified, and the allelic effects were analyzed in the MCC panel based on the 90K array. Results revealed that the genes CYP97A3 (Chr. 6B) and CCD1 (Chr. 5A) were significantly associated with LUC. Additionally, the gene PSY3 (QLuc.5A.1) and several candidate genes involved in the methylerythritol 4-phosphate (MEP) pathways colocalized with stable QTL regions. The present study provides potential targets for future functional gene exploration and molecular breeding in common wheat.

Identification of Genetic Loci Affecting Flag Leaf Chlorophyll in Wheat Grown under Different Water Regimes.

Chlorophyll content of the flag leaf is an important trait for drought resistance in wheat under drought stress. Understanding the regulatory mechanism of flag leaf chlorophyll content could accelerate breeding for drought resistance. In this study, we constructed a recombinant inbred line (RIL) population from a cross of drought-sensitive variety DH118 and drought-resistant variety Jinmai 919, and analyzed the chlorophyll contents of flag leaves in six experimental locations/years using the Wheat90K single-nucleotide polymorphism array. A total of 29 quantitative trait loci (QTLs) controlling flag leaf chlorophyll were detected with contributions to phenotypic variation ranging from 4.67 to 23.25%. Twelve QTLs were detected under irrigated conditions and 18 were detected under dryland (drought) conditions. Most of the QTLs detected under the different water regimes were different. Four major QTLs (Qchl.saw-3B.2, Qchl.saw-5A.2, Qchl.saw-5A.3, and Qchl.saw-5B.2) were detected in the RIL population. Qchl.saw-3B.2, possibly more suitable for marker-assisted selection of genotypes adapted to irrigated conditions, was validated by a tightly linked kompetitive allele specific PCR (KASP) marker in a doubled haploid population derived from a different cross. Qchl.saw-5A.3, a novel stably expressed QTL, was detected in the dryland environments and explained up to 23.25% of the phenotypic variation, and has potential for marker-assisted breeding of genotypes adapted to dryland conditions. The stable and major QTLs identified here add valuable information for understanding the genetic mechanism underlying chlorophyll content and provide a basis for molecular marker-assisted breeding.

Genome-wide association study of grain hardness and novel Puroindoline alleles in common wheat.

Grain hardness (HI) is a key trait for wheat milling and end-use quality. Puroindoline genes (PINs) are the major genes responsible for grain hardness, but other QTLs also contribute to the trait. Therefore, it is essential to identify loci associated with the HI and allelic variations of PINs in wheat. In the present study, 287 accessions from Shanxi province representing 70 years of wheat breeding were grown in one rainfed and two irrigated conditions to study grain hardness. Genome-wide association analysis (GWAS) was performed using the 15 K array, and the variability of PIN alleles was investigated. Among the accessions, hard wheat was most common. The broad-sense heritability (H2) among the three environments was 99.5%, suggesting HI was mainly affected by heredity. GWAS identified nine significant marker-trait associations (MTAs), including that PINs, which explained 7.03% to 17.70% of phenotypic variation. Four MTAs on chromosome 2A, 2B, 5A, and 7A were novel loci. As for diversity of PINs, a total of 11 PINs haplotypes were detected, composed of 12 allelic variations of the PIN gene. The most frequent haplotypes were Pina-D1a/Pinb-D1b (43.9%) and Pina-Dla/Pinb-D1p (18.8%), and both the frequency of Pina-D1a/Pinb-D1b and the HI value increased with breeding years were related to local dietary habits probably. A novel double deletion allele of the PINs haplotype was found in Donghei1206. These results will be useful not only in understanding of the genetics of the HI but also in breeding for improved grain texture. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01303-x.

Analysis of Wheat Wax Regulation Mechanism by Liposome and Transcriptome.

As a barrier for plants to contact with the outside world, epidermal wax plays an important role in resisting biotic and abiotic stresses. In this study, we analyzed the effect of wax content on leaf permeability by measuring the wax loss rate in the leaf. To further clarify the wax composition of the wheat epidermis and its molecular regulation mechanism, we applied untargeted lipidomic and transcriptome analysis on the leaf epidermis wax of Jimai 22 low-wax mutant (waxless) and multi-wax mutant (waxy). Our research showed that the mutant waxy has a slow loss rate, which can maintain higher leaf water content. 31 lipid subclasses and 1,367 lipid molecules were identified. By analyzing the wax differences of the two mutants, we found that the main lipid components of leaf epidermis wax in Jimai 22 were WE (C19-C50), DG (C27-C53), MG (C31-C35), and OAHFA (C31-C52). Carbon chain length analysis showed that, in wheat epidermis wax, WE was dominated by C44 molecules, DG was mainly concentrated in C47, C45, C37, and C31 molecules, C48 played a leading role in OAHFA, and C35 and C31 played a major role in MG. Among them, DG, MG, and OAHFA were detected in wheat leaf wax for the first time, and they were closely related to stress resistance. Compared with the waxy, 6,840 DEGs were detected in the mutant waxless, 3,181 DEGs were upregulated, and 3,659 DEGs were downregulated. The metabolic pattern of main waxy components in the wheat epidermis was constructed according to KEGG metabolic pathway and 46 related genes were screened, including KSC, TER, FAR, WSD1, CER1, MAH1, ALDH7A1, CYP704B1, ACOT1_2_4, CYP86, MGLL, GPAT, ALDH, DPP1, dgkA, plsC, and E2.3.1.158 related genes. The screened wax-related genes were confirmed to be highly reliable by qRT-PCR. In addition, we found TER gene TraesCS6B03G1132900LC in wheat mutant waxless leaves for the first time, which inhibited the synthesis of long-chain acyl-CoA (n+2) by downregulating its expression. These results provide valuable reference information for further study of wheat epidermis wax heredity and molecular regulation.

Insights Into Walnut Lipid Metabolism From Metabolome and Transcriptome Analysis.

Walnut oil is an excellent source of essential fatty acids. Systematic evaluation of walnut lipids has significance for the development of the nutritional and functional value of walnut. Ultra-performance liquid chromatography/Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap HRMS) was used to characterize the lipids of walnut. A total of 525 lipids were detected and triacylglycerols (TG) (18:2/18:2/18:3) and diacylglycerols (DG) (18:2/18:2) were the main glycerolipids present. Essential fatty acids, such as linoleic acid and linolenic acid, were the main DG and TG fatty acid chains. Many types of phospholipids were observed with phosphatidic acid being present in the highest concentration (5.58%). Using a combination of metabolome and transcriptome analysis, the present study mapped the main lipid metabolism pathway in walnut. These results may provide a theoretical basis for further study and specific gene targets to enable the development of walnut with increased oil content and modified fatty acid composition.

Comparative Analysis of Genomic and Transcriptome Sequences Reveals Divergent Patterns of Codon Bias in Wheat and Its Ancestor Species.

The synonymous codons usage shows a characteristic pattern of preference in each organism. This codon usage bias is thought to have evolved for efficient protein synthesis. Synonymous codon usage was studied in genes of the hexaploid wheat Triticum aestivum (AABBDD) and its progenitor species, Triticum urartu (AA), Aegilops tauschii (DD), and Triticum turgidum (AABB). Triticum aestivum exhibited stronger usage bias for G/C-ending codons than did the three progenitor species, and this bias was especially higher compared to T. turgidum and Ae. tauschii. High GC content is a primary factor influencing codon usage in T. aestivum. Neutrality analysis showed a significant positive correlation (p<0.001) between GC12 and GC3 in the four species with regression line slopes near zero (0.16-0.20), suggesting that the effect of mutation on codon usage was only 16-20%. The GC3s values of genes were associated with gene length and distribution density within chromosomes. tRNA abundance data indicated that codon preference corresponded to the relative abundance of isoaccepting tRNAs in the four species. Both mutation and selection have affected synonymous codon usage in hexaploid wheat and its progenitor species. GO enrichment showed that GC biased genes were commonly enriched in physiological processes such as photosynthesis and response to acid chemical. In some certain gene families with important functions, the codon usage of small parts of genes has changed during the evolution process of T. aestivum.

Genome-Wide Association Study of Grain Number in Common Wheat From Shanxi Under Different Water Regimes.

Water availability is a crucial environmental factor on grain number in wheat, which is one of the important yield-related traits. In this study, a diverse panel of 282 wheat accessions were phenotyped for grain number per spike (GNS), spikelet number (SN), basal sterile spikelet number (BSSN), and apical sterile spikelet number (ASSN) under different water regimes across two growing seasons. Correlation analysis showed that GNS is significantly correlated with both SN and BSSN under two water regimes. A total of 9,793 single nucleotide polymorphism (SNP) markers from the 15 K wheat array were employed for genome-wide association study (GWAS). A total of 77 significant marker-trait associations (MTAs) for investigated traits as well as 8 MTAs for drought tolerance coefficient (DTC) were identified using the mixed linear model. Favored alleles for breeding were inferred according to their estimated effects on GNS, based on the mean difference of varieties. Frequency changes in favored alleles associated with GNS in modern varieties indicate there is still considerable genetic potential for their use as markers for genome selection of GNS in wheat breeding.

Analysis of Genetic Regions Related to Field Grain Number per Spike From Chinese Wheat Founder Parent Linfen 5064.

Wheat founder parents have been important in the development of new wheat cultivars. Understanding the effects of specific genome regions on yield-related traits in founder variety derivatives can enable more efficient use of these genetic resources through molecular breeding. In this study, the genetic regions related to field grain number per spike (GNS) from the founder parent Linfen 5064 were analyzed using a doubled haploid (DH) population developed from a cross between Linfen 5064 and Nongda 3338. Quantitative trait loci (QTL) for five spike-related traits over nine experimental locations/years were identified, namely, total spikelet number per spike (TSS), base sterile spikelet number per spike (BSSS), top sterile spikelet number per spike (TSSS), fertile spikelet number per spike (FSS), and GNS. A total of 13 stable QTL explaining 3.91-19.51% of the phenotypic variation were found. The effect of six of these QTL, Qtss.saw-2B.1, Qtss.saw-2B.2, Qtss.saw-3B, Qfss.saw-2B.2, Qbsss.saw-5A.1, and Qgns.saw-1A, were verified by another DH population (Linfen 5064/Jinmai 47), which showed extreme significance (P < 0.05) in more than three environments. No homologs of reported grain number-related from grass species were found in the physical regions of Qtss.saw-2B.1 and Qtss.saw-3B, that indicating both of them are novel QTL, or possess novel-related genes. The positive alleles of Qtss.saw-2B.2 from Linfen 5064 have the larger effect on TSS (3.30%, 0.62) and have 66.89% in Chinese cultivars under long-term artificial selection. This study revealed three key regions for GNS in Linfen 5064 and provides insights into molecular marker-assisted breeding.

Effects of HMW-GSs on quality related traits in wheat (Triticum aestivum L.) under different water regimes.

Alleles at the Glu-1 loci play important roles in the functional properties of wheat flour. The effects of various high-molecular-weight glutenin subunit (HMW-GS) compositions on quality traits and bread-making properties were evaluated using 235 doubled haploid lines (DHs). The experiment was conducted in a split plot design with two water regimes as the main plot treatment, and DH lines as the subplot treatments. Results showed that the presence of subunit pair 5+10 at the Glu-D1 locus, either alone or in combination with others, appears to provide an improvement in quality and bread-making properties. At the Glu-A1 locus, subunit 1 produced a higher Zeleny sedimentation value (Zel) and stretch area (SA) than subunit 2* when subunits 14+15 and 5+10 were expressed at the Glu-B1 and Glu-D1 loci, and 2* had a positive effect on the maximum dough resistance (Rmax) when subunits 14+15 and 5'+12 were expressed at the Glu-B1 and Glu-D1 loci, respectively. Given subunit 1 at the Glu-A1 locus and 5'+12 at the Glu-D1 locus, the effects of Glu-B1 subunits 14+15 on the tractility (Tra), dough stability time (ST), and dough development time (DT) under the well-watered regime were significantly higher than those of Glu-B1 subunits 13+16. However, 13+16 had a positive effect on SA under the rain-fed regime when subunits 2* and 5+10 were expressed at the Glu-A1 and Glu-D1 loci, respectively. Multiple comparisons analysis revealed that the Zel and Rmax of the six subunits and eight HMW-GS compositions were stable under different water regimes. Overall, subunit compositions 1, 13+16 and 5+10 and 1, 14+15 and 5+10 had higher values for quality traits and bread-baking properties under the two water regimes. These results could play a positive guiding role in selecting and popularizing varieties suitable for production and cultivation in local areas.