Introgression of chromosome 5P from Agropyron cristatum enhances grain weight in a wheat background.

KEY MESSAGE: A grain weight locus from Agropyron cristatum chromosome 5P increases grain weight in different wheat backgrounds and is localized to 5PL (bin 7-12). Thousand-grain weight is an important trait in wheat breeding, with a narrow genetic basis being the main factor limiting improvement. Agropyron cristatum, a wild relative of wheat, harbors many desirable genes for wheat improvement. Here, we found that the introduction of the 5P chromosome from A. cristatum into wheat significantly increased the thousand-grain weight by 2.55-7.10 g, and grain length was the main contributor to grain weight. An increase in grain weight was demonstrated in two commercial wheat varieties, indicating that the grain weight locus was not affected by the wheat background. To identify the chromosome segment harboring the grain weight locus, three A. cristatum 5P deletion lines, two wheat-A. cristatum 5P translocation lines and genetic populations of these lines were used to evaluate agronomic traits. We found that the translocation lines harboring the long arm of A. cristatum chromosome 5P (5PL) exhibited high grain weight and grain length, and the genetic locus associated with increased grain weight was mapped to 5PL (bin 7-12). An increase in grain weight did not adversely affect other agronomic traits in translocation line 5PT2, which is a valuable germplasm resource. Overall, we identified a grain weight locus from chromosome 5PL and provided valuable germplasm for improving wheat grain weight.

Chromosomal mapping of a major genetic locus from Agropyron cristatum chromosome 6P that influences grain number and spikelet number in wheat.

KEY MESSAGE: A novel locus on Agropyron cristatum chromosome 6P that increases grain number and spikelet number was identified in wheat-A. cristatum derivatives and across 3 years. Agropyron cristatum (2n = 4x = 28, PPPP), which has the characteristics of high yield with multiple flowers and spikelets, is a promising gene donor for wheat high-yield improvement. Identifying the genetic loci and genes that regulate yield could elucidate the genetic variations in yield-related traits and provide novel gene sources and insights for high-yield wheat breeding. In this study, cytological analysis and molecular marker analysis revealed that del10a and del31a were wheat-A. cristatum chromosome 6P deletion lines. Notably, del10a carried a segment of the full 6PS and 6PL bin (1-13), while del31a carried a segment of the full 6PS and 6PL bin (1-8). The agronomic characterization and genetic population analysis confirmed that the 6PL bin (9-13) brought about an increase in grain number per spike (average increase of 10.43 grains) and spikelet number per spike (average increase of 3.67) over the three growing seasons. Furthermore, through resequencing, a multiple grain number locus was mapped to the physical interval of 593.03-713.89 Mb on chromosome 6P of A. cristatum Z559. The RNA-seq analysis revealed the expression of 537 genes in the del10a young spike tissue, with the annotation indicating that 16 of these genes were associated with grain number and spikelet number. Finally, a total of ten A. cristatum-specific molecular markers were developed for this interval. In summary, this study presents novel genetic material that is useful for high-yield wheat breeding initiatives to meet the challenge of global food security through enhanced agricultural production.

The Genomic Variation and Differentially Expressed Genes on the 6P Chromosomes in Wheat-Agropyron cristatum Addition Lines 5113 and II-30-5 Confer Different Desirable Traits.

Wild relatives of wheat are essential gene pools for broadening the genetic basis of wheat. Chromosome rearrangements and genomic variation in alien chromosomes are widespread. Knowledge of the genetic variation between alien homologous chromosomes is valuable for discovering and utilizing alien genes. In this study, we found that 5113 and II-30-5, two wheat-A. cristatum 6P addition lines, exhibited considerable differences in heading date, grain number per spike, and grain weight. Genome resequencing and transcriptome analysis revealed significant differences in the 6P chromosomes of the two addition lines, including 143,511 single-nucleotide polymorphisms, 62,103 insertion/deletion polymorphisms, and 757 differentially expressed genes. Intriguingly, genomic variations were mainly distributed in the middle of the chromosome arms and the proximal centromere region. GO and KEGG analyses of the variant genes and differentially expressed genes showed the enrichment of genes involved in the circadian rhythm, carbon metabolism, carbon fixation, and lipid metabolism, suggesting that the differential genes on the 6P chromosome are closely related to the phenotypic differences. For example, the photosynthesis-related genes PsbA, PsbT, and YCF48 were upregulated in II-30-5 compared with 5113. ACS and FabG are related to carbon fixation and fatty acid biosynthesis, respectively, and both carried modification variations and were upregulated in 5113 relative to II-30-5. Therefore, this study provides important guidance for cloning desirable genes from alien homologous chromosomes and for their effective utilization in wheat improvement.

Comparative Transcriptome Analysis Reveals the Gene Expression and Regulatory Characteristics of Broad-Spectrum Immunity to Leaf Rust in a Wheat-Agropyron cristatum 2P Addition Line.

Wheat leaf rust (caused by Puccinia triticina Erikss.) is among the major diseases of common wheat. The lack of resistance genes to leaf rust has limited the development of wheat cultivars. Wheat-Agropyron cristatum (A. cristatum) 2P addition line II-9-3 has been shown to provide broad-spectrum immunity to leaf rust. To identify the specific A. cristatum resistance genes and related regulatory pathways in II-9-3, we conducted a comparative transcriptome analysis of inoculated and uninoculated leaves of the resistant addition line II-9-3 and the susceptible cultivar Fukuhokomugi (Fukuho). The results showed that there were 66 A. cristatum differentially expressed genes (DEGs) and 1389 wheat DEGs in II-9-3 during P. triticina infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and gene set enrichment analysis (GSEA) revealed that the DEGs of II-9-3 were associated with plant-pathogen interaction, MAPK signaling pathway-plant, plant hormone signal transduction, glutathione metabolism, and phenylpropanoid biosynthesis. Furthermore, many defense-related A. cristatum genes, such as two NLR genes, seven receptor kinase-encoding genes, and four transcription factor-encoding genes, were identified. Our results indicated that the key step of resistance to leaf rust involves, firstly, the gene expression of chromosome 2P upstream of the immune pathway and, secondly, the effect of chromosome 2P on the co-expression of wheat genes in II-9-3. The disease resistance regulatory pathways and related genes in the addition line II-9-3 thus could play a critical role in the effective utilization of innovative resources for leaf rust resistance in wheat breeding.

Chromosomal mapping of a locus associated with adult-stage resistance to powdery mildew from Agropyron cristatum chromosome 6PL in wheat.

KEY MESSAGE: The powdery mildew resistance locus was mapped to A. cristatum chromosome 6PL bin (0.27-0.51) and agronomic traits evaluation indicated that this locus has potential breeding application value. Agropyron cristatum (2n = 4x = 28, PPPP) is a wild relative of wheat with an abundance of biotic and abiotic stress resistance genes and is considered one of the best exogenous donor relatives for wheat breeding. A number of wheat-A. cristatum derived lines have been generated, including addition lines, translocation lines and deletion lines. In this study, the 6P disomic addition line 4844-12 (2n = 2x = 44) was confirmed to have genetic effects on powdery mildew resistance. Four 6P deletion lines (del16a, del19b, del21 and del27) and two translocation lines (WAT638a and WAT638b), derived from radiation treatment of 4844-12, were used to further assess the 6P powdery mildew resistance locus by powdery mildew resistance assessment, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and 6P specific sequence-tagged-site (STS) markers. Collectively, the locus harboring the powdery mildew resistance gene was genetically mapped to a 6PL bin (0.27-0.51). The genetic effects of this chromosome segment on resistance to powdery mildew were further confirmed by del16a and del27 BC3F2 lines. Comprehensive evaluation of agronomic traits revealed that the powdery mildew resistance locus of 6PL (0.27-0.51) has potential application value in wheat breeding. A total of 22 resistant genes were annotated and 3 specific gene markers were developed for detecting chromatin of the resistant region based on genome re-sequencing. In summary, this study could broaden the powdery mildew resistance gene pool for wheat genetic improvements.

ATP-dependent DNA helicase (TaDHL), a Novel Reduced-Height (Rht) Gene in Wheat.

In wheat, a series of dwarf and semi-dwarf plant varieties have been developed and utilized worldwide since the 1960s and caused the 'Green Revolution'. To date, 25 reduced-height (Rht) genes have been identified, but only several genes for plant height (PH) have been isolated previously. In this study, we identified a candidate gene, ATP-dependent DNA helicase (TaDHL-7B), for PH via QTL mapping and genome-wide association study (GWAS) methods. We knocked out this gene using the CRISPR/Cas9 system in variety 'Fielder'. Two homozygous mutant genotypes, AAbbDD (-5 bp) and AAbbDD (-1 bp), were obtained in the T2 generation. The PH values of AAbbDD (-5 bp) and AAbbDD (-1 bp) were significantly reduced compared with the wild-type (WT, 'Fielder'), indicating that TaDHL-7B is a novel Rht gene that controls the PH. This is the first time that a PH gene of wheat has been isolated with a non-hormone pathway, providing a new insight into the genetic control of PH. The TaDHL gene reduced the PH without a yield penalty. It could be used to improve the lodging resistance and yield in wheat breeding programs.

CRISPR/Cas9-Mediated Disruption of Xylanase inhibitor protein (XIP) Gene Improved the Dough Quality of Common Wheat.

The wheat dough quality is of great significance for the end-use of flour. Some genes have been cloned for controlling the protein fractions, grain protein content, starch synthase, grain hardness, etc. Using a unigene map of the recombinant inbred lines (RILs) for "TN 18 × LM 6," we mapped a quantitative trait locus (QTL) for dough stability time (ST) and SDS-sedimentation values (SV) on chromosome 6A (QSt/Sv-6A-2851). The peak position of the QTL covered two candidate unigenes, and we speculated that TraesCS6A02G077000 (a xylanase inhibitor protein) was the primary candidate gene (named the TaXip gene). The target loci containing the three homologous genes TaXip-6A, TaXip-6B, and TaXip-6D were edited in the variety "Fielder" by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9). Two mutant types in the T2:3 generation were obtained (aaBBDD and AAbbdd) with about 120 plants per type. The SVs of aaBBDD, AAbbdd, and WT were 31.77, 27.30, and 20.08 ml, respectively. The SVs of the aaBBDD and AAbbdd were all significantly higher than those of the wild type (WT), and the aaBBDD was significantly higher than the AAbbdd. The STs of aaBBDD, AAbbdd, and WT were 2.60, 2.24, and 2.25 min, respectively. The ST for the aaBBDD was significantly higher than that for WT and was not significantly different between WT and AAbbdd. The above results indicated that XIP in vivo can significantly affect wheat dough quality. The selection of TaXip gene should be a new strategy for developing high-quality varieties in wheat breeding programs.

QTL mapping for quality traits using a high-density genetic map of wheat.

Protein- and starch-related quality traits, which are quantitatively inherited and significantly influenced by the environment, are critical determinants of the end-use quality of wheat. We constructed a high-density genetic map containing 10,739 loci (5,399 unique loci) using a set of 184 recombinant inbred lines (RILs) derived from a cross of 'Tainong 18 × Linmai 6' (TL-RILs). In this study, a quantitative trait loci (QTLs) analysis was used to examine the genetic control of grain protein content, sedimentation value, farinograph parameters, falling number and the performance of the starch pasting properties using TL-RILs grown in a field for three years. A total of 106 QTLs for 13 quality traits were detected, distributed on the 21 chromosomes. Of these, 38 and 68 QTLs for protein- and starch-related traits, respectively, were detected in three environments and their average values (AV). Twenty-six relatively high-frequency QTLs (RHF-QTLs) that were detected in more than two environments. Twelve stable QTL clusters containing at least one RHF-QTL were detected and classified into three types: detected only for protein-related traits (type I), detected only for starch-related traits (type II), and detected for both protein- and starch-related traits (type III). A total of 339 markers flanked with 11 QTL clusters (all except C6), were found to be highly homologous with 282 high confidence (HC) and 57 low confidence (LC) candidate genes based on IWGSC RefSeq v 1.0. These stable QTLs and RHF-QTLs, especially those grouped into clusters, are credible and should be given priority for QTL fine-mapping and identification of candidate genes with which to explain the molecular mechanisms of quality development and inform marker-assisted breeding in the future.