Pre-harvest Sprouting Tolerance Research Articles

Preharvest Sprouting in Quinoa: A New Screening Method Adapted to Panicles and GWAS Components.

The introduction of quinoa into new growing regions and environments is of interest to farmers, consumers, and stakeholders around the world. Many plant breeding programs have already started to adapt quinoa to the environmental and agronomic conditions of their local fields. Formal quinoa breeding efforts in Washington State started in 2010, led by Professor Kevin Murphy out of Washington State University. Preharvest sprouting appeared as the primary obstacle to increased production in the coastal regions of the Pacific Northwest. Preharvest sprouting (PHS) is the undesirable sprouting of seeds that occurs before harvest, is triggered by rain or humid conditions, and is responsible for yield losses and lower nutrition in cereal grains. PHS has been extensively studied in wheat, barley, and rice, but there are limited reports for quinoa, partly because it has only recently emerged as a problem. This study aimed to better understand PHS in quinoa by adapting a PHS screening method commonly used in cereals. This involved carrying out panicle-wetting tests and developing a scoring scale specific for panicles to quantify sprouting. Assessment of the trait was performed in a diversity panel (N = 336), and the resulting phenotypes were used to create PHS tolerance rankings and undertake a GWAS analysis (n = 279). Our findings indicate that PHS occurred at varying degrees across a subset of the quinoa germplasm tested and that it is possible to access PHS tolerance from natural sources. Ultimately, these genotypes can be used as parental lines in future breeding programs aiming to incorporate tolerance to PHS.

Exploring Genetic Diversity in Black Gram (Vigna mungo (L.) Hepper) for Pre-Harvest Sprouting Tolerance

Pre-harvest sprouting (PHS) is a condition triggered by environmental factors, particularly prevalent in humid conditions, leading to substantial yield losses in black gram. While the potential for genotypic PHS tolerance exists, it has not been thoroughly assessed in black gram. Hence, the present study aimed to delve into the genetic variation for PHS tolerance in diverse black gram (Vigna mungo (L.) Hepper) germplasm, and also to comprehend the impact of various physical and physiological traits on PHS. A diverse set of 112 black gram accessions collected across the phytogeographical zones of India were examined for their seed and pod characteristics. Water absorption by pods and seeds and fresh-seed germination was calculated by following the standard procedure given by the International Seed Testing Association. The alpha-amylase activity was measured on dry seeds (0 h), 24 h, 48 h, and 72 h after germination of each accession, using a UV-VIS spectrophotometer, and hard-seededness was measured using a texture analyzer machine. The results showed a wide range in PHS tolerance and FSG, and 13 accessions were found to be PHS-tolerant (PHS value < 10%). An indicator of PHS, seed germination in a pod, ranged from 2.75% in IC485641 (highly tolerant to PHS) to 95.85% in IC530501 (highly susceptible to PHS). Correlation and multivariate analysis revealed that PHS was positively correlated with water imbibition by pod and seed, fresh-seed germination and alpha-amylase activity. PHS-tolerant accessions showed a slow increase in alpha-amylase activity, in contrast to PHS-susceptible accessions. The utilization of alpha-amylase activity as a biochemical marker has the potential for evaluating PHS tolerance across various black gram accessions. The identified PHS-tolerant accessions can be used as donors in crop improvement programs aimed at developing PHS-tolerant black gram varieties.

Polyphenols and phytohormones profiling of pre-harvest sprouting resistant and susceptible wheat genotypes

Pre-harvest sprouting (PHS) is one of the major threats to global food security as it significantly affects cereals’ production and quality. PHS tolerance depends on diverse factors, among which phytohormones and germination-inhibitory substances (GISs) play essential roles. However, in wheat, information related to GIS in the glume is scarce. Thus, we applied LC–MS/MS-based metabolomics analysis to explore the polyphenols and phytohormones profiles of two contrasting wheat genotypes, Lincang Hulled Wheat (LHW, PHS-resistant) and Yunmai53 (Yun53, PHS-highly susceptible). Physiological tests revealed that LHW PHS-resistance is not associated with the ability of its spikelet to prevent water absorption. The total polyphenol content of the spikes and shells of LHW was significantly higher than that of Yun53, respectively. In total, 214 phenolic compounds classified mainly into phenolic acids (42.52%), flavones (23.83%), and flavonoid carbonosides (16.36%) were identified. 180 differentially accumulated polyphenols (DAPs) were uncovered, including 168 up-regulated in LHW. 24 most up-regulated DAPs (Log2FC ≥ 8) were unveiled as potential candidate GISs. Of the identified phytohormones, abscisic acid, salicylic acid, and jasmonic acid exhibited significantly higher content in LHW compared to Yun53. Whereas, Yun53 contained significantly higher levels of ethylene and gibberellin than LHW. Our findings offer new resources for PHS control in wheat.

Fine-Mapping Analysis of the Genes Associated with Pre-Harvest Sprouting Tolerance in Rice (Oryza sativa L.)

Pre-harvest sprouting (PHS) of rice (Oryza sativa L.) causes severe economic problems due to reduced grain quality and yield. Fine mapping was carried out to identify genes associated with PHS; the detected quantitative trait locus (QTL) was narrowed down to 50 Kbp using F3:4 populations, four polymorphic insertion and deletion (InDel) markers, and two cleaved amplified polymorphic sequence (CAPS) markers. In one region, five candidate genes were detected, and the SNP and InDel in each gene (Os01g0111400 and Os01g0111600) were confirmed to show the differences and resulting amino acid changes between parent plants. Based on haplotype, expression, and co-segregation analysis, the InDel in Os01g0111600 was confirmed to be associated with the PHS trait. The results of this study could be applied to improve the PHS tolerance of Japonica rice varieties, and they also improved our understanding of the genetic basis underlying PHS tolerance.

Streamlined alpha‐amylase assays for wheat preharvest sprouting and late maturity alpha‐amylase detection

AbstractLate maturity alpha‐amylase (LMA) and preharvest sprouting (PHS) lead to elevated alpha‐amylase in wheat (Triticum aestivum L.) grain. Risk of poor end‐product quality due to elevated alpha‐amylase is detected in the wheat industry using the Hagberg–Perten falling number (FN) method. In breeding programs, selection for PHS and LMA tolerance requires higher throughput methods requiring a smaller sample size than the 7 g required for the FN method. Specifically, LMA can only be screened only using detection of alpha‐amylase activity or protein after cold treatment of individual wheat spikes at a specific stage of grain development resulting in very small samples (≤1 g). This study developed and evaluated a high throughput 96‐well method for the Phadebas alpha‐amylase enzyme assay for small wheat grain samples and compared this method to FN and the Megazyme Alpha‐Amylase SD (Sprout Damage) Assay Kit performed on the automated Awareness Technology ChemWell‐T Analyzer. In parallel, the efficacy of low‐cost small‐scale milling methods was evaluated relative to traditional larger scale mills. The Phadebas enzyme activity was highly reproducible and showed a strong correlation to the SD enzyme assay and FN method regardless of which mill was used to process the grain. The SD assay offers simpler standardization and calculation of enzyme activity, whereas the Phadebas assay offers higher sensitivity and lower expense. Both the 96‐well Phadebas and automated Megazyme SD assays are suitable for alpha‐amylase detection from small samples, and the use of low‐cost coffee grinders to process small samples did not significantly impact assay performance.

Pre-Harvest Sprouting Tolerance in 36 Bread Wheat Genotypes

Pre-harvest sprouting (PHS), promoted by rainfall during crop maturity, is a high problem in many wheat-producing regions of the world. Considering its importance in Brazil, 36 national and international varieties and advanced lines of wheat were evaluated for their tolerance to PHS. For this purpose, two experiments were conducted over three years. Seed pericarp rupture was used as an indicator of the beginning of germination. The data were analyzed using analysis of variance, the Scott-Knott test, and the Lin and Binns method. The wide range of germination percentage values allowed the genotypes to be classified as tolerant (in experiment 1 - ND 674 and Grandin*2/RL 4137 and experiment 2 - Frontana and Grandin) and moderately tolerant (Alsen, CD 114, and Milan/3/Attila//Fang 69/CIMMYT 3 in Experiment 1; Avante, BRS 177, IAC 5-Maringá, Onix, OR 1, RL 4137, and Rubi in Experiment 2). Because tolerance to PHS is under genetic control and can be improved through breeding programs, the challenge for wheat breeders is to combine increased PHS tolerance with other requirements to meet market demands.

Transcriptome and Proteome Co-Profiling Offers an Understanding of Pre-Harvest Sprouting (PHS) Molecular Mechanisms in Wheat (Triticum aestivum).

While wheat (Triticum aestivum L.) is a widely grown and enjoyed crop, the diverse and complex global situation and climate are exacerbating the instability of its supply. In particular, pre-harvest sprouting (PHS) is one of the major abiotic stresses that frequently occurs due to irregular climate conditions, causing serious damage to wheat and its quality. In this study, transcriptomic analysis with RNA-seq and proteomic analysis with LC-MS/MS were performed in PHS-treated spikes from two wheat cultivars presenting PHS sensitivity and tolerance, respectively. A total of 13,154 differentially expressed genes (DEGs) and 706 differentially expressed proteins (DEPs) were identified in four comparison groups between the susceptible/tolerant cultivars. Gene function and correlation analysis were performed to determine the co-profiled genes and proteins affected by PHS treatment. In the functional annotation of each comparative group, similar functions were confirmed in each cultivar under PHS treatment; however, in Keumgang PHS+7 (K7) vs. Woori PHS+7 (W7), functional annotations presented clear differences in the "spliceosome" and "proteasome" pathways. In addition, our results indicate that alternative splicing and ubiquitin-proteasome support the regulation of germination and seed dormancy. This study provides an advanced understanding of the functions involved in transcription and translation related to PHS mechanisms, thus enabling specific proposals for the further analysis of germination and seed dormancy mechanisms and pathways in wheat.

Genotypic variability studies and identification of pre-harvest sprouting tolerant wild Vigna

Seed dormancy is the main component of domesticated traits, loss of which results into rapid and uniform germination, synchronous maturity and ease in harvesting. However, breakdown or loss of fresh seed dormancy (FSD) has made seeds vulnerable to pre-harvest sprouting (PHS). Though, seeds of Vigna species are protected by pod, these are still susceptible to PHS. Identification of donor(s) having short duration of FSD (30-45 days) has become utmost important to transfer the trait to high yielding varieties. The wild progenitors and wild relatives of the cultivated Vigna offer a source of several useful traits; therefore, the experiment was initiated to understand the level of variation in PHS and fresh seed germination (FSG) in wild accessions of Vigna species. PHS and FSG among 59 Vigna accessions ranged between 0-100%, with a mean of 55.86% and 61.19%, respectively. Five accessions (IC276983, IC349701, Trichy Local 1, LRM/13-33 and LRM/13-26) of Vigna trilobata and one accession (IC251440) of Vigna sublobata recorded no pod loss due to PHS with 0% FSG (2.5% in V. sublobata). Seed size showed a positive association with PHS, where smaller seeds (100-SW <1 g) recorded comparably low PHS (30.82%) than bold seeds (69.06 and 62.94% with 100-SW between 1-2 g and > 2 g, respectively). The accessions identified, especially V. sublobata (crossing compatible) could be used as a source of PHS tolerance to transfer dormancy in Vigna species, particularly in mungbean and urdbean.

Genome-wide association mapping and genomic prediction for pre‑harvest sprouting resistance, low α-amylase and seed color in Iranian bread wheat

BackgroundPre-harvest sprouting (PHS) refers to a phenomenon, in which the physiologically mature seeds are germinated on the spike before or during the harvesting practice owing to high humidity or prolonged period of rainfall. Pre-harvest sprouting (PHS) remarkably decreases seed quality and yield in wheat; hence it is imperative to uncover genomic regions responsible for PHS tolerance to be used in wheat breeding. A genome-wide association study (GWAS) was carried out using 298 bread wheat landraces and varieties from Iran to dissect the genomic regions of PHS tolerance in a well-irrigated environment. Three different approaches (RRBLUP, GBLUP and BRR) were followed to estimate prediction accuracies in wheat genomic selection.Results Genomes B, A, and D harbored the largest number of significant marker pairs (MPs) in both landraces (427,017, 328,006, 92,702 MPs) and varieties (370,359, 266,708, 63,924 MPs), respectively. However, the LD levels were found the opposite, i.e., genomes D, A, and B have the highest LD, respectively. Association mapping by using GLM and MLM models resulted in 572 and 598 marker-trait associations (MTAs) for imputed SNPs (− log10 P > 3), respectively. Gene ontology exhibited that the pleitropic MPs located on 1A control seed color, α-Amy activity, and PHS. RRBLUP model indicated genetic effects better than GBLUP and BRR, offering a favorable tool for wheat genomic selection.ConclusionsGene ontology exhibited that the pleitropic MPs located on 1A can control seed color, α-Amy activity, and PHS. The verified markers in the current work can provide an opportunity to clone the underlying QTLs/genes, fine mapping, and genome-assisted selection.Our observations uncovered key MTAs related to seed color, α-Amy activity, and PHS that can be exploited in the genome-mediated development of novel varieties in wheat.

Genomic Regions Influencing Preharvest Sprouting Tolerance in Two Doubled-Haploid Wheat Populations (Triticum aestivum L.)

The current and projected climate change that is represented by increasing temperatures and humidity levels and irregular rainfall patterns promotes the occurrence of preharvest sprouting (PHS) in wheat. PHS results in significant economic losses, globally, which necessitates the need for high-yielding cultivars with increased PHS tolerance; hence, this study was conducted. The current study evaluated two doubled-haploid (DH) wheat populations of Tugela-Dn × Elands and Elands × Flamink across six environments in the Free State Province of South Africa to select genotypes with increased PHS tolerance and further map the underlying loci. Significant effects of DH lines (194) and environments (6) were observed for PHS tolerance. The results of this study validate previous findings that PHS is only expressed when environmental conditions are conducive. Quantitative trait loci (QTL) mapping using single-nucleotide polymorphism (SNP) and silicoDArT markers revealed three additive QTLs with major effects on chromosomes 5B and 7B, and these QTLs were detected more than once, when conditions were favourable. These QTLs explained a phenotypic variation (PVE) varying between 10.08% and 20.30% (LOD = 2.73–3.11). About 16.50% of DH lines performed to the level of Elands (the PHS-tolerant parent) and are recommended for further selection in a pre-breeding or breeding programme. The findings of this study are expected to expedite the on-going breeding efforts for PHS tolerance in winter wheat, which will facilitate the development of PHS-tolerant cultivars adapted to the South African environment.

Differential Expression Analysis of Phytohormone-Related Genes of Korean Wheat (Triticum aestivum) in Response to Preharvest Sprouting and Abscisic Acid (ABA)

Preharvest sprouting (PHS) is a key global issue in production and end-use quality of cereals, particularly in regions where the rainfall season overlaps the harvest. To investigate transcriptomic changes in genes affected by PHS-induction and ABA-treatment, RNA-seq analysis was performed in two wheat cultivars that differ in PHS tolerance. A total of 123 unigenes related to hormone metabolism and signaling for abscisic acid (ABA), gibberellic acid (GA), indole-3-acetic acid (IAA), and cytokinin were identified and 1862 of differentially expressed genes were identified and divided into 8 groups by transcriptomic analysis. DEG analysis showed the majority of genes were categorized in sugar related processes, which interact with ABA signaling in PHS tolerant cultivar under PHS-induction. Thus, genes related to ABA are key regulators of dormancy and germination. Our results give insight into global changes in expression of plant hormone related genes in response to PHS.

Targeted amplicon sequencing + next-generation sequencing\u2013based bulked segregant analysis identified genetic loci associated with preharvest sprouting tolerance in common buckwheat (Fagopyrum esculentum)

BackgroundCommon buckwheat (2n = 2x = 16) is an outcrossing pseudocereal whose seeds contain abundant nutrients and potential antioxidants. As these beneficial compounds are damaged by preharvest sprouting (PHS) and PHS is likely to increase with global warming, it is important to find efficient ways to develop new PHS-tolerant lines. However, genetic loci and selection markers associated with PHS in buckwheat have not been reported.ResultsBy next-generation sequencing (NGS) of whole-genome of parental lines, we developed a genome-wide set of 300 markers. By NGS- based bulked segregant analysis (NGS-BSA), we developed 100 markers linked to PHS tolerance. To confirm the effectiveness of marker development from NGS-BSA data, we developed 100 markers linked to the self-compatibility (SC) trait from previous NGS-BSA data. Using these markers, we developed genetic maps with AmpliSeq technology, which can quickly detect polymorphisms by amplicon-based multiplex targeted NGS, and performed quantitative trait locus (QTL) analysis for PHS tolerance in combination with NGS-BSA. QTL analysis detected two major and two minor QTLs for PHS tolerance in a segregating population developed from a cross between the PHS-tolerant ‘Kyukei 29’ and the self-compatible susceptible ‘Kyukei SC7’. We found different major and minor QTLs in other segregating populations developed from the PHS-tolerant lines ‘Kyukei 28’ and ‘NARO-FE-1’. Candidate markers linked to PHS developed by NGS-BSA were located near these QTL regions. We also investigated the effectiveness of markers linked to these QTLs for selection of PHS-tolerant lines among other segregating populations.ConclusionsWe efficiently developed genetic maps using a method combined with AmpliSeq technology and NGS-BSA, and detected QTLs associated with preharvest sprouting tolerance in common buckwheat. This is the first report to identify QTLs for PHS tolerance in buckwheat. Our marker development system will accelerate genetic research and breeding in common buckwheat.

ASSOCIATION BETWEEN FALLING NUMBER AND GRAIN CHARACTERISTICS TO EVALUATE PREHARVEST SPROUTING IN WHEAT

The falling number (FN) is a widely used parameter to evaluate sprouting damage in wheat. Despite its importance, many breeding programs lack facilities to determine the FN routinely. This study was conducted to elucidate the relationship of the falling number with germination percentage, visual sprouting score, hectoliter weight and the grain weight per spike, to improve the selection criteria in the pre-harvest sprouting (PHS) trials. Thirteen genotypes differing in PHS tolerance were evaluated in a rain simulator. The statistical analysis was conducted to understand the relationship of FN with grain characteristics. The path coefficient analysis revealed the highest association of the FN with the percentage of sprouted grains. Also, regression analysis determined that samples with more than 46% of sprouted grains had FN below 200s. The grains with 32 to 46% of germination resulted in FN values between 200 and 250s, and those with 19 to 32% of germination had FN between 250 and 300 s. Only those samples with less than 19% of germination resulted in FN values higher than 300s. The strong association between the germination percentage and the FN allows the selection of promising lines and the elimination of undesirable ones at an early stage of breeding.

Pre-harvest Sprouting Tolerance of Triticale Genotypes in Brazil

Pre-harvest sprouting (PHS) represents one of the main factors, which causes yield, technological and physiological losses in triticale seeds (X Triticosecale Wittmack). This work aimed to rate the variability and identify potential pre-harvest sprouting tolerant sources in triticale genotypes. Based on that, 32 triticale and three wheat genotypes were sown in 2016, 2017 and 2018 growing seasons, in Londrina-PR, Brazil. After the ears harvesting, these were submitted to simulate raining, for sprouting induction, through nebulization in a greenhouse. After nebulization, ears were sun dried, later hand threshed to determain, grain germination percentage (GERM) and hectoliter weight (HW). Additionally, it was determined grains HW of ears, whom were not submitted to nebulization, as well as the whole meal flour falling number (FN). The experiment design was completely randomized design, with two replications, and the experimental unit was made of 20 ears. The data collected were subjected to analysis of variance (ANOVA) and Scott-Knott test. Frontana, ND 674 and Quartzo are a source of tolerance to PHS in wheat. In triticale genotypes, genetic variability was observed for GERM, FN, and HW prior and after nebulization. The triticale genotypes BRS Netuno, BRS Saturno, TCL 15116, X 092181, Tiguera 1 and Tiguera 8, where tolerant towards PHS.

Transcription Factors Rc and OsVP1 Coordinately Regulate Preharvest Sprouting Tolerance in Red Pericarp Rice.

Red pericarp associates with seed dormancy or preharvest sprouting (PHS) tolerance in crops. To identify this association's molecular mechanism, a PHS mutant Osviviparous1 (Osvp1) was characterized in rice and crossed with Kasalath, a red pericarp cultivar with Rc (red coleoptiles) genotype. Among the dehulled seeds of F2 progenies, RcRcvp1vp1 seeds performed a lower PHS rate than rcrcvp1vp1 seeds and showed shallower pigmentation than RcRcVP1VP1 seeds. Kasalath and SL9 (an RcRcVP1VP1 substitution line with Nipponbare background) showed more ABA sensitivity than the Nipponbare (rcrcVP1VP1) by the germination assay, and the transcriptional abundance of ABA signal genes OsABI2, OsSnRK2, OsVP1, ABI5, and especially OsVP1 increased in the red pericarp line SL9. Moreover, OsVP1 can directly bind Rc (bHLH) promoter by yeast one-hybrid, which activates Rc and OsLAR expression in red pericarp rice. Furthermore, a luciferase complementation imaging assay showed that OsVP1 interacts with transcriptions factors Rc and OsC1. These results indicate that OsVP1 promotes proanthocyanidin accumulation through the interaction among OsVP1, Rc, and OsC1 and then increases the plant's ABA sensitivity and PHS resistance.

Transcriptome profiling identifies transcription factors and key homologs involved in seed dormancy and germination regulation of Chenopodium quinoa

Chenopodium quinoa, a halophytic crop belonging to the Amaranthaceae, has remarkable resistance to harsh growth conditions and produces seed with excellent nutritional value. This makes it a suitable crop for marginal soils. However, to date most of the commercial cultivars are susceptible to preharvest sprouting (PHS). Meanwhile, understanding of the PHS regulatory mechanisms is still limited. Abscisic acid (ABA) has been demonstrated to be tightly associated with seed dormancy and germination regulation in many crops. Whether ABA metabolism pathway could be manipulated to prevent PHS in quinoa is worth investigating. In the present study, we tested the inhibitory effects of exogenous ABA on quinoa seed germination. By RNA-seq analysis we investigated the global gene expression changes during seed germination, and obtained 1066 ABA-repressed and 392 ABA-induced genes. Cis-elements enrichment analysis indicated that the promoters of these genes were highly enriched in motifs “AAAAAAAA” and “ACGTGKC (K = G/T)”, the specific binding motifs of ABI3/VP1 and ABI5. Transcription factor annotation showed that 13 genes in bHLH, MADS-box, G2-like and NF-YB, and five genes in B3, bZIP, GATA and LBD families were specifically ABA-repressed and -induced, respectively. Furthermore, expression levels of 53 key homologs involved in seed dormancy and germination regulation were markedly changed. Hence, we speculated that the 18 transcription factors and the homologs were potential candidates involved in ABA-mediated seed dormancy and germination regulation, which could be manipulated for molecular breeding of quinoa elites with PHS tolerance in future.

Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype

Key messageUsing bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A.Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat ‘Zak.’ ERA8 was mapped to chromosome 4A in a Zak/‘ZakERA8’ backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an ‘Otis’/ZakERA8 but not in a ‘Louise’/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.

Genome-wide association study of pre-harvest sprouting tolerance using a 90K SNP array in common wheat (Triticum aestivum L.).

Three major loci for pre-harvest sprouting tolerance (PHST) were mapped on chromosomes 1AL, 3BS, and 6BL, and two CAPS and one dCAPS markers were validated. Sixteen lines with favorable alleles and increased PHST were identified. Pre-harvest sprouting (PHS) significantly affects wheat grain yield and quality. In the present study, the PHS tolerance (PHST) of 192 wheat varieties (lines) was evaluated by assessment of field sprouting, seed germination index, and period of dormancy in different environments. A high-density Illumina iSelect 90K SNP array was used to genotype the panel. A genome-wide association study (GWAS) based on single- and multi-locus mixed linear models was used to detect loci for PHST. The single-locus model identified 23 loci for PHST (P < 0.0001) and explained 6.0-18.9% of the phenotypic variance. Twenty loci were consistent with known quantitative trait loci (QTLs). Three single-nucleotide polymorphism markers closely linked with three major loci (Qphs.ahau-1A, Qphs.ahau-3B, and Qphs.ahau-6B) on chromosomes 1AL, 3BS, and 6BL, respectively, were converted to two cleaved amplified polymorphic sequences (CAPS) and one derived-CAPS markers, and validated in 374 wheat varieties (lines). The CAPS marker EX06323 for Qphs.ahau-6B co-segregated with a novel major QTL underlying PHST in a recombinant inbred line population raised from the cross Jing 411 × Wanxianbaimaizi. Linear regression showed a clear dependence of PHST on the number of favorable alleles. Sixteen varieties showing an elevated degree of PHST were identified and harbored more than 16 favorable alleles. The multi-locus model detected 39 marker-trait associations for PHST (P < 0.0001), of which five may be novel. Six loci common to the two models were identified. The combination of the two GWAS methods contributes to efficient dissection of the complex genetic mechanism of PHST.

Genome-Wide Association Mapping for Tolerance to Preharvest Sprouting and Low Falling Numbers in Wheat.

Preharvest sprouting (PHS), the germination of grain on the mother plant under cool and wet conditions, is a recurring problem for wheat farmers worldwide. α-amylase enzyme produced during PHS degrades starch resulting in baked good with poor end-use quality. The Hagberg-Perten Falling Number (FN) test is used to measure this problem in the wheat industry, and determines how much a farmer's wheat is discounted for PHS damage. PHS tolerance is associated with higher grain dormancy. Thus, breeding programs use germination-based assays such as the spike-wetting test to measure PHS susceptibility. Association mapping identified loci associated with PHS tolerance in U.S. Pacific Northwest germplasm based both on FN and on spike-wetting test data. The study was performed using a panel of 469 white winter wheat cultivars and elite breeding lines grown in six Washington state environments, and genotyped for 15,229 polymorphic markers using the 90k SNP Illumina iSelect array. Marker-trait associations were identified using the FarmCPU R package. Principal component analysis was directly and a kinship matrix was indirectly used to account for population structure. Nine loci were associated with FN and 34 loci associated with PHS based on sprouting scores. None of the QFN.wsu loci were detected in multiple environments, whereas six of the 34 QPHS.wsu loci were detected in two of the five environments. There was no overlap between the QTN detected based on FN and PHS, and there was little correlation between the two traits. However, both traits appear to be PHS-related since 19 of the 34 QPHS.wsu loci and four of the nine QFN.wsu loci co-localized with previously published dormancy and PHS QTL. Identification of these loci will lead to a better understanding of the genetic architecture of PHS and will help with the future development of genomic selection models.

Targeted Haplotype Comparisons between South African Wheat Cultivars Appear Predictive of Pre-harvest Sprouting Tolerance.

Pre-harvest sprouting (PHS) has been a serious production constraint for over two decades, especially in the summer rainfall wheat production regions of South Africa. It is a complex genetic trait controlled by multiple genes, which are significantly influenced by environmental conditions. This complicates the accurate prediction of a cultivar's stability in terms of PHS tolerance. A number of reports have documented the presence of major QTL on chromosomes 3A and 4A of modern bread wheat cultivars, which confer PHS tolerance. In this study, the SSR marker haplotype combination of chromosomes 3A and 4A of former and current South African cultivars were compared with the aim to select for improved PHS tolerance levels in future cultivars. A total of 101 wheat cultivars, including a susceptible cultivar and five international tolerant sources, were used in this study. These cultivars and donors were evaluated for their PHS tolerance by making use of a rain simulator. In addition, five seeds of each entry were planted out into seedling trays and leaf material harvested for DNA isolation. A modified CTAB extraction method was used before progressing to downstream PCR applications. Eight SSR markers targeted from the well-characterized 3A and 4A QTL regions associated with PHS tolerance, were used to conduct targeted haplotype analysis. Additionally, recently published KASP SNP markers, which identify the casual SNP mutations within the TaPHS1 gene, were used to genotype the germplasm. The haplotype marker data and phenotypic PHS data were compared across all cultivars and different production regions. A relative change in observed phenotypic variation percentage was obtained per marker allele and across marker haplotype combinations when compared to the PHS susceptible cultivar, Tugela-DN. Clear favorable haplotypes, contributing 40–60% of the variation for PHS tolerance, were identified for QTL 3A and 4A. Initial analyses show haplotype data appear to be predictive of PHS tolerance status and germplasm can now be selected to improve PHS tolerance. These haplotype data are the first of its kind for PHS genotyping in South Africa. In future, this can be used as a tool to predict the possible PHS tolerance range of a new cultivar.