Diagnostic KASP markers differentiate Vanilla planifolia, V. odorata, V. pompona, and their hybrids using leaf or cured pod tissues.

BACKGROUND: Vanilla is a globally important spice crop used in a variety of food, cosmetic, and pharmaceutical products. V. planifolia is the primary commercial species with V. x tahitensis also permissible for food use. Other aromatic species, including V. pompona, are used for food throughout Central and South America. Supply chain complexity hinders the vanilla bean industry and can lead to false claims of genetic and geographical origins to obtain higher prices. Beans of some species can be differentiated by experienced buyers, but hybrids and morphological differences caused by environmental variability or disease would best be resolved by diagnostic tests. METHODS AND RESULTS: Kompetitive Allele Specific Polymerase Chain Reaction is a widely used molecular marker that can genotype single nucleotide polymorphisms efficiently and inexpensively. Assays were designed to differentiate V. planifolia, V. x tahitensis, and V. pompona using publicly available vanilla genomics data. Ten KASP assays on chromosomes 1 through 7, the ITS region, and plastid-encoded rbcL gene successfully differentiated V. planifolia, V. odorata, and V. x tahitensis. Additional KASP assays on chromosomes 1 through 4, the ITS region, and rbcL gene successfully differentiated V. planifolia and V. pompona. Further, a method for extracting KASP-quality DNA from cured vanilla bean seeds was developed and successfully differentiated V. planifolia, V. odorata, V. x tahitensis, V. pompona, and their hybrids. CONCLUSION: The methods and results from this study can be used to identify interspecific hybrids, ensure the authenticity of cured vanilla beans, and reduce abuse within the vanilla supply chain.

QTL mapping of yield components and kernel traits in wheat cultivars TAM 112 and Duster.

In the Southern Great Plains, wheat cultivars have been selected for a combination of outstanding yield and drought tolerance as a long-term breeding goal. To understand the underlying genetic mechanisms, this study aimed to dissect the quantitative trait loci (QTL) associated with yield components and kernel traits in two wheat cultivars `TAM 112' and `Duster' under both irrigated and dryland environments. A set of 182 recombined inbred lines (RIL) derived from the cross of TAM 112/Duster were planted in 13 diverse environments for evaluation of 18 yield and kernel related traits. High-density genetic linkage map was constructed using 5,081 single nucleotide polymorphisms (SNPs) from genotyping-by-sequencing (GBS). QTL mapping analysis detected 134 QTL regions on all 21 wheat chromosomes, including 30 pleiotropic QTL regions and 21 consistent QTL regions, with 10 QTL regions in common. Three major pleiotropic QTL on the short arms of chromosomes 2B (57.5 - 61.6 Mbps), 2D (37.1 - 38.7 Mbps), and 7D (66.0 - 69.2 Mbps) colocalized with genes Ppd-B1, Ppd-D1, and FT-D1, respectively. And four consistent QTL associated with kernel length (KLEN), thousand kernel weight (TKW), plot grain yield (YLD), and kernel spike-1 (KPS) (Qklen.tamu.1A.325, Qtkw.tamu.2B.137, Qyld.tamu.2D.3, and Qkps.tamu.6A.113) explained more than 5% of the phenotypic variation. QTL Qklen.tamu.1A.325 is a novel QTL with consistent effects under all tested environments. Marker haplotype analysis indicated the QTL combinations significantly increased yield and kernel traits. QTL and the linked markers identified in this study will facilitate future marker-assisted selection (MAS) for pyramiding the favorable alleles and QTL map-based cloning.

Genotyping-by-sequencing and genomic selection applications in hexaploid triticale.

Triticale, a hybrid species between wheat and rye, is one of the newest additions to the plant kingdom with a very short history of improvement. It has very limited genomic resources because of its large and complex genome. Objectives of this study were to generate dense marker data, understand genetic diversity, population structure, linkage disequilibrium (LD), and estimate accuracies of commonly used genomic selection (GS) models on forage yield of triticale. Genotyping-by-sequencing (GBS), using PstI and MspI restriction enzymes for reducing genome complexity, was performed on a triticale diversity panel (n = 289). After filtering for biallelic loci with more than 70% genome coverage, and minor allele frequency (MAF) > 0.05, de novo variant calling identified 16,378 single nucleotide polymorphism (SNP) markers. Sequences of these variants were mapped to wheat and rye reference genomes to infer their homologous groups and chromosome positions. About 45% (7430), and 58% (9500) of the de novo identified SNPs were mapped to the wheat and rye reference genomes, respectively. Interestingly, 28.9% (2151) of the 7430 SNPs were mapped to the D genome of hexaploid wheat, indicating substantial substitution of the R genome with D genome in cultivated triticale. About 27% of marker pairs were in significant LD with an average r2 > 0.18 (P < 0.05). Genome-wide LD declined rapidly to r2 < 0.1 beyond 10 kb physical distance. The three sub-genomes (A, B, and R) showed comparable LD decay patterns. Genetic diversity and population structure analyses identified five distinct clusters. Genotype grouping did not follow prior winter vs spring-type classification. However, one of the clusters was largely dominated by winter triticale. GS accuracies were estimated for forage yield using three commonly used models with different training population sizes and marker densities. GS accuracy increased with increasing training population size while gain in accuracy tended to plateau with marker densities of 2000 SNPs or more. Average GS accuracy was about 0.52, indicating the potential of using GS in triticale forage yield improvement.

Genome-Wide Association Mapping of Seedling Drought Tolerance in Winter Wheat.

In the southern Great Plains of the United States, winter wheat grown for dual-purpose is often planted early, which puts it at risk for drought stress at the seedling stage in the autumn. To map quantitative trait loci (QTL) associated with seedling drought tolerance, a genome-wide association study (GWAS) was performed on a hard winter wheat association mapping panel. Two sets of plants were planted in the greenhouse initially under well-watered conditions. At the five-leaf stage, one set continued to receive the optimum amount of water, whereas watering was withdrawn from the other set (drought stress treatment) for 14 days to mimic drought stress. Large phenotypic variation was observed in leaf chlorophyll content, leaf chlorophyll fluorescence, shoot length, number of leaves per seedling, and seedling recovery. A mixed linear model analysis detected multiple significant QTL associated with seedling drought tolerance-related traits on chromosomes 1B, 2A, 2B, 2D, 3A, 3B, 3D, 4B, 5A, 5B, 6B, and 7B. Among those, 12 stable QTL responding to drought stress for various traits were identified. Shoot length and leaf chlorophyll fluorescence were good indicators in responding to drought stress because most of the drought responding QTL detected using means of these two traits were also detected in at least two experimental repeats. These stable QTL are more valuable for use in marker-assisted selection during wheat breeding. Moreover, different traits were mapped on several common chromosomes, such as 1B, 2B, 3B, and 6B, and two QTL clusters associated with three or more traits were located at 107-130 and 80-83 cM on chromosomes 2B and 6B, respectively. Furthermore, some QTL detected in this study co-localized with previously reported QTL for root and shoot traits at the seedling stage and canopy temperature at the grain-filling stage of wheat. In addition, several of the mapped chromosomes were also associated with drought tolerance during the flowering or grain-filling stage in wheat. Some significant single-nucleotide polymorphisms (SNPs) were aligned to candidate genes playing roles in plant abiotic stress responses. The SNP markers identified in this study will be further validated and used for marker-assisted breeding of seedling drought tolerance during dual-purpose wheat breeding.

Crop breeding has increased the productivity and leaf wax n-alkane concentration in a series of five winter wheat cultivars developed over the last 60 years.

Plant wax n-alkanes are a major constituent of the leaf and grain surface. In this study, we explored what can be learned from the abundance and carbon isotopic composition (δ13C) of n-alkanes in historical winter wheat cultivars. We investigated leaf and grain wax n-alkane concentration (ΣalkLand ΣalkG) and carbon isotopes (δ13CalkL and δ13CalkG) on C29 as well as bulk leaf and grain carbon isotopes (δ13CbulkL and δ13CbulkG) to assess if these wax components changed across five wheat cultivars released from the 1950s to the early 2010s. Results showed that ΣalkL and grain yield increased, while δ13CalkL and δ13CbulkL decreased across the historical wheat cultivars. We found a significant correlation between ΣalkL and shoot biomass at the early growth stage, and a strong correlation between ΣalkL at the grain-filling stage and grain yield. Grain measures, including ΣalkG, δ13CalkG, and δ13CbulkG did not correlate with crop production. Although δ13CalkL and grain yield were not correlated at the flowering stage, they were correlated at the grain-filling stage under dry conditions. Our results indicate that increased ΣalkL has been indirectly selected in breeding efforts to improve crop production in winter wheat, suggesting that greater leaf waxiness confers advantages for crop growth.

Genome-Wide Association Mapping of Seedling Heat Tolerance in Winter Wheat.

Heat stress during the seedling stage of early-planted winter wheat (Triticum aestivum L.) is one of the most abiotic stresses of the crop restricting forage and grain production in the Southern Plains of the United States. To map quantitative trait loci (QTLs) and identify single-nucleotide polymorphism (SNP) markers associated with seedling heat tolerance, a genome-wide association mapping study (GWAS) was conducted using 200 diverse representative lines of the hard red winter wheat association mapping panel, which was established by the Triticeae Coordinated Agricultural Project (TCAP) and genotyped with the wheat iSelect 90K SNP array. The plants were initially planted under optimal temperature conditions in two growth chambers. At the three-leaf stage, one chamber was set to 40/35°C day/night as heat stress treatment, while the other chamber was kept at optimal temperature (25/20°C day/night) as control for 14 days. Data were collected on leaf chlorophyll content, shoot length, number of leaves per seedling, and seedling recovery after removal of heat stress treatment. Phenotypic variability for seedling heat tolerance among wheat lines was observed in this study. Using the mixed linear model (MLM), we detected multiple significant QTLs for seedling heat tolerance on different chromosomes. Some of the QTLs were detected on chromosomes that were previously reported to harbor QTLs for heat tolerance during the flowering stage of wheat. These results suggest that some heat tolerance QTLs are effective from the seedling to reproductive stages in wheat. However, new QTLs that have never been reported at the reproductive stage were found responding to seedling heat stress in the present study. Candidate gene analysis revealed high sequence similarities of some significant loci with candidate genes involved in plant stress responses including heat, drought, and salt stress. This study provides valuable information about the genetic basis of seedling heat tolerance in wheat. To the best of our knowledge, this is the first GWAS to map QTLs associated with seedling heat tolerance targeting early planting of dual-purpose winter wheat. The SNP markers identified in this study will be used for marker-assisted selection (MAS) of seedling heat tolerance during dual-purpose wheat breeding.