Vijay K. Tiwari

Mapping of Quantitative Trait Loci for Grain Iron and Zinc Concentration in Diploid A Genome Wheat

Micronutrients, especially iron (Fe) and zinc (Zn), are deficient in the diets of people in underdeveloped countries. Biofortification of food crops is the best approach for alleviating the micronutrient deficiencies. Identification of germplasm with high grain Fe and Zn and understanding the genetic basis of their accumulation are the prerequisites for manipulation of these micronutrients. Some wild relatives of wheat were found to have higher grain Fe and Zn concentrations compared with the cultivated bread wheat germplasm. One accession of Triticum boeoticum (pau5088) that had relatively higher grain Fe and Zn was crossed with Triticum monococcum (pau14087), and a recombinant inbred line (RIL) population generated from this cross was grown at 2 locations over 2 years. The grains of the RIL population were evaluated for Fe and Zn concentration using atomic absorption spectrophotometer. The grain Fe and Zn concentrations in the RIL population ranged from 17.8 to 69.7 and 19.9 to 64.2 mg/kg, respectively. A linkage map available for the population was used for mapping quantitative trait loci (QTL) for grain Fe and Zn accumulation. The QTL analysis led to identification of 2 QTL for grain Fe on chromosomes 2A and 7A and 1 QTL for grain Zn on chromosome 7A. The grain Fe QTL were mapped in marker interval Xwmc382-Xbarc124 and Xgwm473-Xbarc29, respectively, each explaining 12.6% and 11.7% of the total phenotypic variation and were designated as QFe.pau-2A and QFe.pau-7A. The QTL for grain Zn, which mapped in marker interval Xcfd31-Xcfa2049, was designated as QZn.pau-7A and explained 18.8% of the total phenotypic variation.

SNP Discovery for mapping alien introgressions in wheat.

BackgroundMonitoring alien introgressions in crop plants is difficult due to the lack of genetic and molecular mapping information on the wild crop relatives. The tertiary gene pool of wheat is a very important source of genetic variability for wheat improvement against biotic and abiotic stresses. By exploring the 5Mg short arm (5MgS) of Aegilops geniculata, we can apply chromosome genomics for the discovery of SNP markers and their use for monitoring alien introgressions in wheat (Triticum aestivum L).ResultsThe short arm of chromosome 5Mg of Ae. geniculata Roth (syn. Ae. ovata L.; 2n = 4x = 28, UgUgMgMg) was flow-sorted from a wheat line in which it is maintained as a telocentric chromosome. DNA of the sorted arm was amplified and sequenced using an Illumina Hiseq 2000 with ~45x coverage. The sequence data was used for SNP discovery against wheat homoeologous group-5 assemblies. A total of 2,178 unique, 5MgS-specific SNPs were discovered. Randomly selected samples of 59 5MgS-specific SNPs were tested (44 by KASPar assay and 15 by Sanger sequencing) and 84% were validated. Of the selected SNPs, 97% mapped to a chromosome 5Mg addition to wheat (the source of t5MgS), and 94% to 5Mg introgressed from a different accession of Ae. geniculata substituting for chromosome 5D of wheat. The validated SNPs also identified chromosome segments of 5MgS origin in a set of T5D-5Mg translocation lines; eight SNPs (25%) mapped to TA5601 [T5DL · 5DS-5MgS(0.75)] and three (8%) to TA5602 [T5DL · 5DS-5MgS (0.95)]. SNPs (gsnp_5ms83 and gsnp_5ms94), tagging chromosome T5DL · 5DS-5MgS(0.95) with the smallest introgression carrying resistance to leaf rust (Lr57) and stripe rust (Yr40), were validated in two released germplasm lines with Lr57 and Yr40 genes.ConclusionThis approach should be widely applicable for the identification of species/genome-specific SNPs. The development of a large number of SNP markers will facilitate the precise introgression and monitoring of alien segments in crop breeding programs and further enable mapping and cloning novel genes from the wild relatives of crop plants.

Development of Triticum turgidum subsp. durum – Aegilops longissima amphiploids with high iron and zinc content through unreduced gamete formation in F1 hybrids

Four different interspecific hybrids involving three different accessions of Aegilops longissima Schweinf. & Muschl. with high grain iron and zinc content and three Triticum turgidum L. subsp. durum (Desf.) Husn. cultivars with low micronutrient content were made for durum wheat biofortification and investigated for chromosome pairing, fertility, putative amphiploidy, and micronutrient content. The chromosome pairing in the 21-chromosome F1 hybrids (ABSl) consisted of 0-6 rod bivalents and occasionally 1 trivalent. All the F1 hybrids, however, unexpectedly showed partial but variable fertility. The detailed meiotic investigation indicated the simultaneous occurrence of two types of aberrant meiotic divisions, namely first-division restitution and single-division meiosis, leading to regular dyads and unreduced gamete formation and fertility. The F2 seeds, being putative amphiploids (AABBSlSl), had nearly double the chromosome number (40-42) and regular meiosis and fertility. The F1 hybrids were intermediate between the two parents for different morphological traits. The putative amphiploids with bold seed size had higher grain ash content and ash iron and zinc content than durum wheat cultivars, suggesting that Ae. longissima possesses a better genetic system(s) for uptake and seed sequestration of iron and zinc, which could be transferred to elite durum and bread wheat cultivars and exploited.

De Novo Transcriptome Assembly and Analyses of Gene Expression during Photomorphogenesis in Diploid Wheat Triticum monococcum

Background Triticum monococcum (2n) is a close ancestor of T. urartu, the A-genome progenitor of cultivated hexaploid wheat, and is therefore a useful model for the study of components regulating photomorphogenesis in diploid wheat. In order to develop genetic and genomic resources for such a study, we constructed genome-wide transcriptomes of two Triticum monococcum subspecies, the wild winter wheat T. monococcum ssp. aegilopoides (accession G3116) and the domesticated spring wheat T. monococcum ssp. monococcum (accession DV92) by generating de novo assemblies of RNA-Seq data derived from both etiolated and green seedlings. Principal Findings The de novo transcriptome assemblies of DV92 and G3116 represent 120,911 and 117,969 transcripts, respectively. We successfully mapped ∼90% of these transcripts from each accession to barley and ∼95% of the transcripts to T. urartu genomes. However, only ∼77% transcripts mapped to the annotated barley genes and ∼85% transcripts mapped to the annotated T. urartu genes. Differential gene expression analyses revealed 22% more light up-regulated and 35% more light down-regulated transcripts in the G3116 transcriptome compared to DV92. The DV92 and G3116 mRNA sequence reads aligned against the reference barley genome led to the identification of ∼500,000 single nucleotide polymorphism (SNP) and ∼22,000 simple sequence repeat (SSR) sites. Conclusions De novo transcriptome assemblies of two accessions of the diploid wheat T. monococcum provide new empirical transcriptome references for improving Triticeae genome annotations, and insights into transcriptional programming during photomorphogenesis. The SNP and SSR sites identified in our analysis provide additional resources for the development of molecular markers.

Bioavailability of Iron from Wheat Aegilops Derivatives Selected for High Grain Iron and Protein Contents

A coupled in vitro digestion/Caco-2 model was employed to assess iron bioavailability from wheat Aegilops derivatives selected for high iron and protein contents. The iron content in wheat genotypes used in this study correlated to a great extent with both protein (r = 0.80) and phytate (r = 0.68) contents. The iron bioavailability was based on Caco-2 cell ferritin formation from cooked digests of these derivatives (relative to WL711 control) and correlated positively with dialyzable iron (r = 0.63) and total iron content (r = 0.38) but not with the phytate content. The apparently decreased phytate/iron molar ratios, however, correlated negatively (r = -0.42) with the iron bioavailability, justifying the utilization of these parameters in biofortification programs. Iron bioavailability in the derivatives increased up to 1.5-fold, corresponding to a 1.5-2.2-fold increase observed in iron content over control. These data suggest that biofortification for iron proportionately leading to higher iron bioavailability will be the most feasible and cost-effective approach to combat micronutrient deficiency.

Endosperm tolerance of paternal aneuploidy allows radiation hybrid mapping of the wheat D-genome and a measure of γ ray-induced chromosome breaks.

Physical mapping and genome sequencing are underway for the ≈17 Gb wheat genome. Physical mapping methods independent of meiotic recombination, such as radiation hybrid (RH) mapping, will aid precise anchoring of BAC contigs in the large regions of suppressed recombination in Triticeae genomes. Reports of endosperm development following pollination with irradiated pollen at dosages that cause embryo abortion prompted us to investigate endosperm as a potential source of RH mapping germplasm. Here, we report a novel approach to construct RH based physical maps of all seven D-genome chromosomes of the hexaploid wheat ‘Chinese Spring’, simultaneously. An 81-member subset of endosperm samples derived from 20-Gy irradiated pollen was genotyped for deletions, and 737 markers were mapped on seven D-genome chromosomes. Analysis of well-defined regions of six chromosomes suggested a map resolution of ∼830 kb could be achieved; this estimate was validated with assays of markers from a sequenced contig. We estimate that the panel contains ∼6,000 deletion bins for D-genome chromosomes and will require ∼18,000 markers for high resolution mapping. Map-based deletion estimates revealed a majority of 1–20 Mb interstitial deletions suggesting mutagenic repair of double-strand breaks in pollen provides a useful resource for RH mapping and map based cloning studies.

Development and characterization of Triticum aestivum – Aegilops kotschyi amphiploids with high grain iron and zinc contents

Synthetic amphiploids between Triticum aestivum (AABBDD) landrace Chinese Spring ( Ph I ) and cultivar WL711 with different accessions of Aegilops kotschyi (UUS l S l ) were developed through colchicine treatment of sterile hybrids. The F 1 hybrids and amphiploid plants were intermediate between the parents for plant morphology and spike characteristics. Meiotic metaphase chromosome analysis of the F 1 hybrids (ABDUS l ) showed the expected chromosome number (35) and very little but variable homoeologous chromosome pairing. The amphiploids (AABBDDUUS l S l ), however, had variable frequency of univalents at meiotic metaphase-I. The SDS–PAGE of high molecular weight glutenin subunits of amphiploids along with the parents showed the presence and expression of all the parental genomes in the amphiploids. The amphiploids with seeds as large as that of wheat cultivars had higher grain, flag leaf and grain ash iron and zinc concentrations than the wheat parents and comparable with those of their Ae. kotschyi parents suggest that Ae. kotschyi possesses a distinctive genetic system for the micronutrient uptake, translocation and sequestration than the wheat cultivars. This could, however, be demonstrated unequivocally only with comprehensive data on biomass, grain yield and harvest index of the Aegilops donors and the synthetic amphiploids, which is not feasible due to their shattering and hard threshing. The use of amphiploids for the transfer of high iron and zinc concentrations and development of alien addition and substitution lines in wheat is in progress.

A whole-genome, radiation hybrid mapping resource of hexaploid wheat.

Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole-genome using these approaches is nearly impossible. We developed a whole-genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high-density single nucleotide polymorphism (SNP) array. At the whole-genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR1500 with a total map length of 6866 cR1500 . The 7296 unique mapping bins provided a five- to eight-fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species.

A Method to Produce Radiation Hybrids for the D-Genome Chromosomes of Wheat (Triticum aestivum L.)

Radiation hybrid (RH) mapping is based on radiation-induced chromosome breakage rather than meiotic recombination, as a means to induce marker segregation for mapping. To date, the implementation of this mapping approach in hexaploid (Triticum aestivum L.; 2n = 6x = 42; AABBDD) and tetraploid (T. turgidum L.; 2n = 4x = 28; AABB) wheat has concentrated on the production of mapping panels for individual chromosomes. In order to extend the usefulness of this approach, we have devised a method to produce panels for the simultaneous mapping of all chromosomes of the D subgenome of hexaploid wheat. In this approach, seeds of hexaploid wheat (AABBDD) are irradiated and the surviving plants are crossed to tetraploid wheat (AABB) to produce a mapping panel based on quasi-pentaploids (AABBD). Chromosome lesions in the A and B genomes are largely masked in the quasi-pentaploids due to the presence of A- and B-genome chromosomes from the tetraploid parent. On the other hand, the chromosomes from the D-genome are present in one copy (hemizygous) and allow radiation hybrid mapping of all D-genome chromosomes simultaneously. Our analyses showed that transmission of D-genome chromosomes was apparently normal and that radiation-induced chromosome breakage along D-genome chromosomes was homogeneous. Chromosome breakage levels between D-genome chromosomes were comparable except for chromosome 6D which suffered greater chromosome breakage. These results demonstrate the feasibility of constructing D-genome radiation hybrids (DGRHs) in wheat.

Evaluation and identification of wheat-Aegilops addition lines controlling high grain iron and zinc concentration and mugineic acid production

Iron and zinc deficiency affects more than half of the world population due to low inherent micronutrient content of cereals and other staple foods. The micronutrient deficiency is further aggravated by poor availability of these minerals in calcareous soils and their uptake by crop plants. Series of available wheat-Aegilops addition lines were evaluated for identification of alien chromosomes carrying genes for high grain iron and zinc concentrations and release of mugineic acid(s) facilitating micronutrient uptake under their deficient conditions. Addition lines of chromosome 2Sv, 2Uv and 7Uv of Ae. peregrina, 2Sl and 7Sl of Ae. longissima and 2U of Ae. umbellulata were found to carry genes for high grain iron whereas the group 7 chromosomes had genes for higher grain zinc. Higher release of mugineic acid (MA) under iron deficient condition was observed in addition lines of chromosome 2Sv, 2Uv, 4Uv and 7Sv of Ae. peregrina, 2Sl and 6Sl of Ae. longissima and 2U and 5U of Ae. umbellulata. Higher grain and...