Introgression of the Powdery Mildew Resistance Genes Pm60 and Pm60b from Triticum urartu to Common Wheat Using Durum as a ‘Bridge’

Powdery mildew, caused by the fungus Blumeria graminis f. sp. tritici (Bgt), has limited wheat yields in many major wheat-production areas across the world. Introducing resistance genes from wild relatives into cultivated wheat can enrich the genetic resources for disease resistance breeding. The powdery mildew resistance gene Pm60 was first identified in diploid wild wheat Triticum urartu (T. urartu). In this study, we used durum as a ‘bridge’ approach to transfer Pm60 and Pm60b into hexaploid common wheat. Synthetic hexaploid wheat (SHW, AABBAuAu), developed by crossing T. urartu (AuAu) with durum (AABB), was used for crossing and backcrossing with common wheat. The Pm60 alleles were tracked by molecular markers and the resistance to powdery mildew. From BC1F1 backcross populations, eight recombinant types were identified based on five Pm60-flanking markers, which indicated different sizes of the introgressed chromosome segments from T. urartu. Moreover, we have selected two resistance-harboring introgression lines with high self-fertility, which could be easily used in wheat breeding system. Our results showed that the durum was an excellent ‘bridge’ for introducing the target gene from diploid T. urartu into the hexaploid cultivated wheat. Moreover, these introgression lines could be deployed in wheat resistance breeding programs, together with the assistance of the molecular markers for Pm60 alleles.

Analysis of Salinity Tolerance in Tomato Introgression Lines Based on Morpho-Physiological and Molecular Traits

The development of salt-tolerant tomato genotypes is a basic requirement to overcome the challenges of tomato production under salinity in the field or soil-free farming. Two groups of eight tomato introgression lines (ILs) each, were evaluated for salinity tolerance. Group-I and the group-II resulted from the following crosses respectively: Solanum lycopersicum cv-6203 × Solanum habrochaites and Solanum lycopersicum M82 × Solanum pennellii. Salt tolerance level was assessed based on a germination percentage under NaCl (0, 75, 100 mM) and in the vegetative stage using a hydroponic growing system (0, 120 mM NaCl). One line from group I (TA1648) and three lines from group II (IL2-1, IL2-3, and IL8-3) were shown to be salt-tolerant since their germination percentages were significantly higher at 75 and 100 mM NaCl than that of their respective cultivated parents cvE6203 and cvM82. Using the hydroponic system, IL TA1648 and IL 2-3 showed the highest value of plant growth traits and chlorophyll concentration. The expression level of eight salt-responsive genes in the leaves and roots of salt-tolerant ILs (TA1648 and IL 2-3) was estimated. Interestingly, SlSOS1, SlNHX2, SlNHX4, and SlERF4 genes were upregulated in leaves of both TA1648 and IL 2-3 genotypes under NaCl stress. While SlHKT1.1, SlNHX2, SlNHX4, and SlERF4 genes were upregulated under salt stress in the roots of both TA1648 and IL 2-3 genotypes. Furthermore, SlSOS2 and SlSOS3 genes were upregulated in TA1648 root and downregulated in IL 2-3. On the contrary, SlSOS1 and SlHKT1.2 genes were upregulated in the IL 2-3 root and downregulated in the TA1648 root. Monitoring of ILs revealed that some of them have inherited salt tolerance from S. habrochaites and S. pennellii genetic background. These ILs can be used in tomato breeding programs to develop salt-tolerant tomatoes or as rootstocks in grafting techniques under saline irrigation conditions.

Whole genome sequencing uncovers the structural and transcriptomic landscape of hexaploid wheat/Am. muticum introgression lines

Wheat is a globally vital crop, but its limited genetic variation creates a challenge for breeders aiming to maintain or accelerate agricultural improvements over time. Introducing novel genes and alleles from wheat's wild relatives into the wheat breeding pool via introgression lines is an important component of overcoming this low variation but is limited by poor genomic resolution and limited understanding of the genomic impact of introgression breeding. By sequencing 17 hexaploid wheat/Ambylopyrum muticum introgression lines and the parent lines, we have precisely pinpointed the borders of introgressed segments. We report a genome assembly and annotation of Am. muticum that has facilitated the identification of Am. muticum resistance genes commonly introgressed in lines resistant to stripe rust. Our analysis has identified an abundance of structural disruption and homoeologous pairing across the introgression lines, likely caused by the suppressed Ph1 locus. mRNAseq analysis of six of these introgression lines revealed that introgressed genes tend to be downregulated, shifting the expression balance of triads towards suppression of the introgressed region, with no discernible compensation in the expression of the homoeologous copies. This analysis explores the genomic impact of introgression breeding and provides an affordable way for breeders to better characterise introgression lines and more effectively deploy wild relative variation.

Unravelling genetic variability for moisture stress tolerance in Indian mustard and identification of high breeding value donor lines

Aim: Identification of high breeding value donor lines harbouring tolerance to moisture stress from diversity stock of 443 genotypes of Brassica juncea. Methodology: Germplasm stock of 443 Indian mustard (Brassica juncea L.) genotypes, comprising introgression lines, land races, old cultivars and advance breeding lines, were evaluated under two environments viz., irrigated and rainfed conditions to check the variability for yield and related traits. Statistical software META-Rver 6.0 (Multi Environment Trial Analysis using R) was used for computation of Best Linear Unbiased Predictions, Analysis of Variance (ANOVA), Genetic Correlations and Heritability. Drought susceptibility index and per cent seed yield reduction under rainfed conditions were used as indicators to determine moisture stress tolerance in genotypes. Results: The variability for moisture stress tolerance has been unravelled in a diverse genetic stock of Brassica juncea under two environments: irrigated and rainfed. For high prediction accuracy, the ranking of genotypes was done based on Best Linear Unbiased Predictions for seed yield and its component traits. PBR-378, RGN-329, RB-73, RB-50 and PBR-422 and Giriraj were identified as moisture stress tolerant genotypes. One land race Sahib 36 and three introgression lines viz., MCP 12-211, PTJ-3-69 and MSC-3 have also been identified as potential genetic resources for moisture stress tolerance. Principal component analysis based on biplots depicted specific distribution of variables for each environment. Interpretation: This study led to the identification of potential donors for moisture stress tolerance with high predictive accuracy. Low drought susceptibility index and high breeding value in a land race and three introgression lines derived from Erucastrum cardaminoides, B. tournefortii and B. carinata emphasized their utilization as potential genetic resources to breed for moisture stress tolerance in B. juncea.

Detection and Verification of QTL for Salinity Tolerance at Germination and Seedling Stages Using Wild Barley Introgression Lines

Salinity is one of the major environmental factors that negatively affect crop development, particularly at the early growth stage of a plant and consequently the final yield. Therefore, a set of 50 wild barley (Hordeum vulgare ssp. spontaneum, Hsp) introgression lines (ILs) was used to detect QTL alleles improving germination and seedling growth under control, 75 mM, and 150 mM NaCl conditions. Large variation was observed for germination and seedling growth related traits that were highly heritable under salinity stress. In addition, highly significant differences were obtained for five salinity tolerance indices and between treatments as well. A total of 90 and 35 significant QTL were identified for ten investigated traits and for tolerance indices, respectively. The Hsp introgression alleles are involved in improving salinity tolerance at forty (43.9%) out of 90 QTL including introgression lines S42IL-109 (2H), S42IL-116 (4H), S42IL-132 (6H), S42IL-133 (7H), S42IL-148 (6H), and S42IL-176 (5H). Interestingly, seven exotic QTL alleles were successfully validated in the wild barley ILs including S42IL-127 (5H), 139 (7H), 125 (5H), 117 (4H), 118 (4H), 121 (4H), and 137 (7H). We conclude that the barley introgression lines contain numerous germination and seedling growth-improving novel QTL alleles, which are effective under salinity conditions.

A High-Throughput Skim-sequencing Approach for Genotyping, Dosage Estimation and Identifying Translocations

The development of next generation sequencing (NGS) enabled a shift from array-based genotyping to high-throughput genotyping by directly sequencing genomic libraries. Even though whole genome sequencing was initially too costly for routine analysis in large populations, such as those utilized for breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to utilize whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage, a limitation comes in the time and high cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on amount of data required and extended to 3,072 samples or more. Panels of double haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T. aestivum x Hordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1x down to 0.01x per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the low coverage skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

A novel approach to develop wheat chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in doubled haploid introgression lines

Many wild relative species are being used in pre-breeding programmes to increase the genetic diversity of wheat. Genotyping tools such as single nucleotide polymorphism (SNP)-based arrays and molecular markers have been widely used to characterise wheat-wild relative introgression lines. However, due to the polyploid nature of the recipient wheat genome, it is difficult to develop SNP-based KASP markers that are codominant to track the introgressions from the wild species. Previous attempts to develop KASP markers have involved both exome- and PCR-amplicon-based sequencing of the wild species. But chromosome-specific KASPs assays have been hindered by homoeologous SNPs within the wheat genome. This study involved whole genome sequencing of the diploid wheat wild relative Amblyopyrum muticum and development of a SNP discovery pipeline that generated ~38,000 SNPs in single-copy wheat genome sequences. New assays were designed to increase the density of Am. muticum polymorphic KASP markers. With a goal of one marker per 60 Mbp, 335 new KASP assays were validated as functional. Together with assays validated in previous studies, 498 well distributed chromosome-specific markers were used to recharacterize previously genotyped wheat-Am. muticum doubled haploid (DH) introgression lines. The chromosome specific nature of the KASP markers allowed clarification of which wheat chromosomes were involved with recombination events or substituted with Am. muticum chromosomes and the higher density of markers allowed detection of new small introgressions in these DH lines.

Assessment of introgression wheat lines for yield, protein yield and resistance to diseases

Aim. To determine the value of breeding traits from new sources by studying the resistance to diseases, protein content and their relationship with the productivity of introgression wheat lines. Methods. Growing plants under infection backgrounds of wheat diseases was combined with the laboratory methods for determining protein content and weight of 1000 kernels. Results. Disease resistance has depended on the pathogen species and the source of alien variability. Prolonged resistance to stem rust has been observed only among the derivatives of the collection sample H74/90-245, containing the translocation 1BL.1RS in the karyotype and T. timopheevii in the pedigree. Derivatives Ae. tauschii gradually lost the resistance to the time of full maturation. Conclusions. The parameters of plant resistance to diseases, protein content and yield, absolute protein content in 1000 kernels should be used for the material selection. The best lines have been selected from 736 introgression lines. They characterized by high productivity in some years, large grain, high protein content, disease resistance, adaptability to local environments and devoid of the wild species negative qualities. The lines are of interest for further breeding work in the south of Ukraine. Lines with leaf pubescence from T. timopheevii and with modified translocation 1BL.1RSm were characterized by low productivity.Keywords: wheat, introgression lines, disease resistance, protein content, productivity.