Development of white-grain pre-harvest sprouting tolerant and pyramided protein-rich leaf rust resistant wheats using molecular breeding

The present study was undertaken for developing pre-harvest sprouting tolerant (PHST) wheat genotypes using marker-assisted backcross breeding (MABB). A major QTL for PHST was introgressed into an elite Indian wheat cv. Lok1 that is PHS susceptible. These PHST lines were also pyramided with one gene each for high grain protein content (Gpc-B1) and leaf rust resistance (Lr24). For introgression of PHST QTL, initially Lok1 was separately crossed with each of the two donors (PHS tolerant white-grained AUS1408 and CN19055). Backcrossing in each generation was followed by foreground and background selections using SSR markers. In advanced lines, KASP assay was also carried out for the candidate gene TaMKK3-A underlying the PHST QTL. The MAS derived lines homozygous for PHST QTL were screened for PHS using simulated rain chambers resulting in the selection of 10 PHST lines. For pyramiding of three QTL/genes (PHST QTL, Gpc-B1, and Lr24), MABB derived BC4F2 plants (from the cross Lok1/CN19055) were crossed with a MAS derived BC2F5 line [Lok1 (Gpc-B1 + Lr24)] developed earlier by us in the same background of Lok1. After foreground MAS followed by PHS screening, four advanced lines carrying all the three QTL/genes in homozygous condition were selected. These lines exhibited high level of PHST (PHS score 2–3) associated with significant improvement in GPC with no yield penalty and resistance against leaf rust under artificial epiphytotic conditions.

Development of Breeder-Friendly KASP Markers for Low Concentration of Kunitz Trypsin Inhibitor in Soybean Seeds

Trypsin inhibitors (TI), a common anti-nutritional factor in soybean, prevent animals’ protein digestibility reducing animal growth performance. No commercial soybean cultivars with low or null concentration of TI are available. The availability of a high throughput genotyping assay will be beneficial to incorporate the low TI trait into elite breeding lines. The aim of this study is to develop and validate a breeder friendly Kompetitive Allele Specific PCR (KASP) assay linked to low Kunitz trypsin inhibitor (KTI) in soybean seeds. A total of 200 F3:5 lines derived from PI 547656 (low KTI) X Glenn (normal KTI) were genotyped using the BARCSoySNP6K_v2 Beadchip. F3:4 and F3:5 lines were grown in Blacksburg and Orange, Virginia in three years, respectively, and were measured for KTI content using a quantitative HPLC method. We identified three SNP markers tightly linked to the major QTL associated to low KTI in the mapping population. Based on these SNPs, we developed and validated the KASP assays in a set of 93 diverse germplasm accessions. The marker Gm08_44814503 has 86% selection efficiency for the accessions with low KTI and could be used in marker assisted breeding to facilitate the incorporation of low KTI content in soybean seeds.

Development and Utilization of the Functional Co-dominant KASP Marker for Thermo-sensitive Genic Male Sterility in Rice

Thermos-sensitive genic male sterility (TGMS) is the important genetic resource in two-line hybrid rice breeding. The pollen fertility of TGMS lines is regulated by a single point mutation on the TGMS genes. Based on the single nucleotide polymorphism (SNP) and indel, KASP markers were developed and utilized in rice molecular breeding due to high-through detection with low cost and save time. In this study, we convert the SNPs on TGMS genes including p/tms12-1 and tms9-1 gene to the functional co-dominant KASP marker and used in two-line hybrid rice breeding. We can differentiate the TGMS lines conferring p/tms12-1 or tms9-1 from another TGMS lines by KASP assay. Of them, the genotype of Pei'ai64S and Hua201S containing p/tms12-1 is homozygous GG genotype with blue signal, and the genotype of HengnongS-1 containing tms9-1 is homozygous CC genotype with red signal. KASP assay for tms9-1 gene was proved that the genetic mode is fit to one recessive Mendalian trait. The test of seed purity was performed by KASP marker for the two-line hybrid varieties of Liangyoupeijiu and Hualiangyou1206, which is consistent with the previous dCAPS marker. Moreover, the tms12-1 and tms9-1 gene were pyramided in the same genetic background and the new TGMS lines were generated. Therefore, the KASP marker for TGMS genes developed in this study can be widely used in two-line hybrid rice breeding. It will provide a visual convenient toolkit for breeders to select the target individual plant by the high-throughput detection in the different two-line rice breeding program.

New Transcriptome-Based SNP Markers for Noug (Guizotia abyssinica) and Their Conversion to KASP Markers for Population Genetics Analyses

The development and use of genomic resources are essential for understanding the population genetics of crops for their efficient conservation and enhancement. Noug (Guizotia abyssinica) is an economically important oilseed crop in Ethiopia and India. The present study sought to develop new DNA markers for this crop. Transcriptome sequencing was conducted on two genotypes and 628 transcript sequences containing 959 single nucleotide polymorphisms (SNPs) were developed. A competitive allele-specific PCR (KASP) assay was developed for the SNPs and used for genotyping of 24 accessions. A total of 554 loci were successfully genotyped across the accessions, and 202 polymorphic loci were used for population genetics analyses. Polymorphism information content (PIC) of the loci varied from 0.01 to 0.37 with a mean of 0.24, and about 49% of the loci showed significant deviation from the Hardy-Weinberg equilibrium. The mean expected heterozygosity was 0.27 suggesting moderately high genetic variation within accessions. Low but significant differentiation existed among accessions (FST = 0.045, p < 0.0001). Landrace populations from isolated areas may have useful mutations and should be conserved and used in breeding this crop. The genomic resources developed in this study were shown to be useful for population genetics research and can also be used in, e.g., association genetics.

Presence of the HPPD Inhibitor Sensitive 1 Gene and ALSS653N Mutation in Weedy Oryza sativa Sensitive to Benzobicyclon

Benzobicyclon has shown varying results in controlling weedy rice, including those with imidazolinone (IMI) resistance. Tolerance to benzobicyclon in cultivated japonica rice, but not indica or aus-like cultivars, is conferred by a fully functional HPPD Inhibitor Sensitive 1 (HIS1) gene. Herein, a diagnostic Kompetitive Allele Specific PCR (KASP) assay was developed to predict the HIS1 genotype of weedy rice plants from 37 accessions and correlated to their response to benzobicyclon in the field. Two-thirds of the 693 weedy rice plants screened were tolerant to benzobicyclon (371 g ai ha−1, SC formulation) at 30 days after treatment (DAT). Thirty-four percent of plants were homozygous for the HIS1 allele and 98% of these plants exhibited field tolerance. However, the his1 genotype did not always correlate with field data. Only 52% of his1 plants were considered sensitive, indicating that the single nucleotide polymorphisms (SNPs) chosen in the KASP assay are not a reliable tool in predicting his1 homozygous plants. In an additional experiment, 86% of the 344 plants with at least one copy of the ALSS653N trait harbored a HIS1 allele, suggesting fields infested with IMI herbicide-resistant weedy rice are unlikely to be controlled with benzobicyclon.

KASP Genotyping as a Molecular Tool for Diagnosis of Cassava-Colonizing Bemisia tabaci

Bemisia tabaci is a cryptic species complex that requires the use of molecular tools for identification. The most widely used approach for achieving this is the partial sequencing of the mitochondrial DNA cytochrome oxidase I gene (COI). A more reliable single nucleotide polymorphism (SNP)-based genotyping approach, using Nextera restriction-site-associated DNA (NextRAD) sequencing, has demonstrated the existence of six major haplogroups of B. tabaci on cassava in Africa. However, NextRAD sequencing is costly and time-consuming. We, therefore, developed a cheaper and more rapid diagnostic using the Kompetitive Allele-Specific PCR (KASP) approach. Seven sets of primers were designed to distinguish the six B. tabaci haplogroups based on the NextRAD data. Out of the 152 whitefly samples that were tested using these primer sets, 151 (99.3%) produced genotyping results consistent with NextRAD. The KASP assay was designed using NextRAD data on whiteflies from cassava in 18 countries across sub-Saharan Africa. This assay can, therefore, be routinely used to rapidly diagnose cassava B. tabaci by laboratories that are researching or monitoring this pest in Africa. This is the first study to develop an SNP-based assay to distinguish B. tabaci whiteflies on cassava in Africa, and the first application of the KASP technique for insect identification.

Phylogeny and distribution of Y-chromosomal haplotypes in domestic, ancient and wild goats

The male-specific part of the Y-chromosome is in mammalian and many other species the longest haplotype that is inherited without recombination. By its paternal transmission it has a small effective population size in species with dominant males. In several species, Y-chromosomal haplotypes are sensitive markers of population history and introgression. Previous studies have identified in domestic goats four major Y-chromosomal haplotypes Y1A, Y1B, Y2A and Y2B with a marked geographic differentiation and several regional variants. In this study we used published whole-genome sequences of 70 male goats from 16 modern breeds, 11 ancient-DNA samples and 29 samples from seven wild goat species. We identified single-copy male-specific SNPs in four scaffolds, containing SRY, ZFY, DBY with SSX3Y and UTY, and USP9Y with UMN2001, respectively. Phylogenetic analyses indicated haplogroups corresponding to the haplotypes Y1B, Y2A and Y2B, respectively, but Y1A was split into Y1AA and Y1AB. All haplogroups were detected in ancient DNA samples from southeast Europe and, with the exception of Y1AB, in the bezoar goat, which is the wild ancestor of the domestic goats. Combining these data with those of previous studies and with genotypes obtained by Sanger sequencing or the KASP assay yielded haplogroup distributions for 132 domestic breeds or populations. The phylogeographic differentiation indicated paternal population bottlenecks on all three continents. This possibly occurred during the Neolithic introductions of domestic goats to those continents with a particularly strong influence in Europe along the Danubian route. This study illustrates the power of the Y-chromosomal haplotype for the reconstructing the history of mammalian species with a wide geographic range.

KASPspoon: an in vitro and in silico PCR analysis tool for high-throughput SNP genotyping

Motivation Fine mapping becomes a routine trial following quantitative trait loci (QTL) mapping studies to shrink the size of genomic segments underlying causal variants. The availability of whole genome sequences can facilitate the development of high marker density and predict gene content in genomic segments of interest. Correlations between genetic and physical positions of these loci require handling of different experimental genetic data types, and ultimately converting them into positioning markers using a routine and efficient tool. Results To convert classical QTL markers into KASP assay primers, KASPspoon simulates a PCR by running an approximate-match searching analysis on user-entered primer pairs against the provided sequences, and then comparing in vitro and in silico PCR results. KASPspoon reports amplimers close to or adjoining genes/SNPs/simple sequence repeats and those that are shared between in vitro and in silico PCR results to select the most appropriate amplimers for gene discovery. KASPspoon compares physical and genetic maps, and reports the primer set genome coverage for PCR-walking. KASPspoon could be used to design KASP assay primers to convert QTL acquired by classical molecular markers into high-throughput genotyping assays and to provide major SNP resource for the dissection of genotypic and phenotypic variation. In addition to human-readable output files, KASPspoon creates Circos configurations that illustrate different in silico and in vitro results. Availability and implementation Code available under GNU GPL at (http://www.ageri.sci.eg/index.php/facilities-services/ageri-softwares/kaspspoon). Supplementary information Supplementary data are available at Bioinformatics online.