Genome-wide association study for cashmere traits in Inner Mongolia cashmere goat population reveals new candidate genes and haplotypes.

BACKGROUND: The cashmere goat industry is one of the main pillars of animal husbandry in Inner Mongolia Autonomous Region, and plays an irreplaceable role in local economic development. With the change in feeding methods and environment, the cashmere produced by Inner Mongolia cashmere goats shows a tendency of coarser, and the cashmere yield can not meet the consumption demand of people. However, the genetic basis behind these changes is not fully understood. We measured cashmere traits, including cashmere yield (CY), cashmere diameter (CD), cashmere thickness (CT), and fleece length (FL) traits for four consecutive years, and utilized Genome-wide association study of four cashmere traits in Inner Mongolia cashmere goats was carried out using new genomics tools to infer genomic regions and functional loci associated with cashmere traits and to construct haplotypes that significantly affect cashmere traits. RESULTS: We estimated the genetic parameters of cashmere traits in Inner Mongolia cashmere goats. The heritability of cashmere yield, cashmere diameter, and fleece length traits of Inner Mongolia cashmere goats were 0.229, 0.359, and 0.250, which belonged to the medium heritability traits (0.2 ~ 0.4). The cashmere thickness trait has a low heritability of 0.053. We detected 151 genome-wide significantly associated SNPs with four cashmere traits on different chromosomes, which were very close to the chromosomes of 392 genes (located within the gene or within ± 500 kb). Notch3, BMPR1B, and CCNA2 have direct functional associations with fibroblasts and follicle stem cells, which play important roles in hair follicle growth and development. Based on GO functional annotation and KEGG enrichment analysis, potential candidate genes were associated with pathways of hair follicle genesis and development (Notch, P13K-Akt, TGF-beta, Cell cycle, Wnt, MAPK). We calculated the effective allele number of the Inner Mongolia cashmere goat population to be 1.109-1.998, the dominant genotypes of most SNPs were wild-type, the polymorphic information content of 57 SNPs were low polymorphism (0 < PIC < 0.25), and the polymorphic information content of 79 SNPs were moderate polymorphism (0.25 < PIC < 0.50). We analyzed the association of SNPs with phenotypes and found that the homozygous mutant type of SNP1 and SNP3 was associated with the highest cashmere yield, the heterozygous mutant type of SNP30 was associated with the lowest cashmere thickness, the wild type of SNP76, SNP77, SNP78, SNP80, and SNP81 was associated with the highest cashmere thickness, and the wild type type of SNP137 was associated with the highest fleece length. 21 haplotype blocks and 68 haplotype combinations were constructed. Haplotypes A2A2, B2B2, C2C2, and D4D4 were associated with increased cashmere yield, haplotypes E2E2, F1F1, G5G5, and G1G5 were associated with decreased cashmere fineness, haplotypes H2H2 was associated with increased cashmere thickness, haplotypes I1I1, I1I2, J1J4, L5L3, N3N2, N3N3, O2O1, P2P2, and Q3Q3 were associated with increased cashmere length. We verified the polymorphism of 8 SNPs by KASP, and found that chr7_g.102631194A > G, chr10_g.82715068 T > C, chr1_g.124483769C > T, chr24_g.12811352C > T, chr6_g.114111249A > G, and chr6_g.115606026 T > C were significantly genotyped in verified populations (P < 0.05). CONCLUSIONS: In conclusion, the genetic effect of single SNP on phenotypes is small, and SNPs are more inclined to be inherited as a whole. By constructing haplotypes from SNPs that are significantly associated with cashmere traits, it will help to reveal the complex and potential causal variations in cashmere traits of Inner Mongolia cashmere goats. This will be a valuable resource for genomics and breeding of the cashmere goat.

Identification of two major QTLs for pod shell thickness in peanut (Arachis hypogaea L.) using BSA-seq analysis.

BACKGROUND: Pod shell thickness (PST) is an important agronomic trait of peanut because it affects the ability of shells to resist pest infestations and pathogen attacks, while also influencing the peanut shelling process. However, very few studies have explored the genetic basis of PST. RESULTS: An F2 segregating population derived from a cross between the thick-shelled cultivar Yueyou 18 (YY18) and the thin-shelled cultivar Weihua 8 (WH8) was used to identify the quantitative trait loci (QTLs) for PST. On the basis of a bulked segregant analysis sequencing (BSA-seq), four QTLs were preliminarily mapped to chromosomes 3, 8, 13, and 18. Using the genome resequencing data of YY18 and WH8, 22 kompetitive allele-specific PCR (KASP) markers were designed for the genotyping of the F2 population. Two major QTLs (qPSTA08 and qPSTA18) were identified and finely mapped, with qPSTA08 detected on chromosome 8 (0.69-Mb physical genomic region) and qPSTA18 detected on chromosome 18 (0.15-Mb physical genomic region). Moreover, qPSTA08 and qPSTA18 explained 31.1-32.3% and 16.7-16.8% of the phenotypic variation, respectively. Fifteen genes were detected in the two candidate regions, including three genes with nonsynonymous mutations in the exon region. Two molecular markers (Tif2_A08_31713024 and Tif2_A18_7198124) that were developed for the two major QTL regions effectively distinguished between thick-shelled and thin-shelled materials. Subsequently, the two markers were validated in four F2:3 lines selected. CONCLUSIONS: The QTLs identified and molecular markers developed in this study may lay the foundation for breeding cultivars with a shell thickness suitable for mechanized peanut shelling.

Phenotyping and Exploitation of Kompetitive Allele-Specific PCR Assays for Genes Underpinning Leaf Rust Resistance in New Spring Wheat Mutant Lines.

Leaf rust (Puccinia triticina Eriks) is a wheat disease causing substantial yield losses in wheat production globally. The identification of genetic resources with permanently effective resistance genes and the generation of mutant lines showing increased levels of resistance allow the efficient incorporation of these target genes into germplasm pools by marker-assisted breeding. In this study, new mutant (M3 generation) lines generated from the rust-resistant variety Kazakhstanskaya-19 were developed using gamma-induced mutagenesis through 300-, 350-, and 400-Gy doses. In field trials after leaf rust inoculation, 75 mutant lines showed adult plant resistance. These lines were evaluated for resistance at the seedling stage via microscopy in greenhouse experiments. Most of these lines (89.33%) were characterized as resistant at both developmental stages. Hyperspectral imaging analysis indicated that infected leaves of wheat genotypes showed increased relative reflectance in visible and near-infrared light compared to the non-infected genotypes, with peak means at 462 and 644 nm, and 1936 and 2392 nm, respectively. Five spectral indexes, including red edge normalized difference vegetation index (RNDVI), structure-insensitive pigment index (SIPI), ratio vegetation index (RVSI), water index (WI), and normalized difference water index (NDWI), demonstrated significant potential for determining disease severity at the seedling stage. The most significant differences in reflectance between susceptible and resistant mutant lines appeared at 694.57 and 987.51 nm. The mutant lines developed were also used for the development and validation of KASP markers for leaf rust resistance genes Lr1, Lr2a, Lr3, Lr9, Lr10, and Lr17. The mutant lines had high frequencies of "a" resistance alleles (0.88) in all six Lr genes, which were significantly associated with seedling resistance and suggest the potential of favorable haplotype introgression through functional markers. Nine mutant lines characterized by the presence of "b" alleles in Lr9 and Lr10-except for one line with allele "a" in Lr9 and three mutant lines with allele "a" in Lr10-showed the progressive development of fungal haustorial mother cells 72 h after inoculation. One line from 300-Gy-dosed mutant germplasm with "b" alleles in Lr1, Lr2a, Lr10, and Lr17 and "a" alleles in Lr3 and Lr9 was characterized as resistant based on the low number of haustorial mother cells, suggesting the contribution of the "a" alleles of Lr3 and Lr9.

Genome-wide association study and development of molecular markers for yield and quality traits in peanut (Arachis hypogaea L.).

BACKGROUND: This study aims to decipher the genetic basis governing yield components and quality attributes of peanuts, a critical aspect for advancing molecular breeding techniques. Integrating genotype re-sequencing and phenotypic evaluations of seven yield components and two grain quality traits across four distinct environments allowed for the execution of a genome-wide association study (GWAS). RESULTS: The nine phenotypic traits were all continuous and followed a normal distribution. The broad heritability ranged from 88.09 to 98.08%, and the genotype-environment interaction effects were all significant. There was a highly significant negative correlation between protein content (PC) and oil content (OC). The 10× genome re-sequencing of 199 peanut accessions yielded a total of 631,988 high-quality single nucleotide polymorphisms (SNPs), with 374 significant SNP loci identified in association with the nine traits of interest. Notably, 66 of these pertinent SNPs were detected in multiple environments, and 48 of them were linked to multiple traits of interest. Five loci situated on chromosome 16 (Chr16) exhibited pleiotropic effects on yield traits, accounting for 17.64-32.61% of the observed phenotypic variation. Two loci on Chr08 were found to be strongly associated with protein and oil contents, accounting for 12.86% and 14.06% of their respective phenotypic variations, respectively. Linkage disequilibrium (LD) block analysis of these seven loci unraveled five nonsynonymous variants, leading to the identification of one yield-related candidate gene and two quality-related candidate genes. The correlation between phenotypic variation and SNP loci in these candidate genes was validated by Kompetitive allele-specific PCR (KASP) marker analysis. CONCLUSIONS: Overall, molecular markers were developed for genetic loci associated with yield and quality traits through a GWAS investigation of 199 peanut accessions across four distinct environments. These molecular tools can aid in the development of desirable peanut germplasm with an equilibrium of yield and quality through marker-assisted breeding.

Novel allelic variations in Tannin1 and Tannin2 contribute to tannin absence in sorghum.

Sorghum is an important food crop commonly used for brewing, feed, and bioenergy. Certain genotypes of sorghum contain high concentrations of condensed tannins in seeds, which are beneficial, such as protecting grains from herbivore bird pests, but also impair grain quality and digestibility. Previously, we identified Tannin1 and Tannin2, each with three recessive causal alleles, regulate tannin absence in sorghum. In this study, via characterizing 421 sorghum accessions, we further identified three novel recessive alleles from these two genes. The tan1-d allele contains a 12-bp deletion at position 659 nt and the tan1-e allele contains a 10-bp deletion at position 771 nt in Tannin1. The tan2-d allele contains a C-to-T transition, which results in a premature stop codon before the bHLH domain in Tannin2, and was predominantly selected in China. We further developed KASP assays targeting these identified recessive alleles to efficiently genotype large populations. These studies provide new insights in sorghum domestication and convenient tools for breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01463-y.

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

A pilot study of p53 immunohistochemistry in atypical squamous lesions, using a vulvar scoring system.

BACKGROUND: Histopathologic overlap between cutaneous squamous cell carcinoma (cSCC) and its indolent mimics likely leads to the overdiagnosis of cSCC. OBJECTIVE: To perform a pilot study of the p53 immunohistochemical scoring system developed on vulvar squamous lesions in cSCC. METHODS: The consistency and reliability of p53 immunostaining using a scoring system developed on vulvar cases, as compared with TP53 genomic sequencing, was studied in an initial cohort of 28 cutaneous cases. p53 labeling was further assessed in an additional 63 cases of atypical squamous lesions, including 20 atypical squamous lesions classified by the authors as benign, 22 cases diagnosed as cSCC without high-risk features, and 21 cases of high-risk cSCC (cSCC-HR). RESULTS: The concordance of p53 labeling and TP53 sequencing was 82.1%. Four positive patterns of p53 mutation were identified: basal, parabasal/diffuse, null, and cytoplasmic. p53 positivity in atypical, benign squamous lesions (10%) was significantly lower than that of low-risk cSCC (63.6%, p = 0.0004) or cSCC-HR (90.5%, p < 0.0001). p53 positivity in low-risk cSCC versus cSCC-HR was not statistically significant (p = 0.07). CONCLUSION: p53 Labeling may be a helpful biomarker to support the diagnosis of cSCC and distinguish cSCC from atypical but benign mimics.

Genotypic and Resistance Profile Analysis of Two Oat Crown Rust Differential Sets Urge Coordination and Standardization.

Puccinia coronata f. sp. avenae is the causal agent of the disease known as crown rust, which represents a bottleneck in oat production worldwide. Characterization of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared the virulence profiles of 25 P. coronata f. sp. avenae isolates from Australia using two host differential sets, one from Australia and one from the United States. These differential sets were also genotyped using diversity arrays technology sequencing technology. Phenotypic and genotypic discrepancies were detected on 8 out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91-linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterize materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance in the Uzbekistani Wheat Landrace Teremai Bugdai.

Leaf rust, caused by Puccinia triticina, is a major cause of wheat yield losses globally, and novel leaf rust resistance genes are needed to enhance wheat leaf rust resistance. Teremai Bugdai is a landrace from Uzebekistan that is highly resistant to many races of P. triticina in the United States. To unravel leaf rust resistance loci in Teremai Bugdai, a recombinant inbred line (RIL) population of Teremai Bugdai × TAM 110 was evaluated for response to P. triticina race Pt54-1 (TNBGJ) and genotyped using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). Quantitative trait loci (QTL) analysis using 5,130 high-quality GBS-SNPs revealed three QTLs, QLr-Stars-2DS, QLr-Stars-6BL, and QLr.Stars-7BL, for leaf rust resistance in two experiments. QLr-Stars-2DS, which is either a new Lr2 allele or a new resistance locus, was delimited to an ∼19.47-Mb interval between 46.4 and 65.9 Mb on 2DS and explained 31.3 and 33.2% of the phenotypic variance in the two experiments. QLr-Stars-6BL was mapped in an ∼84.0-kb interval between 719.48 and 719.56 Mb on 6BL, accounting for 33 to 36.8% of the phenotypic variance in two experiments. QLr.Stars-7BL was placed in a 350-kb interval between 762.41 and 762.76 Mb on 7BL and explained 4.4 to 5.3% of the phenotypic variance. Nine GBS-SNPs flanking these QTLs were converted to kompetitive allele specific PCR (KASP) markers, and these markers can be used to facilitate their introgression into locally adapted wheat lines.

Three novel er1 alleles and their functional markers for breeding resistance to powdery mildew (Erysiphe pisi) in pea.

Powdery mildew caused by Erysiphe pisi DC is a global notorious disease on peas. Deploying resistance pea cultivars is the most efficient and environmentally friendly method for the disease control. This study focuses on revealing the resistance genes in three pea germplasms and developing their functional markers for resistance breeding. The identification of resistance genes involved genetic mapping and the sequencing of the PsMLO1 gene. To confirm the hereditary in three reisistant germplasms, they were crossed with susceptible cultivars to generate F1, F2, and F2:3 populations. The F1 generation exhibited susceptibility to E. pisi, while segregation patterns in subsequent generations adhered to the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, indicating that powdery mildew resistance was governed by single recessive gene in each germplasm. Analysis of er1-linked markers and genetic mapping suggested that the resistance genes could be er1 alleles in these germplasms. The multiple clone sequencing results of the three homologous PsMLO1 genes showed they were novel er1 alleles, named er1-15, er1-16, and er1-17, respectively. The er1-15 and er1-16 were caused by 1-bp deletion at position 335 (A) and 429 (T) in exon 3, respectively, while er1-17 was caused a 1-bp insertion at position 248 in exon 3, causing a frame-shift mutation and premature termination of PsMLO1 protein translation. Their respective functional markers KASP-er1-15, KASP-er1-16 and KASP-er1-17 were successfully developed and validated in respective mapping populations and pea germplasms. These results provide valuable tools for pea breeding resistance to E pisi.

Genetic Diversity and Genome-Wide Association Analysis of the Hulled/Naked Trait in a Barley Collection from Shanghai Agricultural Gene Bank.

Barley is one of the most important cereal crops in the world, and its value as a food is constantly being revealed, so the research into and the use of barley germplasm are very important for global food security. Although a large number of barley germplasm samples have been collected globally, their specific genetic compositions are not well understood, and in many cases their origins are even disputed. In this study, 183 barley germplasm samples from the Shanghai Agricultural Gene Bank were genotyped using genotyping-by-sequencing (GBS) technology, SNPs were identified and their genetic parameters were estimated, principal component analysis (PCA) was preformed, and the phylogenetic tree and population structure of the samples were also analyzed. In addition, a genome-wide association study (GWAS) was carried out for the hulled/naked grain trait, and a KASP marker was developed using an associated SNP. The results showed that a total of 181,906 SNPs were identified, and these barley germplasm samples could be roughly divided into three categories according to the phylogenetic analysis, which was generally consistent with the classification of the traits of row type and hulled/naked grain. Population structure analysis showed that the whole barley population could be divided into four sub-populations (SPs), the main difference from previous classifications being that the two-rowed and the hulled genotypes were sub-divided into two SPs. The GWAS analysis of the hulled/naked trait showed that many associated loci were unrelated to the Nud/nud locus, indicating that there might be new loci controlling the trait. A KASP marker was developed for one exon-type SNP on chromosome 7. Genotyping based on the KASP assay was consistent with that based on SNPs, indicating that the gene of this locus might be associated with the hulled/naked trait. The above work not only lays a good foundation for the future utilization of this barley germplasm population but it provides new loci and candidate genes for the hulled/naked trait.

Screening Indels from the whole genome to identify the candidates and their association with economic traits in several goat breeds.

In the present study, the re-sequencing of our previous whole-genome sequencing (WGS) for selected individuals of Dazu-black goat (DBG) and Inner-Mongolia Cashmere goat (IMCG) breeds was used to detect and compare the differentiation in Indels depending on the reference genome of goat. Then, three selected candidate Indels rs668795676, rs657996810, and rs669452874 of the three genes SUFU, SYCP2L and GLIPR1L1, respectively, have been chosen, based on the results of prior GWAS across the genome, and examined for their association with body weight and dimensions (body height, body length, heart girth, chest width, cannon circumference, and chest depth) by kompetitive allele specific PCR assay for 342 goats from the three studied goat breeds (DBG, n = 203; ♂99, ♀104), IMCG (n = 65; 15♂, 50♀), and Hechuan white goat (HWG, n = 74; 34♂, 40♀) breeds. The analysis of 192.747 Gb WGS revealed an average 334,151 Indels in the whole genome of DBG and IMCG breeds. Chromosome 1 had a maximum number of mutations (Indels) of 58,497 and 55,527 for IMCG and DBG, respectively, while chromosome 25 had the least number of mutations of 15,680 and 16,103 for IMCG and DBG, respectively. The majority of Indels were either Ins or Del of short fragments of 1-5 bp, which covered 79.06 and 71.78% of the total number of Indels mutations in IMCG and DBG, respectively. Comparing the differences of Indels between the studied goat breeds revealed 100 and 110 unique Indels for IMCG and DBG, respectively. The Indels loci in the intron region were unique for both studied goat breeds which were related to 30 and 38 candidate genes in IMCG and DBG, respectively, including SUFU, SYCP2L, and GLIPR1L1 genes. Concerning rs669452874 locus, body height and body length of Del/Del genotype in DBG were significantly higher (P < 0.05) than that of Ins/Del genotype, while body height and body length of Del/Del genotype in IMCG were significantly higher (P < 0.01) than those of Ins/Ins genotype, whereas body height and body length and heart girth of Del/Del genotype in HWG were significantly higher (P < 0.01) than those of the Ins/Del and Ins/Ins genotypes. Thus, Del/Del genotype of rs669452874 locus can be used as a candidate molecular marker related to the body dimensions in the studied goat breeds.

Biochemical and phenological characterization of diverse wheats and their association with drought tolerance genes.

Drought is one of the most important wheat production limiting factor, and can lead to severe yield losses. This study was designed to examine the effect of drought stress on wheat physiology and morphology under three different field capacities (FC) viz. 80% (control), 50% (moderate) and 30% (severe drought stress) in a diverse collection of wheat germplasm including cultivars, landraces, synthetic hexaploid and their derivatives. Traits like grain weight, thousand grain weight and biomass were reduced by 38.23%, 18.91% and 26.47% respectively at 30% FC, whereas the reduction rate for these traits at 50% FC were 19.57%, 8.88% and 18.68%. In principal component analysis (PCA), the first two components PC1 and PC2 accounted for 58.63% of the total variation and separated the cultivars and landraces from synthetic-based germplasm. Landraces showed wide range of phenotypic variations at 30% FC compared to synthetic-based germplasm and improved cultivars. However, least reduction in grain weight was observed in improved cultivars which indicated the progress in developing drought resilient cultivars. Allelic variations of the drought-related genes including TaSnRK2.9-5A, TaLTPs-11, TaLTPs-12, TaSAP-7B-, TaPPH-13, Dreb-B1 and 1fehw3 were significantly associated with the phenological traits under drought stress in all 91 wheats including 40 landraces, 9 varieties, 34 synthetic hexaploids and 8 synthetic derivatives. The favorable haplotypes of 1fehw3, Dreb-B1, TaLTPs-11 and TaLTPs-12 increased grain weight, and biomass. Our results iterated the fact that landraces could be promising source to deploy drought adaptability in wheat breeding. The study further identified drought tolerant wheat genetic resources across various backgrounds and identified favourable haplotypes of water-saving genes which should be considered to develop drought tolerant varieties.

Conservatively transmitted alleles of key agronomic genes provide insights into the genetic basis of founder parents in bread wheat (Triticum aestivum L.).

BACKGROUND: Founder parents play extremely important roles in wheat breeding. Studies into the genetic basis of founder parents and the transmission rules of favorable alleles are of great significance in improving agronomically important traits in wheat. RESULTS: Here, a total of 366 founder parents, widely grown cultivars, and derivatives of four representative founder parents were genotyped based on efficient kompetitive allele-specific PCR (KASP) markers in 87 agronomically important genes controlling yield, quality, adaptability, and stress resistance. Genetic composition analysis of founder parents and widely grown cultivars showed a consistently high frequency of favorable alleles for yield-related genes. This analysis further showed that other alleles favorable for resistance, strong gluten, dwarf size, and early heading date were also subject to selective pressure over time. By comparing the transmission of alleles from four representative founder parents to their derivatives during different breeding periods, it was found that the genetic composition of the representative founder parents was optimized as breeding progressed over time, with the number and types of favorable alleles carried gradually increasing and becoming enriched. There are still a large number of favorable alleles in wheat founder parents that have not been fully utilized in breeding selection. Eighty-seven agronomically important genes were used to construct an enrichment map that shows favorable alleles of four founder parents, providing an important theoretical foundation for future identification of candidate wheat founder parents. CONCLUSIONS: These results reveal the genetic basis of founder parents and allele transmission for 87 agronomically important genes and shed light on breeding strategies for the next generation of elite founder parents in wheat.

Combined QTL-sequencing, linkage mapping, and RNA-sequencing identify candidate genes and KASP markers for low-temperature germination in Oryza sativa L. ssp. Japonica.

MAIN CONCLUSION: Through QTL-seq, QTL mapping and RNA-seq, six candidate genes of qLTG9 can be used as targets for cold tolerance functional characterization, and six KASP markers can be used for marker-assisted breeding to improve the germination ability of japonica rice at low temperature. The development of direct-seeded rice at high latitudes and altitudes depends on the seed germination ability of rice under a low-temperature environment. However, the lack of regulatory genes for low-temperature germination has severely limited the application of genetics in improving the breeds. Here, we used cultivars DN430 and DF104 with significantly different low-temperature germination (LTG) and 460 F2:3 progeny derived from them to identify LTG regulators by combining QTL-sequencing, linkage mapping, and RNA-sequencing. The QTL-sequencing mapped qLTG9 within a physical interval of 3.4 Mb. In addition, we used 10 Kompetitive allele-specific PCR (KASP) markers provided by the two parents, and qLTG9 was optimized from 3.4 Mb to a physical interval of 397.9 kb and accounted for 20.4% of the phenotypic variation. RNA-sequencing identified qLTG9 as eight candidate genes with significantly different expression within the 397.9 kb interval, six of which possessed SNPs on the promoter and coding regions. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) completely validated the results of these six genes in RNA-sequencing. Subsequently, six non-synonymous SNPs were designed using variants in the coding region of these six candidates. Genotypic analysis of these SNPs in 60 individuals with extreme phenotypes indicated these SNPs determined the differences in cold tolerance between parents. The six candidate genes of qLTG9 and the six KASP markers could be used together for marker-assisted breeding to improve LTG.

Variable fragment length allele-specific polymerase chain reaction (VFLASP), a method for simple and reliable genotyping.

Single-nucleotide polymorphism (SNP) is a substitution of a single nucleotide at a specific position in the genome. Until now, 585 million SNPs have been identified in the human genome, and therefore, a widely applicable method is desirable to detect a specific SNP. Herein we report a simple and reliable genotyping assay, which seems to be suitable for medium and small size laboratories, as well, to easily genotype most of the SNPs. In our study, all of the possible base variations (A-T, A-G, A-C, T-G, T-C, G-C) were tested to prove the general feasibility of our technique. The basis of the assay is a fluorescent PCR, in which both allele-specific primers, differing only at the 3' end according to the sequence of the SNP, were present, and the length of one of them was modified with 3 bp by adding an adapter sequence to the 5' end of that primer. The competitive presence of both allele-specific primers excludes the false amplification of the absent allele (which can happen in simple allele-specific PCR (AS-PCR)) and ensures the amplification of the proper allele(s). Unlike other complicated genotyping methods that use of manipulation of fluorescent dyes for genotyping, we apply an approach based on the length of amplicons from different alleles to differentiate between them. In our experiment (named variable fragment length allele-specific polymerase chain reaction (VFLASP)), the investigated six SNPs, containing the six available base variations, gave clear and reliable results after detecting the amplicons by capillary electrophoresis.

Robust markers associated with floral traits in roses are suitable for marker-assisted selection across gene pools.

We investigated the potential of markers associated with floral traits for parental selection in a cut rose breeding program. We analysed six Kompetitive Allele Specific PCR (KASP) markers for three important floral traits, petal length, petal number and scent, derived from experiments in a garden rose population. The six markers were applied to genotype a collection of 384 parental genotypes used for commercial cut rose breeding. We phenotyped a selection of progeny derived from pairs of parents having either high or low dosages of (contrasting) marker alleles associated with these traits. Significant differences were found between the contrasting progeny groups for each of the traits, although parents with the optimal allele dosage combinations could not always be used for the crosses. This not only supports the robustness of these marker‒trait associations but also demonstrates their potential for commercial rose breeding. It also demonstrates the use of marker information generated in garden rose populations for cut rose breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01438-5.

Development and validation of KASP markers for resistance to Phytophthora capsici in Capsicum annuum L.

Resistance of Capsicum annuum to Phytophthora blight is dependent on the genetic background of the resistance source and the Phytophthora capsici isolate, which poses challenges for development of generally applicable molecular markers for marker-assisted selection. In this study, the resistance to P. capsici of C. annuum was genetically mapped to chromosome 5 within a 1.68-Mb interval by genome-wide association study analysis of 237 accessions. In this candidate region, 30 KASP markers were developed using genome resequencing data for a P. capsici-resistant line (0601 M) and a susceptible line (77,013). Seven of these KASP markers, located in the coding region of a probable leucine-rich repeats receptor-like serine/threonine-protein kinase gene (Capana05g000704), were validated in the 237 accessions, which showed an average accuracy of 82.7%. The genotyping of the seven KASP markers strongly corresponded with the phenotype of 42 individual plants in a pedigree family (PC83-163) developed from the P. capsici-resistant line CM334. This research provides a set of efficient and high-throughput KASP markers for marker-assisted selection of resistance to P. capsici in C. annuum. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01367-3.

Identification of a Novel Pm65 Allele Conferring a Wide Spectrum of Resistance to Powdery Mildew in Wheat Accession PI 351817.

Powdery mildew is caused by the highly adaptive biotrophic fungus Blumeria graminis f. sp. tritici infecting wheat worldwide. Novel powdery mildew resistance genes are urgently needed that can be used rapidly in wheat cultivar development with minimal disruption of trait advances elsewhere. PI 351817 is a German cultivar exhibiting a wide spectrum of resistance to B. graminis f. sp. tritici isolates collected from different wheat-growing regions of the United States. Evaluation of an F2 population and 237 F2:3 lines derived from OK1059060-2C14 × PI 351817 for responses to B. graminis f. sp. tritici isolate OKS(14)-B-3-1 identified a single dominant gene, designated Pm351817, for powdery mildew resistance in PI 351817. Using bulked segregant analysis (BSA) and simple sequence repeat (SSR) markers, Pm351817 was mapped in the terminal region of the long arm of chromosome 2A. Deep sequencing of the genotyping-by-sequencing libraries of the two parental lines identified a set of single-nucleotide polymorphism (SNP) markers in the 2AL candidate gene region. Those SNP markers was subsequently converted to Kompetitive allele-specific PCR (KASP) markers for genotyping the mapping population. Linkage analysis delimited Pm351817 to a 634-kb interval between Stars-KASP656 (771,207,512 bp) and Stars-KASP662 (771,841,609 bp) on 2AL, based on the Chinese Spring reference sequence IWGSC RefSeq v 2.1. Tests of allelism indicated that Pm351817 is located at the Pm65 locus. Pm351817 shows resistance to all B. graminis f. sp. tritici isolates used in this study and can be used to enhance powdery mildew resistance in the United States. KASP markers flanking Pm351817 can be used to select Pm351817 in wheat breeding programs after further tests for polymorphism.

Identification of a Major-Effect Quantitative Trait Loci Associated with Begomovirus Resistance in Cucurbita moschata.

Begomoviruses, viz. squash leaf curl China virus and tomato leaf curl New Delhi virus causative diseases are major concerns of quantitative and qualitative losses in pumpkin (Cucurbita moschata) worldwide. Punjab Agricultural University (PAU) in India has identified a resistant source (PVR-1343) against mixed infection (MI-Sq/To) of these begomoviruses. Introgression of resistance in diverse genetic backgrounds requires the identification of quantitative trait loci (QTLs) associated with MI-Sq/To resistance. Phenotyping of 229 F2:3 progenies derived from the PVR-1343 × P-135 cross revealed digenic recessive inheritance against MI-Sq/To resistance in PVR-1343. To identify the genomic region, resistant and susceptible bulks were subjected to whole-genome resequencing along with their parents. The whole-genome resequence analysis of parents and bulks using QTLseq/QTLseqr approaches identified an overlapping 1.52 Mb region on chromosome 7 (qMI-Sq/To7.1), while chromosomal region spanning 0.87 Mb on chromosome17 (qMI-Sq/To17.1) was additionally identified by QTLseqr. However, the highest peak value on chromosome 7 with three algorithms {G', ∆(SNP-index) and -log10 (P value)} highlighted the major contribution of qMI-Sq/To7.1 in MI-Sq/To resistance. Nine polymorphic SNPs identified within the highly significant qMI-Sq/To7.1 region were converted into KASP markers. KASP genotyping of F2 individuals narrowed down the qMI-Sq/To7.1 interval to 103 kb region flanked by two markers, Cmo3914729 and Cmo4018182, which contained 16 annotated genes and accounted for 59.84% of phenotypic variation. The Cmo4018182 KASP marker accurately predicted disease reaction in 91% of diverse Cucurbita genotypes and showed nonsynonym substitutions in the coding region of putative candidate SYNTAXIN-121 gene. These findings pave the way for marker-assisted breeding and elucidating the underlying mechanism of begomovirus resistance in C. moschata.