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A causal gene for MALE STERILITY 4 (MS4), the CjTKPR1 gene, was recently identified in Cryptomeria japonica. To apply marker-assisted selection (MAS) to breeding of male-sterile C. japonica with MS4, we developed a Kompetitive Allele-Specific polymerase chain reaction (KASP) marker, as a highly cost-effective and accurate genotyping method based on the CjTKPR1 sequence. The accuracy of genotyping using the KASP marker was verified with a backcross family for MS4, and MS4 genotypes were accurately identified. Next, we attempted to select individuals carrying ms4 from 1,500 breeding materials collected throughout Japan. No new individuals with ms4 were detected beyond those already identified. Individuals with ms4 were estimated to be geographically restricted and distributed at extremely low frequency (0.13%). We also attempted to produce new breeding materials with double heterozygotes for MS1 and MS4 (Ms1/ms1, MS4/ms4). Artificial crossings were performed between 'Shindai 11' (ms1/ms1, Ms4/Ms4) or 'Shindai 12' (ms1/ms1, Ms4/Ms4) and 'S8-94_sb1' (Ms1/Ms1, Ms4/ms4). Of the 56 and 35 progenies produced from each family, 31 and 13 double-heterozygous individuals with MS1 and MS4 (Ms1/ms1, Ms4/ms4) were selected, respectively. The segregation ratio of genotypes for MS4 did not deviate from the expected 1:1 ratio in either family. The KASP marker developed in this study facilitates accurate genotyping that is less labor-intensive and less costly than conventional methods. This method is anticipated to contribute to greater efficiency in the breeding and seed production of male-sterile C. japonica with MS4. [ABSTRACT FROM AUTHOR]

A validated marker system is crucial to running an effective genomics-assisted breeding program. We used 36 Kompetitive Allele-Specific PCR (KASP) markers to genotype 376 clones from the biofortified cassava pipeline, and fingerprinted 93 of these clones with DArTseq markers to characterize breeding materials and evaluate their relationships. The discriminating ability of the 36-quality control (QC) KASP and 6602 DArTseq markers was assessed using 92 clones genotyped in both assays. In addition, trait-specific markers were used to determine the presence or absence of target genomic regions. Hierarchical clustering identified two major groups, and the clusters were consistent with the breeding program origins. There was moderate genetic differentiation and a low degree of variation between the identified groups. The general structure of the population was similar using both assays. Nevertheless, KASP markers had poor resolution when it came to differentiating the genotypes by seed sources and overestimated the prevalence of duplicates. The trait-linked markers did not achieve optimal performance as all markers displayed variable levels of false positive and/or false negative. These findings represent the initial step in the application of genomics-assisted breeding for the biofortified cassava pipeline, and will guide the use of genomic selection in the future. [ABSTRACT FROM AUTHOR]

Large and distinct families of receptor-like kinases (RLKs) play elemental roles in many fundamental processes of plants. The proline-rich extensin-like receptor kinase (PERK) family is one of the most pivotal classes of RLKs. To date, there have been no comprehensive or published studies conducted on the PERK gene family in Glycine max. This research aimed to characterize the role of the PERK gene family in cultivated soybean using a systematic array of bioinformatic and experimental approaches. We identified 16 PERK members in G. max through local BLASTp, using PERK members from Arabidopsis thaliana as a query. Tissue expression of genes, predicted via tissue specific expression analysis from the soybean database "SoyBase", revealed that these PERK genes exhibit differentiated expression patterns in various plant organs. The gene structure was predicted via Gene Structure Display Server (GSDS). Phylogeny was demonstrated through an evolutionary tree employing the neighbor-joining method. Subcellular localization of proteins was identified via "Softberry" and cis-acting elements were identified through PlantCARE. The KASP (Kompetitive Allele Specific PCR (KASP)) marker was developed for the GmPERK-1-C and GmPERK-1-T allele, targeting position 167 nt in the CDS region. Genotyping results indicated that GmPERK-1 exhibits promising potential for utilization in molecular breeding programs for soybean to increase crop yield. Collectively, our findings indicate that G. max accessions harboring the GmPERK-1-C allele exhibit significantly higher thousand grain weight compared to accessions carrying the GmPERK-1-T allele. This research enhances the understanding of the molecular roles of PERK genes in G. max, providing valuable insights for the utilization of favorable genetic variations in soybean molecular breeding programs. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract 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 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

【Objective】Rice blast resistance genes Pi2 and Pita are broad-spectrum resistance genes to rice blast in rice areas in South China, which have very important application value for rice blast resistance breeding. Therefore, the development of a set of efficient identification methods is beneficial to genetic breeding efficiency of rice blast-resistant varieties.【Method】According to the variant GCA GGA/GTG TTA of the 787 and 788 codons of the Pi2 gene in 'Huanghuangyouzhan' (highly resistant to rice blast) and 'Guangluai 4' (highly susceptible to rice blast), and the variant G/T at base 6 640 of Pita gene in Tetp (an international rice germplasm resource with high resistance to rice blast) and Lijiang Xintuan Heigu (a local ermplasm resource with high susceptibility to rice blast), based on the competitive allele PCR (Kompetitive Allele Specific PCR, KASP) labeling technology principle, molecular markers of rice blast resistance genes were developed.【Result】Two KASP markers (W-Pi2 and W-Pita) were developed to detect the functional sites of Pi2 and Pita. The markers were used to detect the conventional rice, aromatic rice and hybrid rice cultivated by the Rice Research Institute of Guangdong Academy of Agricultural Sciences. 19 resistant varieties were screened, including 13 varieties with a single gene tested as a diseaseresistant allele and 6 varieties with two genes tested as disease-resistant alleles. The comparison of the two markers suggested that the frequency of disease-resistance was higher in the varieties carrying Pi2 resistance gene than in the varieties carrying Pita resistance gene. The results showed that the two markers could detect the allele types of rice blast resistance genes Pi2 and Pita in breeding materials in the early stage (seed or seedling stage), and the breeding materials were not required to be planted in the disease nursery, then individual plants carrying resistant alleles could be screened and identified.【Conclusion】The developed KASP functional molecular markers W-Pi2 and W-Pita can better distinguish parental varieties with different resistance, clearly distinguish different allele types among breeding materials, and accurately screen breeding materials.

Rind colour is an important quality attribute of melon appearance. Identifying target genes and developing functional molecular markers is significant for rind colour breeding in melon. This study involved a genetic analysis of the fruit rind colours of two inbred lines: H185 (with black green melon rind) and H160 (with white rind) alongside a fruit rind colour gene, CmAPRR2. The purpose was to discover the variation sites of the CmAPRR2 gene and develop specific molecular marker for rind colour in melon. The results showed that the black green rind is dominant over white, and single gene controls the colours. A mutation of the G856T base occurred in the eighth exon of the CmAPRR2 coding DNA region in H160, suggesting that this mutation is the key factor for the white rind colour. Thus, a codominant molecular marker, FC, for gene CmAPRR2 was developed and used for molecular identification of 189 F2 individuals. The marker revealed a complete correspondence between genotype and phenotype. Additionally, the marker revealed that the other four white rinds had G856T mutations. This study provides a basis for targeted improvement of melon rind colours, offering a technical means for molecular marker‐assisted breeding of melon rind colours. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Matrilineal (MTL) gene is known to induce haploidy because of the 4-bp insertion in the last exon of the gene in maize haploid inducer stock. Orthologue gene search in wheat genome identified TaPLA genes on wheat chromosomes 4A, 4B and 4D. The DNA sequence homology between the maize MTL gene and TaPLA gene orthologues in wheat was 86–88%. The present study involves the selection of EMS mutants with mutations in TaPLA genes from Cadenza (6X) and Kronos (4X) TILLING populations with an aim to identify/develop a haploid inducer stock in wheat. A total of 45 mutants including 24 for Cadenza and 21 for Kronos were selected with 18 having mutations in TaPLA.4A, 17 in TaPLA.4B and 10 in TaPLA.4D gene. On applying KASP assay using mutation-specific markers, 36 markers showed clear polymorphism among the parents and the mutants. On the basis of validated KASP assay and the mutations leading to the changes in the amino acid, their biochemical properties and nature of amino acids, a total of 17 Cadenza and 14 Kronos mutants have been selected to be further used for pyramiding of these mutations. The validated KASP assay will be used to check the true F1s and homozygosity status of the mutant alleles in F2s. The homozygous F2s will be further used to cross with hexaploid or tetraploid wheat to check the haploidy induction ability. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

【Objective】 Rice blast resistance genes Pi2 and Pita are broad-spectrum resistance genes to rice blast in rice areas in South China, which have very important application value for rice blast resistance breeding. Therefore, the development of a set of efficient identification methods is beneficial to genetic breeding efficiency of rice blast-resistant varieties.【Method】According to the variant GCA GGA/GTG TTA of the 787 and 788 codons of the Pi2 gene in ‘Huanghuangyouzhan’ (highly resistant to rice blast) and ‘Guangluai 4’ (highly susceptible to rice blast), and the variant G/T at base 6 640 of Pita gene in Tetp (an international rice germplasm resource with high resistance to rice blast) and Lijiang Xintuan Heigu (a local ermplasm resource with high susceptibility to rice blast), based on the competitive allele PCR (Kompetitive Allele Specific PCR, KASP) labeling technology principle, molecular markers of rice blast resistance genes were developed.【Result】 Two KASP markers (W-Pi2 and W-Pita) were developed to detect the functional sites of Pi2 and Pita. The markers were used to detect the conventional rice, aromatic rice and hybrid rice cultivated by the Rice Research Institute of Guangdong Academy of Agricultural Sciences. 19 resistant varieties were screened, including 13 varieties with a single gene tested as a diseaseresistant allele and 6 varieties with two genes tested as disease-resistant alleles. The comparison of the two markers suggested that the frequency of disease-resistance was higher in the varieties carrying Pi2 resistance gene than in the varieties carrying Pita resistance gene. The results showed that the two markers could detect the allele types of rice blast resistance genes Pi2 and Pita in breeding materials in the early stage (seed or seedling stage), and the breeding materials were not required to be planted in the disease nursery, then individual plants carrying resistant alleles could be screened and identified.【Conclusion】 The developed KASP functional molecular markers W-Pi2 and W-Pita can better distinguish parental varieties with different resistance, clearly distinguish different allele types among breeding materials, and accurately screen breeding materials. [ABSTRACT FROM AUTHOR]

The aim was to investigate the relationship between polymorphisms of gene mutation loci and reproductive traits in local sheep breeds (Duolang Sheep) and introduced sheep breeds (Suffolk, Hu Sheep) in Xinjiang to provide new molecularmarkers for the selection and breeding of high fecundity sheep. The expression pattern of typing successful genes in sheep tissues was investigated by RT-qPCR technology, providing primary data for subsequent verification of gene function. The 26 mutation loci of WWC2, ARHGEF9, SLK, GAB3, and FSHR genes were typed using KASP. Association analyses were performed using SPSS 25.0, and the typing results showed that five genes with six loci,WWC2 (g.14962207 C>T), ARHGEF9 (g.48271079 C>A), SLK (g.27107842 T>C, g.27108855 G>A), GAB3 (g.86134602 G>A), and FSHR (g.80789180 T>G) were successfully typed. The results of the association analyses showed that WWC2 (g.14962207 C>T), SLK (g.27108855 G>A), ARHGEF9 (g.48271079 C>A), and FSHR (g.80789180 T>G) caused significant or extremely significant effects on the litter size in Duolang, Suffolk and Hu Sheep populations. The expression distribution pattern of the five genes in 12 sheep reproduction-related tissues was examined by RTqPCR. The results showed that the expression of the SLK gene in the uterus, the FSHR gene in the ovary, and the ARHGEF9 gene in hypothalamic-pituitarygonadal axis-related tissues were significantly higher than in the tissues of other parts of the sheep. WWC2 and GAB3 genes were highly expressed both in reproductive organs and visceral tissues. In summary, the WWC2 (g.14962207 C>T), SLK (g.27108855 G>A), ARHGEF9 (g.48271079 C>A), and FSHR (g.80789180 T>G) loci can be used as potential molecular markers for detecting differences in reproductive performance in sheep. Due to variations in typing results, the SLK (g.27107842 T>C) and GAB3 (g.86134602 G>A) loci need further validation. [ABSTRACT FROM AUTHOR]

Rice is a staple food for more than half of the global population due to its food security and sustainable development. Weeds compete with crops for sunlight and indispensable nutrients, affecting the yield and quality of crops. Breeding herbicide-tolerant rice varieties paired with herbicide application is expected to help with weed control. In this study, 194 Japonica/Geng rice varieties or lines collected from the Huanghuaihai region of China were screened by Kompetitive Allele-Specific PCR (KASP) markers based on four mutation sites within OsALS1 (LOC_Os02g30630), which is the target of imidazolinone (IMI) herbicides. Only the OsALS1627N haplotype was identified in 18 varieties, including the previously reported Jingeng818 (JG818), and its herbicide resistance was validated by treatment with three IMIs. To investigate the origin of the OsALS1627N haplotype in the identified varieties, six codominant PCR-based markers tightly linked with OsALS1 were developed. PCR analysis revealed that the other 17 IMI-tolerant varieties were derived from JG818. We randomly selected three IMI-tolerant varieties for comparative whole-genome resequencing with known receptor parent varieties. Sequence alignment revealed that more loci from JG818 have been introduced into IMI-tolerant varieties. However, all three IMI-tolerant varieties carried clustered third type single nucleotide polymorphism (SNP) sites from unknown parents, indicating that these varieties were not directly derived from JG818, whereas those from different intermediate improved lines were crossed with JG818. Overall, we found that only OsALS1627N from JG818 has been broadly introduced into the Huanghuaihai region of China. Additionally, the 17 identified IMI-tolerant varieties provide alternative opportunities for improving such varieties along with other good traits. [ABSTRACT FROM AUTHOR]

NCED1 (9-cis-epoxy carotenoid dioxygenase) plays important roles in controlling ABA levels and drought stress tolerance. The relationships between sequence polymorphisms of the TaNCED1 gene and drought resistance in wheat were analysed. Four allelic variations of the TaNCED1-5B generated to drought resistance were identified. Four groups of KASP (kompetitive allele-specific PCR) markers were developed based on the four alleles and were verified in a natural population of 311 wheat cultivars. The distribution of the four alleles of TaNCED1-5B in the natural population was clarified. Among the allelic variations, 84.62% of the wheat varieties with TT/-/C/-, TT/C/C/-, TT/-/C/G or TT/C/C/G haplotypes were dryland cultivars. The common feature of the above four haplotypes is that they all contain the TT (98) and C (343) alleles at the TaNCED1-5B-TT/CG (98) and TaNCED1-5B-C/T (343) loci, which indicated that these two locis had highly positive correlation with drought resistance in wheat. [ABSTRACT FROM AUTHOR]

Copyright of Xinjiang Agricultural Sciences is the property of Xinjiang Agricultural Sciences Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Soybean [Glycine max(L.)Merr.] is a leading oil-bearing crop and cultivated globally over a vast scale. The agricultural landscape in China faces a formidable challenge with drought significantly impacting soybean production. In this study, we treated a natural population of 264 Chinese soybean accessions using 15% PEG-6000 and used GR, GE, GI, RGR, RGE, RGI and ASFV as evaluation index. Using the ASFV, we screened 17 strong drought-tolerant soybean germplasm in the germination stage. Leveraging 2,597,425 high-density SNP markers, we conducted Genome-Wide Association Studies (GWAS) and identified 92 SNPs and 9 candidate genes significantly associated with drought tolerance. Furthermore, we developed two KASP markers for S14_5147797 and S18_53902767, which closely linked to drought tolerance. This research not only enriches the pool of soybean germplasm resources but also establishes a robust foundation for the molecular breeding of drought tolerance soybean varieties. [ABSTRACT FROM AUTHOR]

In order to establish a KASP identification method for the endangered plant Ariocarpus kotschoubeyanus, 13 samples of 6 species of Ariocarpus kotschoubeyanus were selected as materials. According to the single nucleotide polymorphism (SNP) site on the phosphoenolpyruvate carboxylase (PEPC) gene, a specific KASP primer was designed for PCR amplification of Ariocarpus kotschoubeyanus. The test results show that, KASP primer designed by SNP site of PEPC sequence can form obvious genotyping between Ariocarpus kotschoubeyanus and its similar species, can accurately distinguish it from its relatives and other cactus species, and can be used for identification of true and false Ariocarpus kotschoubeyanus and identification of endangered species at entry and exit ports. [ABSTRACT FROM AUTHOR]

Seed commerce is a highly profitable global market. Most commercialized seeds are hybrid seeds originating from a controlled cross between two selected parental lines. The market value of hybrid seeds depends on their hybrid genetic purity. DNA molecular markers are a reliable and widespread tool to genotype plant materials; however, DNA extraction from seeds is challenging, often laborious and expensive. With the ultimate goal of creating a tomato and melon hybrid seeds purity test, various challenges arise. To overcome these problems and with the purpose of crude DNA extraction, a simple, fast, inexpensive and easily scalable adaptation of the hot sodium hydroxide and tris method coupled to a competitive allele-specific PCR genotyping method is proposed.

Increasing wheat yield is a long-term goal for wheat breeders around the world. Exploiting elite genetic resources and dissecting the genetic basis of important agronomic traits in wheat are the necessary methods for high-yield wheat breeding. This study evaluated nine crucial agronomic traits found in a natural population of 156 wheat varieties and 77 landraces from Sichuan, China in seven environments over two years. The results of this investigation of agronomic traits showed that the landraces had more tillers and higher kernel numbers per spike (KNS), while the breeding varieties had higher thousand-kernel weight (TKW) and kernel weight per spike (KWS). The generalized heritability (H2) values of the nine agronomic traits varied from 0.74 to 0.95. Structure analysis suggested that the natural population could be divided into three groups using 43 198 single nucleotide polymorphism (SNP) markers from the wheat 55K SNP chip. A total of 67 quantitative trait loci (QTLs) were identified by the genome-wide association study (GWAS) analysis based on the Q+K method of a mixed linear model. Three important QTLs were analyzed in this study. Four haplotypes of QFTN.sicau-7BL.1 for fertile tillers number (FTN), three haplotypes of QKNS.sicau-1AL.2 for KNS, and four haplotypes of QTKW.sicau-3BS.1 for TKW were detected. FTN-Hap2, KNS-Hap1, and TKW-Hap2 were excellent haplotypes for each QTL based on the yield performance of 42 varieties in regional trials from 2002 to 2013. The varieties with all three haplotypes showed the highest yield compared to those with either two haplotypes or one haplotype. In addition, the KASP-AX-108866053 marker of QTL QKNS.sicau-1AL.2 was successfully distinguished between three haplotypes (or alleles) in 63 varieties based on the number of kernels per spike in regional trials between 2018 and 2021. These genetic loci and reliable makers can be applied in marker-assisted selection or map-based gene cloning for the genetic improvement of wheat yield.

Molecular marker-assisted selection (MAS) can significantly accelerate and improve the efficiency of the breeding process in seedless grape cultivars. In this study, we developed the KASP_VviAGL11 and VviAGL11_410 markers based on a single nucleotide polymorphism (SNP) site (Chr18: 26889437 (A/C)) of the VviAGL11 gene, and compared them with previously reported SSR markers p3_VvAGL11 and 5U_VviAGL11 by testing 101 cultivars and 81 F1 hybrid progenies. The results showed that both of the proposed markers obtained 100% accuracy rates in detecting allele A, which was closely associated with the seedless trait in grapes, while p3_VvAGL11 and 5U_VviAGL11 had lower accuracy rates due to their tendency to produce false positives. After careful evaluation of the technical advantages and disadvantages associated with these markers, we concluded that KASP_VviAGL11 was superior in terms of simplicity, cost-effectiveness, efficiency, and accuracy. Thus, we optimized the process of molecular MAS for seedless grapes, focusing on the KASP_VviAGL11 marker as a central component, to provide key technical support for the development of new seedless grape cultivars.

Wheat sharp eyespot, a stem disease caused by the soilborne fungus Rhizoctonia cerealis van der Hoeven, has become a threat to wheat production worldwide. Exploiting resistance resources from wild relatives of wheat is a promising strategy for controlling this disease. In this study, a new wheat–Dasypyrum villosum T2DS·2V#4L translocation line in the background of Chinese Spring (CS) showed stable resistance to R. cerealis. Introgression of the T2DS·2V#4L chromosome into wheat cultivar Aikang 58 by backcrossing produced a marked increase in sharp eyespot resistance in NIL-T2DS·2V#4L in comparison with NIL-T2DS·2DL, and no detrimental effects of 2V#4L on agronomic traits were observed in the BC2F2, BC2F2:3, and BC2F2:4 generations. Flow-sorted sequencing of 2V#4L yielded 384.3 Mb of assembled sequence, and 8836 genes were predicted of which 6154 had orthologs in at least one of the 2AL, 2BL, and 2DL arms of CS, whereas 1549 genes were unique to 2V#4L. About 100,000 SNPs were detected in genes of 2V#4L and 2DL in 10 sequenced bread wheat cultivars. A Kompetitive Allele Specific Polymerase chain reaction and 30 conserved ortholog sequence markers were developed to trace the 2V#4L chromatin in wheat backgrounds. T2DS·2V#4L compensating translocation lines represent novel germplasm with sharp eyespot resistance and the markers will allow rapid detection in breeding programs.

A validated marker system is crucial to running an effective genomics-assisted breeding program. We used 36 Kompetitive Allele-Specific PCR (KASP) markers to genotype 376 clones from the biofortified cassava pipeline, and fingerprinted 93 of these clones with DArTseq markers to characterize breeding materials and evaluate their relationships. The discriminating ability of the 36-quality control (QC) KASP and 6602 DArTseq markers was assessed using 92 clones genotyped in both assays. In addition, trait-specific markers were used to determine the presence or absence of target genomic regions. Hierarchical clustering identified two major groups, and the clusters were consistent with the breeding program origins. There was moderate genetic differentiation and a low degree of variation between the identified groups. The general structure of the population was similar using both assays. Nevertheless, KASP markers had poor resolution when it came to differentiating the genotypes by seed sources and overestimated the prevalence of duplicates. The trait-linked markers did not achieve optimal performance as all markers displayed variable levels of false positive and/or false negative. These findings represent the initial step in the application of genomics-assisted breeding for the biofortified cassava pipeline, and will guide the use of genomic selection in the future.

Large and distinct families of receptor-like kinases (RLKs) play elemental roles in many fundamental processes of plants. The proline-rich extensin-like receptor kinase (PERK) family is one of the most pivotal classes of RLKs. To date, there have been no comprehensive or published studies conducted on the PERK gene family in Glycine max. This research aimed to characterize the role of the PERK gene family in cultivated soybean using a systematic array of bioinformatic and experimental approaches. We identified 16 PERK members in G. max through local BLASTp, using PERK members from Arabidopsis thaliana as a query. Tissue expression of genes, predicted via tissue specific expression analysis from the soybean database “SoyBase”, revealed that these PERK genes exhibit differentiated expression patterns in various plant organs. The gene structure was predicted via Gene Structure Display Server (GSDS). Phylogeny was demonstrated through an evolutionary tree employing the neighbor-joining method. Subcellular localization of proteins was identified via “Softberry” and cis-acting elements were identified through PlantCARE. The KASP (Kompetitive Allele Specific PCR (KASP)) marker was developed for the GmPERK-1-C and GmPERK-1-T allele, targeting position 167 nt in the CDS region. Genotyping results indicated that GmPERK-1 exhibits promising potential for utilization in molecular breeding programs for soybean to increase crop yield. Collectively, our findings indicate that G. max accessions harboring the GmPERK-1-C allele exhibit significantly higher thousand grain weight compared to accessions carrying the GmPERK-1-T allele. This research enhances the understanding of the molecular roles of PERK genes in G. max, providing valuable insights for the utilization of favorable genetic variations in soybean molecular breeding programs.

Seedlessness is one of the highest valued agronomic traits in grapes. Embryo rescue in combination with marker-assisted selection have been widely applied in seedless grape breeding due to the advantages of increasing the ratio of seedless progenies and shortening the breeding cycle. However, the large number of deformed seedlings produced during embryo rescue and the lack of fast, efficient, and low-cost markers severely inhibit the process of seedless grape breeding. In this study, a total of eighty-three grape cultivars (51 seedless and 32 seeded) with diverse genetic backgrounds and two populations derived from embryo rescue, including 113 F1 hybrid individuals (60 seedless and 53 seeded), were utilized. We screened suitable media for converting malformed seedlings into normal seedlings, analyzed the association between the SNP in VviAGL11 and seeded/seedless phenotype, and developed a KASP marker related to stenospermocarpic seedlessness. Our results indicated that the transformation rate of 37.8% was obtained with MS medium supplemented with 2.0 mg·L−1 of 6-BA and 0.5 mg·L−1 of IBA. The presence of an A nucleotide allele at position chr18:26889437 was further confirmed to be fully associated with the stenospermocarpic seedlessness phenotype. The developed KASP marker, based on the verified SNP locus in VviAGL11, successfully distinguished the seedless and seeded genotypes with high precision and throughput. The results will contribute to enhancing the efficiency of embryo rescue and facilitate parent selection and early selection of seedless offspring with molecular markers, thereby accelerating the breeding process in seedless table grapes. [ABSTRACT FROM AUTHOR]

Donkey milk has high nutritional and medicinal value, but there are few researches in donkey milk traits, especially on genome. The whole lactation of 89 donkeys was recorded and it was found that Xinjiang donkey had good lactation performance while great differences among individuals. In our previous study, four genes including LGALS2, NUMB, ADCY8 and CA8 were identified as milk-associated with Chinese Kazakh house, based on Equine 670k Chip genomic analysis. And then 15 SNPs of the four key genes were conducted for genotyping in Xinjiang donkey in this study, one of Chinese indigenous breed, 14 SNPs were successful classified. And those SNPs were correlation analysis with milk yield of Xinjiang donkeys. The results showed that NUMB g.46709914T > G was significantly correlated with daily milk yield of Xinjiang donkey in the early, middle, and late periods, while ADCY8 g.48366302T > C, CA8 g.89567442T > G and CA8 g.89598328T > A were significantly correlated with lactation in the late periods. These results indicate that NUMB g.46709914T > G can be as markers of candidate genes for lactating traits in donkeys, SNPs of ADCY8 and CA8 as potential. Our findings will not only help confirm key genes for donkey milk traits, but also provide future for genomic selection in donkeys. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Potato cyst nematodes (PCNs), Globodera rostochiensis and G. pallida, are one of the major pathogens of potato and cause significant losses worldwide. These species, which are on the worldwide quarantine list, can survive in infected areas for more than 20 years. Therefore, these species need to be identified accurately and quickly. Although different molecular methods have been developed for the detection of G. rostochiensis and G. pallida, new techniques are being studied to identify these species. Kompetitive Allele-Specific PCR (KASP) based on single nucleotide polymorphisms (SNP) is a novel molecular technique. This technique is used for marker-assisted selection of genes that confer resistance in plants, for determining genetic variation of pathogens and for species identification. In this study, G. rostochiensis and G. pallida specific KASP primers were developed from the available DNA sequences of all Globodera species retrieved from GenBank. The KASP assay successfully identified the species G. rostochiensis and G. pallida. The developed KASP assays were able to distinguish these species from each other and from other nematode species. Results shown that even one cyst of these species can be sufficient for identification. This is the first study to develop KASP primers to identify PCNs. These assays can be used for accurate and rapid identification PCNs in laboratories. [ABSTRACT FROM AUTHOR]

Radish (Raphanus sativus L.) is a vegetable crop with economic value and ecological significance in the genus Radish, family Brassicaceae. In recent years, developed countries have attached great importance to the collection and conservation of radish germplasm resources and their research and utilization, but the lack of population genetic information and molecular markers has hindered the development of the genetic breeding of radish. In this study, we integrated the radish genomic data published in databases for the development of single-nucleotide polymorphism (SNP) markers, and obtained a dataset of 308 high-quality SNPs under strict selection criteria. With the support of Kompetitive Allele-Specific PCR (KASP) technology, we screened a set of 32 candidate core SNP marker sets to analyse the genetic diversity of the collected 356 radish varieties. The results showed that the mean values of polymorphism information content (PIC), minor allele frequency (MAF), gene diversity and heterozygosity of the 32 candidate core SNP markers were 0.32, 0.30, 0.40 and 0.25, respectively. Population structural analysis, principal component analysis and genetic evolutionary tree analysis indicated that the 356 radish materials were best classified into two taxa, and that the two taxa of the material were closely genetically exchanged. Finally, on the basis of 32 candidate core SNP markers we calculated 15 core markers using a computer algorithm to construct a fingerprint map of 356 radish varieties. Furthermore, we constructed a core germplasm population consisting of 71 radish materials using 32 candidate core markers. In this study, we developed SNP markers for radish cultivar identification and genetic diversity analysis, and constructed DNA fingerprints, providing a basis for the identification of radish germplasm resources and molecular marker-assisted breeding as well as genetic research.

Rice is a staple food for more than half of the global population due to its food security and sustainable development. Weeds compete with crops for sunlight and indispensable nutrients, affecting the yield and quality of crops. Breeding herbicide-tolerant rice varieties paired with herbicide application is expected to help with weed control. In this study, 194 Japonica/Geng rice varieties or lines collected from the Huanghuaihai region of China were screened by Kompetitive Allele-Specific PCR (KASP) markers based on four mutation sites within OsALS1 (LOC_Os02g30630), which is the target of imidazolinone (IMI) herbicides. Only the OsALS1627N haplotype was identified in 18 varieties, including the previously reported Jingeng818 (JG818), and its herbicide resistance was validated by treatment with three IMIs. To investigate the origin of the OsALS1627N haplotype in the identified varieties, six codominant PCR-based markers tightly linked with OsALS1 were developed. PCR analysis revealed that the other 17 IMI-tolerant varieties were derived from JG818. We randomly selected three IMI-tolerant varieties for comparative whole-genome resequencing with known receptor parent varieties. Sequence alignment revealed that more loci from JG818 have been introduced into IMI-tolerant varieties. However, all three IMI-tolerant varieties carried clustered third type single nucleotide polymorphism (SNP) sites from unknown parents, indicating that these varieties were not directly derived from JG818, whereas those from different intermediate improved lines were crossed with JG818. Overall, we found that only OsALS1627N from JG818 has been broadly introduced into the Huanghuaihai region of China. Additionally, the 17 identified IMI-tolerant varieties provide alternative opportunities for improving such varieties along with other good traits.

Abstract 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.

A recombinant inbred line population including 371 lines was developed by a high kernel number per spike (KNPS) genotype T1208 and a low KNPS genotype Chuannong18 (CN18). A genetic linkage map consisting of 11,583 markers was constructed by the Wheat55K SNP Array. The quantitative trait loci (QTLs) related to KNPS were detected in three years. Eight, twenty-seven, and four QTLs were identified using the ICIM-BIP, ICIM-MET, and ICIM-EPI methods, respectively. One QTL, QKnps.sau-2D.1, which was mapped on chromosome 2D, can explain 18.10% of the phenotypic variation (PVE) on average and be considered a major and stable QTL for KNPS. This QTL was located in a 0.89 Mb interval on chromosome 2D and flanked by the markers AX-109283238 and AX-111606890. Moreover, KASP-AX-111462389, a Kompetitive Allele-Specific PCR (KASP) marker which closely linked to QKnps.sau-2D.1, was designed. The genetic effect of QKnps.sau-2D.1 on KNPS was successfully confirmed in two RIL populations. The results also showed that the significant increase of KNPS and 1000-kernel weight (TKW) was caused by QKnps.sau-2D.1 overcoming the disadvantage due to the decrease of spike number (SN) and finally lead to a significant increase of grain yield. In addition, within the interval in which QKnps.sau-2D.1 is located in Chinese Spring reference genomes, only fifteen genes were found, and two genes that might associate with KNPS were identified. QKnps.sau-2D.1 may provide a new resource for the high-yield breeding of wheat in the future. [ABSTRACT FROM AUTHOR]

Kompetitive allele-specific PCR (KASP) was a fluorescence-based homogeneous genotyping technique, which could perform the biallelic genotyping detection on the single nucleotide polymorphism sites and insertion/deletion polymorphisms in the genome・ It had the advantages of rapid detection, low cost and high accuracy・ The composition, amplification principle and reaction steps of KASP detection system were described in this paper, as well as its advantages over the traditional SNP detection methods. Then, the current research progresses in animal genetic identification, functional gene analysis and molecular marker-assisted selection (MAS) assisted breeding were summarized, in order to provide reference for the application of KASP technology in animal field. [ABSTRACT FROM AUTHOR]

The Hessian fly (HF) is an invasive insect that has caused millions of dollars in yield losses to southeastern US wheat farms. Genetic resistance is the most sustainable solution to control HF. However, emerging biotypes are quickly overcoming resistance genes in the southeast; therefore, identifying novel sources of resistance is critical. The resistant line "UGA 111729" and susceptible variety "AGS 2038" were crossbred to generate a population of 225 recombinant inbred lines. This population was phenotyped in the growth chamber (GC) during 2019 and 2021 and in field (F) trials in Georgia during the 2021–2022 growing seasons. Visual scoring was utilized in GC studies. The percentage of infested tillers and number of pupae/larvae per tiller, and infested tiller per sample were measured in studies from 2021 to 2022. Averaging across all traits, a major QTL on chromosome 3D explained 42.27% (GC) and 10.43% (F) phenotypic variance within 9.86 centimorgans (cM). SNP marker IWB65911 was associated with the quantitative trait locus (QTL) peak with logarithm of odds (LOD) values of 14.98 (F) and 62.22 (GC). IWB65911 colocalized with resistance gene H32. KASP marker validation verified that UGA 111729 and KS89WGRC06 express H32. IWB65911 may be used for marker-assisted selection. [ABSTRACT FROM AUTHOR]

Guava (Psidium guajava L.) is an important fruit crop of the Indian sub-continent, with potential for improvements in quality and yield. The goal of the present study was to construct a genetic linkage map in an intraspecific cross between the elite cultivar 'Allahabad Safeda' and the Purple Guava landrace to identify the genomic regions responsible for important fruit quality traits, viz., total soluble solids, titratable acidity, vitamin C, and sugars. This population was phenotyped in field trials (as a winter crop) for three consecutive years, and showed moderateto- high values of heterogeneity coefficients along with higher heritability (60.0%-97.0%) and genetic-advance-over-mean values (13.23%-31.17%), suggesting minimal environmental influence on the expression of fruit-quality traits and indicating that these traits can be improved by phenotypic selection methods. Significant correlations and strong associations were also detected among fruit physico-chemical traits in segregating progeny. The constructed linkage map consisted of 195 markers distributed across 11 chromosomes, spanning a length of 1,604.47 cM (average inter-loci distance of 8.80 markers) and with 88.00% coverage of the guava genome. Fifty-eight quantitative trait loci (QTLs) were detected in three environments with best linear unbiased prediction (BLUP) values using the composite interval mapping algorithm of the BIP (biparental populations) module. The QTLs were distributed on seven different chromosomes, explaining 10.95%-17.77% of phenotypic variance, with the highest LOD score being 5.96 for qTSS.AS.pau-6.2. Thirteen QTLs detected across multiple environments with BLUPs indicate stability and utility in a future breeding program for guava. Furthermore, seven QTL clusters with stable or common individual QTLs affecting two or more different traits were located on six linkage groups (LGs), explaining the correlation among fruit-quality traits. Thus, the multiple environmental evaluations conducted here have increased our understanding of the molecular basis of phenotypic variation, providing the basis for future high-resolution fine-mapping and paving the way for marker-assisted breeding of fruit-quality traits. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Barley (Hordeum vulgare L.) is one of the most commonly cultivated cereals worldwide. Its local varieties can represent a valuable source of unique genetic variants useful for crop improvement. The aim of this study was to reveal loci contributing to spike productivity traits in Siberian spring barley and to develop diagnostic DNA markers for marker-assisted breeding programs. For this purpose we conducted a genome-wide association study using a panel of 94 barley varieties. In total, 64 SNPs significantly associated with productivity traits were revealed. Twenty-three SNP markers were validated by genotyping in an independent sample set using competitive allele-specific PCR (KASP). Finally, fourteen markers associated with spike productivity traits on chromosomes 2H, 4H and 5H can be suggested for use in breeding programs. [ABSTRACT FROM AUTHOR]

Simple Summary: In order to improve animals' resistance to mastitis in subsequent generations and to reduce disease prevalence, one feasible approach could be the genomic selection of cows with the most potentially protective immune responses. In this study, 298 cattle were genotyped for 89 single nucleotide polymorphisms through the Kompetitive Allele Specific PCR. Our findings reveal a significant association of a molecular marker in the BOLA-DRB3 gene with mastitis prevalence in the investigated breeds. This supports previous studies that the BOLA-DRB3 gene represents an important locus affecting resistance to mastitis in cattle. These results contribute to an increase in knowledge and available data regarding the genetic variability of SNPs for udder health in cattle. Mastitis is the most common production disease in the dairy sector worldwide, its incidence being associated with both cows' exposure to bacteria and the cows' genetic make-up for resistance to pathogens. The objective of our study was to analyse 89 missense SNPs belonging to six genes (CXCR2, CXCL8, TLR4, BRCA1, LTF, BOLA-DRB3), which were found to be associated with genetic resistance or susceptibility to mastitis. A total of 298 cattle (250 Romanian Spotted and 48 Romanian Brown) were genotyped by Kompetitive Allele Specific PCR (KASP) and a chi-squared test was used for genetic association studies with clinical mastitis. A total of 35 SNPs (39.3%) among the selected 89 SNPs were successfully genotyped, of which 31 markers were monomorphic. The polymorphic markers were found in two genes: TLR4 (rs460053411) and BOLA-DRB3 (rs42309897, rs208816121, rs110124025). The polymorphic SNPs with MAF > 5% and call rates > 95% were used for the association study. The results showed that rs110124025 in the BOLA-DRB3 gene was significantly associated with mastitis prevalence (p ≤ 0.05) in both investigated breeds. Current results show that the SNP rs110124025 in the BOLA-DRB3 gene can be used as a candidate genetic marker in selection for mastitis resistance in Romanian dairy cattle. [ABSTRACT FROM AUTHOR]

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 mose hata) 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 F21 progenies derived from the PVR-1343 P-135 cross revealed digenic recessive inheritance against MI - Sqfro resistance in PVR-1 343. 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 lqMI-Sqf[07. 1), while chromosomal region spanning 0. 87 Mb on chromosomel 7 0!MI-Sq/Toll. 1) was additionally identified by QTLseqr. However, the highest peak value on chromosome 7 with three algorithms {G', 8(SNP-index) and -logio (P value)} high-lighted the major contribution of qMI-Sqn-07. j in MI -Sq/To resistance. Nine polymorphic SNPs identified within the highly significant qM/- Sq/T07. 1 region were converted into KASP markers. KASP genotyping of Fi individuals narrowed down the qMI-scin'07. 1 interval to [03 kb region flanked by two markers, Cnio3914729 and Cm04018182. which contained 16 annotated genes and accounted for 59.84% of phenotypic variation. The Cmo4018182 KASPmarkeraccurately predicteddisease 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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Pesticide resistance is a long‐standing and growing problem in the chemical control of invertebrate pests. Molecular diagnostic methods can facilitate pesticide resistance management by accurately and efficiently detecting resistant mutations and their frequency. In this study, the kompetitive allele specific PCR (KASP) approach, a technology for high‐throughput single nucleotide polymorphism (SNP) genotyping, is validated as a useful method for characterizing genotypes at a pesticide‐resistance locus for the first time. We focus on the spinetoram resistance mutation of G275E in the nicotinic acetylcholine receptor alpha 6 (nAChR α6) subunit gene of Thrips palmi. RESULTS: Of the 341 individuals of Thrips palmi tested, 98.24% were successfully genotyped, with 100% concordance with Sanger sequencing results. We then quantitatively mixed genomic DNA of known genotypes to establish 21 DNA mixtures with a resistant allele frequency ranging from 0 to 100% at steps of 5%. The linear discriminant analysis (LDA) showed that 75.8% of original grouped cases were correctly classified; six groups had no overlap in membership (resistant allele frequency: 0%, 5%, 10–75%, 80–85%, 90–95%, and 100%). When we chose 11 pooled samples with 10% steps for LDA, 84.4% of original grouped cases were correctly classified; seven groups had no overlap in membership (0%, 10%, 20–30%, 40–70%, 80%, 90%, 100%). The results indicated that KASP applied to pooled samples may provide a semi‐quantitative estimate of resistance. CONCLUSIONS: Our study points to the suitability of KASP for high‐throughput genotyping of genotypes affecting pesticide resistance and semi‐quantitative assessments of resistance allele frequencies in populations. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Almond shell hardness is an economically important quality trait that protects the integrity of the kernel. It is directly related with shelling or crack-out percent. In the U.S. and Australian markets, soft to semi-hard shells varieties have been used in the industry, while in the European market, hard shells are dominantly sold and distributed. The origin of a hard shell is from wild almond species. Soft shells in the USA and Australia have been selected for a higher crack-out percent or larger kernel and ease of processing. Here, we examined breeding populations totaling 264 trees that were genotyped with genotyping-by-sequencing (GBS) where shell hardness was measured with a texture analyzer, and shell weight, in-shell weights and shell thickness were measured and crack-out percent was calculated. In a genome-wide association, seven genetic loci were found to be associated with shell hardness, crack-out percent, shell thickness and shell weight. Of these seven, one was shared between multiple traits. Kompetitive allele-specific PCR (KASP) assays were developed and tested in unrelated breeding lines from Australia, Spain, France and Italy. One KASP assay was found to be highly predictive of shell hardness in this diverse panel and can be applied for use in marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Heading date (HD) and total spikelet number per spike (SNS) play important roles in the yield improvement of wheat. HD often influences the formation of total spikelets. Identifying various genes that control HD and SNS is vital for the yield improvement of wheat. Here, we used two recombinant inbred line (RIL) populations derived from crosses of Yangmai 12 (YM12)/Yanzhan 1 (YZ1) and Yangmai 4 (YM4)/YZ1 to construct two genetic linkage maps and identify QTL for HD and SNS. A total of sixteen QTL were detected, among which three QTL for HD and three QTL for SNS detected in the YM12/YZ1 population overlapped those detected in the YM4/YZ1 population, respectively. Two pleiotropic QTL regions (Cluster-2B and Cluster-7D) simultaneously controlling HD and SNS were detected in both two populations. Among the QTL for SNS, only the favored allele of QSns.Y12Y-2A/QSns.Y4Y-2A had a significantly positive effect on grain number per spike without negatively affecting thousand-grain weight. A kompetitive allele-specific PCR (KASP) marker for QSns.Y12Y-2A/QSns.Y4Y-2A had been developed and verified in a set of 256 diverse cultivars/lines to facilitate its applications. Then the genes within the physical interval of QSns.Y12Y-2A/QSns.Y4Y-2A were analyzed based on the spatiotemporal expression patterns, gene annotation, and orthologous search. Six candidate genes were expressed specially in spike or spikelet. These results provide a solid foundation for future fine mapping and cloning of QSns.Y12Y-2A/QSns.Y4Y-2A. [ABSTRACT FROM AUTHOR]

Abstract Cauliflower is one of the most important vegetable crops grown worldwide. However, the lack of genetic diversity information and efficient molecular markers hinders efforts to improve cauliflower. This study aims to construct DNA fingerprints for 329 cauliflower cultivars based on SNP markers and the KASP system. After rigorous filtering, a total of 1662 candidate SNPs were obtained from nearly 17.9 million SNP loci. The mean values of PIC, MAF, heterozygosity and gene diversity of these SNPs were 0.389, 0.419, 0.075, and 0.506, respectively. We developed a program for in silico simulations on 153 core germplasm samples to generate ideal SNP marker sets from the candidates. Finally, 41 highly polymorphic KASP markers were selected and applied to identify 329 cauliflower cultivars, mainly collected from the public market. Furthermore, based on the KASP genotyping data, we performed phylogenetic analysis and population structure analysis of the 329 cultivars. As a result, these cultivars could be classified into three major clusters, and the classification patterns were significantly related to their curd solidity and geographical origin. Finally, fingerprints of the 329 cultivars and 2D barcodes with the genetic information of each sample were generated. The fingerprinting database developed in this study provides a practical tool for identifying the authenticity and purity of cauliflower seeds and valuable genetic information about the current cauliflower cultivars.

Abstract Background Breeding of conventional and hybrid rice (Oryza sativa L.) have solved hunger problems and increased farmers' income in the world. Molecular markers have been widely used in marker-assisted breeding and identification of larger numbers of different bred varieties in the past decades. The recently developed SNP markers are applied for more stable and detectable compared with other markers. But the cost of genotyping lots SNPs is high. So, it is essential to select less representative SNPs and inexpensive detecting methods to lower the cost and accelerate variety identification and breeding process. KASP (Kompetitive Allele-Specific PCR) is a flexible method to detect the SNPs, and large number of KASP markers have been widely used in variety identification and breeding. However, the ability of less KASP markers on massive variety identification and breeding remains unknown. Results Here, 48 KASP markers were selected from 378 markers to classify and analyze 518 varieties including conventional and hybrid rice. Through analyzing the population structure, the 48 markers could almost represent the 378 markers. In terms of variety identification, the 48 KASP markers had a 100% discrimination rate in 53 conventional indica varieties and 193 hybrid varieties, while they could distinguish 89.1% conventional japonica rice from different breeding institutes. Two more markers added would increase the ratio from 68.38 to 77.94%. Additionally, the 48 markers could be used for classification of subpopulations in the bred variety. Also, 8 markers had almost completely different genotypes between japonica and indica, and 3 markers were found to be very important for japonica hybrid rice. In hybrid varieties, the heterozygosity of chromosomes 3, 6 and 11 was relatively higher than others. Conclusions Our results showed that 48 KASP markers could be used to identify rice varieties, and the panel we tested could provide a database for breeders to identify new breeding lines. Also, the specific markers we found were useful for marker-assisted breeding in rice, including conventional and hybrid.

Introduction: Pre-harvest Sprouting (PHS) seriously affects wheat quality and yield. However, to date there have been limited reports. It is of great urgency to breed resistance varieties via quantitative trait nucleotides (QTNs) or genes for PHS resistance in white-grained wheat. Methods: 629 Chinese wheat varieties, including 373 local wheat varieties from 70 years ago and 256 improved wheat varieties were phenotyped for spike sprouting (SS) in two environments and genotyped by wheat 660K microarray. These phenotypes were used to associate with 314,548 SNP markers for identifying QTNs for PHS resistance using several multi-locus genome-wide association study (GWAS) methods. Their candidate genes were verified by RNA-seq, and the validated candidate genes were further exploited in wheat breeding. Results: As a result, variation coefficients of 50% and 47% for PHS in 629 wheat varieties, respectively, in 2020-2021 and 2021-2022 indicated large phenotypic variation, in particular, 38 white grain varieties appeared at least medium resistance, such as Baipimai, Fengchan 3, and Jimai 20. In GWAS, 22 significant QTNs, with the sizes of 0.06% - 38.11%, for PHS resistance were stably identified by multiple multilocus methods in two environments, e.g., AX-95124645 (chr3D:571.35Mb), with the sizes of 36.390% and 45.850% in 2020-2021 and 2021-2022, respectively, was detected by several multi-locus methods in two environments. As compared with previous studies, the AX-95124645 was used to develop Kompetitive Allele-Specific PCR marker QSS.TAF9-3D (chr3D:569.17Mb-573.55Mb) for the first time, especially, it is available in white-grain wheat varieties. Around this locus, nine genes were significantly differentially expressed, and two of them (TraesCS3D01G466100 and TraesCS3D01G468500) were found by GO annotation to be related to PHS resistance and determined as candidate genes. Discussion: The QTN and two new candidate genes related to PHS resistance were identified in this study. The QTN can be used to effectively identify the PHS resistance materials, especially, all the white-grained varieties with QSS.TAF9-3DTT haplotype are resistant to spike sprouting. Thus, this study provides candidate genes, materials, and methodological basis for breeding wheat PHS resistance in the future. [ABSTRACT FROM AUTHOR]

Drought stress is a major yield-limiting factor throughout the world in wheat (Triticum aestivum L.), causing losses of up to 80% of the total yield. The identification of factors affecting drought stress tolerance in the seedling stage is especially important to increase adaptation and accelerate the grain yield potential. In the current study, 41 spring wheat genotypes were tested for their tolerance to drought at the germination stage under two different polyethylene glycol concentrations (PEG) of 25% and 30%. For this purpose, twenty seedlings from each genotype were evaluated in triplicate with a randomized complete block design (RCBD) in a controlled growth chamber. The following nine parameters were recorded: germination pace (GP), germination percentage (G%), number of roots (NR), shoot length (SL), root length (RL), shoot–root length ratio (SRR), fresh biomass weight (FBW), dry biomass weight (DBW), and water content (WC). An analysis of variance (ANOVA) revealed highly significant differences (p < 0.01) among the genotypes, treatments (PEG25%, PEG30%) and genotypes × treatment interaction, for all traits. The broad-sense heritability (H2) estimates were very high in both concentrations. They ranged from 89.4 to 98.9% under PEG25% and from 70.8 to 98.7% under PEG30%. Citr15314 (Afghanistan) was among the best performing genotypes under both concentrations for most of the germination traits. Two KASP markers for TaDreb-B1 and Fehw3 genes were used to screen all genotypes and to study the effect of these on drought tolerance at the germination stage. All genotypes with Fehw3 (only) showed a better performance for most traits under both concentrations compared to other genotypes having TaDreb-B1 or having both genes. To our knowledge, this work is the first report showing the effect of the two genes on germination traits under severe drought stress conditions. [ABSTRACT FROM AUTHOR]

BACKGROUND: The diamondback moth (DBM) Plutella xylostella has developed resistance to almost all insecticides used to control it. Populations of DBM in temperate regions mainly migrate from annual breeding areas. However, the distribution pattern of insecticide resistance of DBM within the context of long-distance migration remains unclear. RESULTS: In this study, we examined the frequency of 14 resistance mutations for 52 populations of DBM collected in 2010, 2011, 2017 and 2018 across China using a high-throughput KASP genotyping method. Mutations L1041F and T929I conferring pyrethroid resistance, and mutations G4946E and E1338D conferring chlorantraniliprole resistance were near fixation in most populations, whereas resistant alleles of F1020S, M918I, A309V and F1845Y were uncommon or absent in most populations. Resistance allele frequencies were relatively stable among different years, although the frequency of two mutations decreased. Principal component analysis based on resistant allele frequencies separated a southern population as an outlier, whereas the immigrants clustered with other populations, congruent with the migration pattern of northern immigrants coming from the Sichuan area of southwestern China. Most resistant mutations deviated from Hardy–Weinberg equilibrium due to a lower than expected frequency of heterozygotes. The deviation index of heterozygosity for resistant alleles was significantly higher than the index obtained from single nucleotide polymorphisms across the genome. These findings suggest heterogeneous selection pressures on resistant mutations. CONCLUSION: Our results provide a picture of resistant mutation patterns in DBM shaped by insecticide usage and migration of this pest, and highlight the widespread distribution of resistance alleles in DBM. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLVs) affect sheep and goats worldwide. The major gene related to SRLV infections is the Transmembrane Protein Gene 154 (TMEM154). We estimated the haplotype frequencies of TMEM154 in the USA (USDA-ARS) and Brazil (Embrapa) Gene Banks by using two different SNP genotyping methodologies, FluidigmTM and KASPTM. We also genotyped the ZNF389_ss748775100 deletion variant in Brazilian flocks. A total of 1040 blood samples and 112 semen samples from 15 Brazilian breeds were genotyped with Fluidigm for the SNP ZNF389_ss748775100 and 12 TMEM154 SNPs. A total of 484 blood samples from the Santa Inês breed and 188 semen samples from 14 North American sheep breeds were genotyped with KASP for 6 TMEM154 SNPs. All the Brazilian samples had the "I/I" genotype for the ZNF389_ss748775100 mutation. There were 25 TMEM154 haplotypes distributed across the Brazilian breeds, and 4 haplotypes in the US breeds. Haplotypes associated with susceptibility were present in almost all breeds, which suggests that genetic testing can help to improve herd health and productivity by selecting non-susceptible animals as founders of the next generations. Fluidigm and KASP are reliable assays when compared with Beadchip arrays. Further studies are necessary to understand the unknown role of TMEM154 mutations, host–pathogen interaction and new genes associated with the clinical condition. [ABSTRACT FROM AUTHOR]