Tools For Marker-assisted Selection Research Articles

Genomic tools for early selection among Thoroughbreds and Polo Argentino horses for practicing polo

The Polo Argentino (PA) horse is a recognized breed, developed originally by mixing crossbred and Thoroughbred (TB) horses to play polo. Early PA selection is difficult due to unreliable performance estimations. This study investigated the usefulness of genomic markers previously linked to morphological and functional traits as a tool for the early selection of PA. To this, we genotyped 520 PA and 30 TB horses using the Equine GGPArray (Illumina, n = 71,778 SNPs). Analyses included a genetic characterization of six genetic markers associated with behavioral (DRD4), muscular development (MSTN), and body size (LCORL, HMGA6, ZFAT, and LASP1) genes. Genetic differences in the DRD4, MSTN, and LCORL SNP were found between the two breeds, in the last two FST index between breeds was 0.13 and 0.6, respectively (p < 0.01). In DRD4, G allele was the more prevalent in PA (0.56 vs 0.45 in TB, p < 0.05), but no differences were observed between the genotypes associated with phenotypes. In MSTN, heterozygous genotypes were the most common in PA (48 %), with a significant decrease in AA (Hardy-Weinberg p < 0.05), suggesting a negative selection against it in polo horses. In body size, HMGA2 was monomorphic in all horses, while ZFAT and LASP1 SNP showed higher variability. Interestingly, 99 % of PA showed a TT genotype in LCORL (only 66 % in TB), demonstrating selection for smaller horses. Our results suggest that empirical selection in PA has generated an incipient genomic differentiation in discrete traits which could be used as a marker-assisted selection tool for early selection of polo horses.

SNPs in microRNA seed region and impact of miR-375 in concurrent regulation of multiple lipid accumulation-related genes

Bovine intramuscular fat (IMF), commonly referred to as marbling, is regulated by lipid metabolism, which includes adipogenesis, lipogenesis, glycerolipid synthesis, and lipolysis. In recent years, breeding researchers have identified single nucleotide polymorphisms (SNPs) as useful marker-assisted selection tools for improving marbling scores in national breeding programs. These included causal SNPs that induce phenotypic variation. MicroRNAs (miRNAs) are small highly conserved non-coding RNA molecules that bind to multiple non-coding regions. They are involved in post-transcriptional regulation. Multiple miRNAs may regulate a given target. Previously, three SNPs in the GPAM 3ʹ UTR and four miRNAs were identified through in silico assays. The aim of this study is to verify the binding ability of the four miRNAs to the SNPs within the 3ʹUTR of GPAM, and to identify the regulatory function of miR-375 in the expression of genes related to lipid metabolism in mammalian adipocytes. It was verified that the four miRNAs bind to the GPAM 3ʹUTR, and identified that the miR-375 sequence is highly conserved. Furthermore, it was founded that miR-375 upregulated the GPAM gene, C/EBPα, PPARγ and lipid metabolism-related genes and promoted lipid droplet accumulation in 3T3-L1 cells. In conclusion, these results suggest that miR-375 is a multifunctional regulator of multiple lipid metabolism-related genes and may aid in obesity research as a biomarker.

Quantitative Trait Loci Analysis and Marker Development for Fruit Rot Resistance in Cranberry Shows Potential Genetic Association with Epicuticular Wax.

Fruit rot is a fungal disease complex that threatens cranberry yields in North American growing operations. Management of fruit rot is especially difficult because of the diversity of the infecting fungal species, and although infections take place early in the season, the pathogens usually remain latent in the ovary until the fruit ripen. Control methods heavily rely on fungicide applications, a practice that may be limited in viability long term. Breeding for fruit rot resistance (FRR) is essential for sustainable production. It is likely that field resistance is multifaceted and involves a myriad of traits that fortify cranberry plants against the biotic and abiotic stresses contributing to fruit rot. In this study, we identified quantitative trait loci (QTL) for FRR in a segregating population. Interestingly, a QTL associated with resistance was found to overlap with one associated with fruit epicuticular wax (ECW). A single-nucleotide polymorphism genotyping assay successfully identified accessions that exhibit the desired phenotypes (i.e., less rot and more ECW), thus making it a useful tool for marker-assisted selection. Candidate genes that may contribute to FRR and ECW were also identified. This work will expedite breeding for improved cranberry fruit quality.

Brinjal: Breeding and Genomics

Brinjal (Solanum melongena L.) also known as eggplant is an important solanaceous vegetable crop grown across tropical and subtropical regions of the world. India is the centre of origin of the crop, large diversity in the morphology of fruit and plant type exists in the country. Also, a large number of wild relatives is being utilized in breeding programs as a source of biotic and abiotic stress tolerance. Conventional breeding programs have focused on improving plant type and yield through the development of high-yielding varieties and hybrids. More recently, breeding for stress tolerance and enhanced nutritional content has gained importance. Genetic inheritance studies, mapping, molecular tools are enriching the crop improvement work. Of late, the availability of high-quality genome sequences has spurred functional analysis at the genome level augmenting molecular tools for marker-assisted selection. In the present article, attempt has been made to highlight the improvement work carried out from relevance of the crop diversity to genomic-level knowledge advancement. Future prospective in brinjal improvement having relevance in Indian context is also highlighted.

Development of novel specific molecular markers for the Sw-5b gene to assist with tomato spotted wilt virus-resistant tomato breeding

Tomato spotted wilt virus (TSWV) is a plant pathogen that causes devastating tomato yield losses worldwide. The Sw-5b gene is one of the most effective resistance genes for TSWV control in tomato plants, and has been widely used in resistance breeding. Molecular markers are specific DNA sequences with known locations on the chromosome; they are indispensable tools in marker-assisted selection, which detects the presence of target genes to expedite breeding. We developed gene-specific molecular markers for Sw-5b to facilitate the accurate distinction of resistance (Sw-5bR) and susceptibility (Sw-5bS) alleles of Sw-5b. Using these markers, we successfully detected Sw-5b and determined its genotype (homozygous Sw-5bR, heterozygous Sw-5bR/S, or homozygous Sw-5bS) in six tomato varieties. Then we successfully applied these markers to 46 commercial tomato cultivars to detect and determine the genotype of Sw-5b. The results revealed a striking absence of the Sw-5bR gene and high TSWV susceptibility among most of the analyzed commercial cultivars. With the assistance of the novel Sw-5b-specific molecular markers, we generated a TSWV-resistant and homozygous Sw-5bR Micro-Tom tomato line, demonstrating the practical application of these markers in plant breeding. In summary, we developed novel gene-specific molecular markers for Sw-5b, and applied them to distinguish Sw-5b alleles for TSWV resistance or susceptibility. This marker set provides a valuable tool for breeding TSWV-resistant tomato varieties.

Genetic variation among elite inbred lines suggests potential to breed for BNI-capacity in maize

Biological nitrification inhibition (BNI) is a plant function where root systems release antibiotic compounds (BNIs) specifically aimed at suppressing nitrifiers to limit soil-nitrate formation in the root zone. Little is known about BNI-activity in maize (Zea mays L.), the most important food, feed, and energy crop. Two categories of BNIs are released from maize roots; hydrophobic and hydrophilic BNIs, that determine BNI-capacity in root systems. Zeanone is a recently discovered hydrophobic compound with BNI-activity, released from maize roots. The objectives of this study were to understand/quantify the relationship between zeanone activity and hydrophobic BNI-capacity. We assessed genetic variability among 250 CIMMYT maize lines (CMLs) characterized for hydrophobic BNI-capacity and zeanone activity, towards developing genetic markers linked to this trait in maize. CMLs with high BNI-capacity and ability to release zeanone from roots were identified. GWAS was performed using 27,085 SNPs (with unique positions on the B73v.4 reference genome, and false discovery rate = 10), and phenotypic information for BNI-capacity and zeanone production from root systems. Eighteen significant markers were identified; three associated with specific BNI-activity (SBNI), four with BNI-activity per plant (BNIPP), another ten were common between SBNI and BNIPP, and one with zeanone release. Further, 30 annotated genes were associated with the significant SNPs; most of these genes are involved in pathways of “biological process”, and one (AMT5) in ammonium regulation in maize roots. Although the inbred lines in this study were not developed for BNI-traits, the identification of markers associated with BNI-capacity suggests the possibility of using these genomic tools in marker-assisted selection to improve hydrophobic BNI-capacity in maize.

Drought stress in rice: morpho-physiological and molecular responses and marker-assisted breeding.

Rice (Oryza Sativa L.) is an essential constituent of the global food chain. Drought stress significantly diminished its productivity and threatened global food security. This review concisely discussed how drought stress negatively influenced the rice's optimal growth cycle and altered its morpho-physiological, biochemical, and molecular responses. To withstand adverse drought conditions, plants activate their inherent drought resistance mechanism (escape, avoidance, tolerance, and recovery). Drought acclimation response is characterized by many notable responses, including redox homeostasis, osmotic modifications, balanced water relations, and restored metabolic activity. Drought tolerance is a complicated phenomenon, and conventional breeding strategies have only shown limited success. The application of molecular markers is a pragmatic technique to accelerate the ongoing breeding process, known as marker-assisted breeding. This review study compiled information about quantitative trait loci (QTLs) and genes associated with agronomic yield-related traits (grain size, grain yield, harvest index, etc.) under drought stress. It emphasized the significance of modern breeding techniques and marker-assisted selection (MAS) tools for introgressing the known QTLs/genes into elite rice lines to develop drought-tolerant rice varieties. Hence, this study will provide a solid foundation for understanding the complex phenomenon of drought stress and its utilization in future crop development programs. Though modern genetic markers are expensive, future crop development programs combined with conventional and MAS tools will help the breeders produce high-yielding and drought-tolerant rice varieties.

Development of InDels markers for the identification of cytoplasmic male sterility in Sorghum by complete chloroplast genome sequences analysis.

Cytoplasmic male sterility (CMS) is predominantly used for F1 hybrid breeding and seed production in Sorghum. DNA markers to distinguish between normal fertile (CMS-N) and sterile (CMS-S) male cytoplasm can facilitate F1 hybrid cultivar development in Sorghum breeding programs. In this study, the complete chloroplast (cp) genome sequences of CMS-S and Korean Sorghum cultivars were obtained using next-generation sequencing. The de novo assembled genome size of ATx623, the CMS-S line of the chloroplast, was 140,644bp. When compared to the CMS-S and CMS-N cp genomes, 19 single nucleotide polymorphisms (SNPs) and 142 insertions and deletions (InDels) were identified, which can be used for marker development for breeding, population genetics, and evolution studies. Two InDel markers with sizes greater than 20 bp were developed to distinguish cytotypes based on the copy number variation of lengths as 28 and 22 bp tandem repeats, respectively. Using the newly developed InDel markers with five pairs of CMS-S and their near isogenic maintainer line, we were able to easily identify their respective cytotypes. The InDel markers were further examined and applied to 1,104 plants from six Korean Sorghum cultivars to identify variant cytotypes. Additionally, the phylogenetic analysis of seven Sorghum species with complete cp genome sequences, including wild species, indicated that CMS-S and CMS-N contained Milo and Kafir cytotypes that might be hybridized from S. propinquum and S. sudanese, respectively. This study can facilitate F1 hybrid cultivar development by providing breeders with reliable tools for marker-assisted selection to breed desirable Sorghum varieties.

Genetic Improvements in Rice Grain Quality: A Review of Elite Genes and Their Applications in Molecular Breeding

High yield and superior quality are the main objectives of rice breeding and research. While innovations in rice breeding have increased production to meet growing demand, the universal issue of balancing high yield and susperior quality has led to a lack of focus on improving rice quality. With rising living standards, improving rice quality has become increasingly important. Rice grain quality is a complex trait influenced by both genetic and environmental factors, with four primary aspects: milling quality, appearance quality, eating and cooking quality, and nutritional quality. While different populations have varying demands for rice quality, the core traits that contribute to rice quality include grain shape and chalkiness in terms of appearance, as well as endosperm composition that influences cooking and sensory evaluation. Researchers have made substantial advancements in discovering genes/QTLs associated with critical traits including appearance, aroma, texture, and nutritional properties. Markers derived from these genetic discoveries have provided an efficient tool for marker-assisted selection to improve rice quality. Thus, this review focuses on elite genes and their applications in breeding practices to quickly develop superior quality rice varieties that meet various market demands.

Pipeline to Design Inbred Lines and F1 Hybrids of Leaf Chicory (Radicchio) Using Male Sterility and Genotyping-by-Sequencing.

Chicory, a horticultural crop cultivated worldwide, presents many botanical varieties and local biotypes. Among these, cultivars of the Italian radicchio group of the pure species Cichorium intybus L. and its interspecific hybrids with Cichorium endivia L.-as the "Red of Chioggia" biotype-includes several phenotypes. This study uses a pipeline to address the marker-assisted breeding of F1 hybrids: it presents the genotyping-by-sequencing results of four elite inbred lines using a RADseq approach and an original molecular assay based on CAPS markers for screening mutants with nuclear male sterility in the radicchio of Chioggia. A total of 2953 SNP-carrying RADtags were identified and used to compute the actual estimates of homozygosity and overall genetic similarity and uniformity of the populations, as well as to determine their genetic distinctiveness and differentiation. Molecular data were further used to investigate the genomic distribution of the RADtags among the two Cichorium species, allowing their mapping in 1131 and 1071 coding sequences in chicory and endive, respectively. Paralleling this, an assay to screen the genotype at the male sterility locus Cims-1 was developed to discriminate wild-type and mutant alleles of the causative gene myb80-like. Moreover, a RADtag mapped close to this genomic region proved the potential application of this method for future marker-assisted selection tools. Finally, after combining the genotype information of the core collection, the best 10 individuals from each inbred line were selected to compute the observed genetic similarity as a measure of uniformity as well as the expected homozygosity and heterozygosity estimates scorable by the putative progenies derived from selfing (pollen parent) and full-sibling (seed parent) or pair-wise crossing (F1 hybrids). This predictive approach was conducted as a pilot study to understand the potential application of RADseq in the fine tuning of molecular marker-assisted breeding strategies aimed at the development of inbred lines and F1 hybrids in leaf chicory.

Fine mapping of reduced height locus RHT26 in common wheat.

We fine mapped RHT26 for plant height in wheat, confirmed its genetic effects in a panel of wheat cultivars and predicted candidate genes. Development of wheat cultivars with appropriate plant height (PH) is an important goal in breeding. Utilization of semi-dwarfing genes Rht-B1b and Rht-D1b triggered wheat Green Resolution in the 1960s. Since these genes also bring unfavorable features, such as reduced coleoptile length and grain weight, it is necessary to identify alternative reduced height genes without yield penalty. Here we constructed a high-density genetic map of a recombinant inbred line population derived from the cross of Zhongmai175 and Lunxuan987 and detected a stable genetic locus for PH, designated RHT26, on chromosome arm 3DL in all of six environments, accounting for 6.8-14.0% of the phenotypic variances. RHT26 was delimited to an approximate 1.4Mb physical interval (517.1-518.5Mb) using secondary mapping populations derived from 22 heterozygous recombinant plants and 24 kompetitive allele-specific PCR markers. Eleven high-confidence genes were annotated in the physical interval according to the Chinese Spring reference genome, and four of them were predicted as candidates for RHT26 based on genome and transcriptome sequencing analyses. We also confirmed that RHT26 had significant effects on PH, but not grain yield in a panel of wheat cultivars; its dwarfing allele has been frequently used in wheat breeding. These findings lay a sound foundation for map-based cloning of RHT26 and provide a breeding-applicable tool for marker-assisted selection.

Mapping of a soybean rust resistance in PI 594756 at the Rpp1 locus.

The online version contains supplementary material available at 10.1007/s11032-023-01358-4.

Genome-Wide Association Study Discovered Favorable Single Nucleotide Poly Morphisms and Candidate Genes Associated with Ramie (Boehmeria Nivea L.) Colloidal Matters

ABSTRACT Ramie (Boehmeria nivea L.) bast fiber is one of the most ancient natural fibers, which is used for textile only after degumming. Hemicellulose, pectin, and hydrotrope are the main colloidal matters in ramie fiber, which are removed when degumming. However, the genetic variation and control loci are poorly understood. Here, we analyzed the genetic variation of the colloidal matters and discovered the favorable single nucleotide poly morphisms (SNP) by genome-wide association study using 319 ramie core germplasms. In total, 21, 5, and 10 SNPs were found to be the significantly key control loci associated with hemicellulose, pectin, and hydrotrope, respectively, which changed the amino acid sequences of the reference genes. Fourteen key genes involved in various sugars biosynthesis and metabolism pathways were identified to regulate the hemicellulose content in ramie. Meanwhile, 7 calcium transporting or binding related genes were identified to involve in the biosynthesis of calcium magnesium pectinate-the main component of pectin. Moreover, 306 genes related to hydrotrope were identified and their function were also discussed. Our study first reveals the genetic variation and candidate genes related to the colloidal matters of ramie bast fiber, and provides tools for marker-assisted selection in improvement of agricultural traits.

QTL Analysis and CAPS Marker Development Linked with Russet in Pear (Pyrus spp.).

The fruit skin types of pear (Pyrus spp.) are divided into russet, smooth, and intermediate. One of the important traits in pear breeding programs is russet on pear fruit skin because it affects the commercial value. In the present study, a high-density genetic linkage map of ‘Whangkeumbae’ (smooth) × ‘Minibae’ (russet) was constructed. In addition, quantitative trait loci (QTL) analysis was performed to identify russet related QTL and develop a cleaved amplified polymorphism sequence (CAPS) marker. Together with SNPs derived from Axiom Pear 70K Genotyping Array and genotyping-by-sequencing derived SNPs and SSRs generated in previous study, an integrated genetic linkage map of ‘Whangkeumbae’ × ‘Minibae’ was constructed. A total of 1263 markers were anchored in 17 linkage groups (LGs) with a total genetic distance of 1894.02 cM and an average marker density of 1.48 cM. The chromosome coverage of ‘Whangkeumbae’ × ‘Minibae’ map was improved because the SNPs derived from Axiom Pear 70K Genotyping Array were anchored. QTL analysis was performed using previous russet phenotype data evaluated with russet coverage and Hunter a. As a result of QTL analysis, russet coverage- and Hunter a-related QTLs were identified in LG8 of the ‘Whangkeumbae’ × ‘Minibae’ map, and SNPs located in the QTL region were heterozygous in the ‘Minibae’. Although the russet coverage- and Hunter a-related QTLs were commonly detected in LG8, the logarithm of odds values of SNPs in the QTL region were higher in QTL related to russet coverage than to Hunter a. The CAPS marker (CBp08ca01) was developed using an array SNP located in the russet coverage related QTL, and the genotype of CBp08ca01 showed a 1:1 ratio in ‘Whangkeumbae’ × ‘Minibae’ (χ2 = 0.65, p > 0.05). ‘Whangkeumbae’ and ‘Minibae’ were thought to have rr and Rr genotypes, respectively, and the genetic factors controlling the russet formation might be located in chromosome 8. The CBp08ca01 was able to select F1 individuals with less than 30% russet coverage. Thus, it will be a useful tool for marker-assisted selection in pears.

Genetic Analysis of Mutagenesis That Induces the Photoperiod Insensitivity of Wild Cotton Gossypium hirsutum Subsp. purpurascens.

Cotton genus Gossypium L., especially its wild species, is rich in genetic diversity. However, this valuable genetic resource is barely used in cotton breeding programs. In part, due to photoperiod sensitivities, the genetic diversity of Gossypium remains largely untapped. Herein, we present a genetic analysis of morphological, cytological, and genomic changes from radiation-mediated mutagenesis that induced plant photoperiod insensitivity in the wild cotton of Gossypium hirsutum. Several morphological and agronomical traits were found to be highly inheritable using the progeny between the wild-type G. hirsutum subsp. purpurascens (El-Salvador) and its mutant line (Kupaysin). An analysis of pollen mother cells (PMCs) revealed quadrivalents that had an open ring shape and an adjoining type of divergence of chromosomes from translocation complexes. Using 336 SSR markers and 157 F2 progenies that were grown with parental genotypes and F1 hybrids in long day and short night conditions, five quantitative trait loci (QTLs) associated with cotton flowering were located on chromosomes At-05, At-11, and Dt-07. Nineteen candidate genes related to the flowering traits were suggested through molecular and in silico analysis. The DNA markers associated with the candidate genes, upon future functional analysis, would provide useful tools in marker-assisted selection (MAS) in cotton breeding programs for early flowering and maturity.

A divide-and-conquer approach for genomic prediction in rubber tree using machine learning

Rubber tree (Hevea brasiliensis) is the main feedstock for commercial rubber; however, its long vegetative cycle has hindered the development of more productive varieties via breeding programs. With the availability of H. brasiliensis genomic data, several linkage maps with associated quantitative trait loci have been constructed and suggested as a tool for marker-assisted selection. Nonetheless, novel genomic strategies are still needed, and genomic selection (GS) may facilitate rubber tree breeding programs aimed at reducing the required cycles for performance assessment. Even though such a methodology has already been shown to be a promising tool for rubber tree breeding, increased model predictive capabilities and practical application are still needed. Here, we developed a novel machine learning-based approach for predicting rubber tree stem circumference based on molecular markers. Through a divide-and-conquer strategy, we propose a neural network prediction system with two stages: (1) subpopulation prediction and (2) phenotype estimation. This approach yielded higher accuracies than traditional statistical models in a single-environment scenario. By delivering large accuracy improvements, our methodology represents a powerful tool for use in Hevea GS strategies. Therefore, the incorporation of machine learning techniques into rubber tree GS represents an opportunity to build more robust models and optimize Hevea breeding programs.

Identification and validation of stable quantitative trait loci for yield component traits in wheat

Grain weight and grain number are important yield component traits in wheat and identification of underlying genetic loci is helpful for improving yield. Here, we identified eight stable quantitative trait loci (QTL) for yield component traits, including five loci for thousand grain weight (TGW) and three for grain number per spike (GNS) in a recombinant inbred line population derived from cross Yangxiaomai/Zhongyou 9507 across four environments. Since grain size is a major determinant of grain weight, we also mapped QTL for grain length (GL) and grain width (GW). QTGW.caas-2D, QTGW.caas-3B, QTGW.caas-5A and QTGW.caas-7A.2 for TGW co-located with those for grain size. QTGW.caas-2D also had a consistent genetic position with QGNS.caas-2D, suggesting that the pleiotropic locus is a modulator of trade-off effect between TGW and GNS. Sequencing and linkage mapping showed that TaGL3-5A and WAPO-A1 were candidate genes of QTGW.caas-5A and QTGW.caas-7A.2, respectively. We developed Kompetitive allele specific PCR (KASP) markers linked with the stable QTL for yield component traits and validated their genetic effects in a diverse panel of wheat cultivars from the Huang-Huai River Valley region. KASP-based genotyping analysis further revealed that the superior alleles of all stable QTL for TGW but not GNS were subject to positive selection, indicating that yield improvement in the region largely depends on increased TGW. Comparative analyses with previous studies showed that most of the QTL could be detected in different genetic backgrounds, and QTGW.caas-7A.1 is likely a new QTL. These findings provide not only valuable genetic information for yield improvement but also useful tools for marker-assisted selection.

Molecular Characterizations of the er1 Alleles Conferring Resistance to Erysiphe pisi in Three Chinese Pea (Pisum sativum L.) Landraces.

Powdery mildew caused by Erysiphe pisi DC. is a major disease affecting pea worldwide. This study aimed to confirm the resistance genes contained in three powdery mildew-resistant Chinese pea landraces (Suoshadabaiwan, Dabaiwandou, and Guiwan 1) and to develop the functional markers of the novel resistance genes. The resistance genes were identified by genetic mapping and PsMLO1 gene sequence identification. To confirm the inheritance of powdery mildew resistance in the three Landraces, the susceptible cultivars Bawan 6, Longwan 1, and Chengwan 8 were crossed with Suoshadabaiwan, Dabaiwandou, and Guiwan 1 to produce F1, F2, and F2:3 populations, respectively. All F1 plants were susceptible to E. pisi, and phenotypic segregation patterns in all the F2 and F2:3 populations fit the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, respectively, indicating powdery mildew resistance in the three Landraces were controlled by a single recessive gene, respectively. The analysis of er1-linked markers and genetic mapping in the F2 populations suggested that the recessive resistance genes in three landraces could be er1 alleles. The cDNA sequences of 10 homologous PsMLO1 cDNA clones from the contrasting parents were obtained. A known er1 allele, er1-4, was identified in Suoshadabaiwan. Two novel er1 alleles were identified in Dabaiwandou and Guiwan 1, which were designated as er1-13 and er1-14, respectively. Both novel alleles were characterized with a 1-bp deletion (T) in positions 32 (exon 1) and 277 (exon 3), respectively, which caused a frame-shift mutation to result in premature termination of translation of PsMLO1 protein. The co-dominant functional markers specific for er1-13 and er1-14, KASPar-er1-13, and KASPar-er1-14 were developed and effectively validated in populations and pea germplasms. Here, two novel er1 alleles were characterized and their functional markers were validated. These results provide powerful tools for marker-assisted selection in pea breeding.

Fine mapping and characterization of a major QTL for grain weight on wheat chromosome arm 5DL.

We fine mapped QTL QTKW.caas-5DL for thousand kernel weight in wheat, predicted candidate genes and developed a breeding-applicable marker. Thousand kernel weight (TKW) is an important yield component trait in wheat, and identification of the underlying genetic loci is helpful for yield improvement. We previously identified a stable quantitative trait locus (QTL) QTKW.caas-5DL for TKW in a Doumai/Shi4185 recombinant inbred line (RIL) population. Here we performed fine mapping of QTKW.caas-5DL using secondary populations derived from 15 heterozygous recombinants and delimited the QTL to an approximate 3.9Mb physical interval from 409.9 to 413.8Mb according to the Chinese Spring (CS) reference genome. Analysis of genomic synteny showed that annotated genes in the physical interval had high collinearity among CS and eight other wheat genomes. Seven genes with sequence variation and/or differential expression between parents were predicted as candidates for QTKW.caas-5DL based on whole-genome resequencing and transcriptome assays. A kompetitive allele-specific PCR (KASP) marker for QTKW.caas-5DL was developed, and genotyping confirmed a significant association with TKW but not with other yield component traits in a panel of elite wheat cultivars. The superior allele of QTKW.caas-5DL was frequent in a panel of cultivars, suggesting that it had undergone positive selection. These findings not only lay a foundation for map-based cloning of QTKW.caas-5DL but also provide an efficient tool for marker-assisted selection.

KASP Markers Specific for the Fertility Restorer Locus Rf1 and Application for Genetic Purity Testing in Sunflowers (Helianthus annuus L.).

Single nucleotide polymorphisms (SNPs) were significantly associated with fertility restoration of cytoplasmic male sterility (CMS) PET1 by the restorer gene Rf1. For these SNPs, four Kompetitive allele-specific PCR (KASP) markers were successfully designed. The KASP markers cover the fertility restorer locus Rf1, spanning about 3 Mb, and clearly differentiate restorer and maintainer lines. For genetic purity testing in sunflower hybrid production, the efficiency for detecting contaminations in samples was simulated using mixtures of hypocotyls or leaves. Contaminations of restorer lines with 1%, 3%, 5%, 10%, and 50% of maintainer lines were screened with all four KASP markers. Contaminations of 10% could be clearly detected in pools of 100 plants. Contaminations below this level require detection on a single plant level. For single plant detections, ethyl methanesulfonate-treated sunflower F1 hybrids, which had been phenotypically evaluated for male sterility (potential mutation in the Rf1 gene) were screened. Nine identified either partially male-sterile or male-sterile plants were analyzed with all four KASP markers and only one proved to be a hybrid with a mutation, seven were male-sterile contaminants in the F1 seeds used (1.6%) and one a recombinant plant. The four KASP markers should be valuable tools for marker-assisted selection (MAS) in sunflower breeding regarding the restorer locus Rf1.