Haploid identification in maize.

Doubled haploid (DH) line production through in vivo maternal haploid induction is widely adopted in maize breeding programs. The established protocol for DH production includes four steps namely in vivo maternal haploid induction, haploid identification, genome doubling of haploid, and self-fertilization of doubled haploids. Since modern haploid inducers still produce relatively small portion of haploids among undesirable hybrid kernels, haploid identification is typically laborious, costly, and time-consuming, making this step the second foremost in the DH technique. This manuscript reviews numerous methods for haploid identification from different approaches including the innate differences in haploids and diploids, biomarkers integrated in haploid inducers, and automated seed sorting. The phenotypic differentiation, genetic basis, advantages, and limitations of each biomarker system are highlighted. Several approaches of automated seed sorting from different research groups are also discussed regarding the platform or instrument used, sorting time, accuracy, advantages, limitations, and challenges before they go through commercialization. The past haploid selection was focusing on finding the distinguishable marker systems with the key to effectiveness. The current haploid selection is adopting multiple reliable biomarker systems with the key to efficiency while seeking the possibility for automation. Fully automated high-throughput haploid sorting would be promising in near future with the key to robustness with retaining the feasible level of accuracy. The system that can meet between three major constraints (time, workforce, and budget) and the sorting scale would be the best option.

Fine mapping of major QTL qshgd1 for spontaneous haploid genome doubling in maize (Zea mays L.).

KEY MESSAGE: A large-effect QTL was fine mapped, which revealed 79 gene models, with 10 promising candidate genes, along with a novel inversion. In commercial maize breeding, doubled haploid (DH) technology is arguably the most efficient resource for rapidly developing novel, completely homozygous lines. However, the DH strategy, using in vivo haploid induction, currently requires the use of mutagenic agents which can be not only hazardous, but laborious. This study focuses on an alternative approach to develop DH lines-spontaneous haploid genome duplication (SHGD) via naturally restored haploid male fertility (HMF). Inbred lines A427 and Wf9, the former with high HMF and the latter with low HMF, were selected to fine-map a large-effect QTL associated with SHGD-qshgd1. SHGD alleles were derived from A427, with novel haploid recombinant groups having varying levels of the A427 chromosomal region recovered. The chromosomal region of interest is composed of 45 megabases (Mb) of genetic information on chromosome 5. Significant differences between haploid recombinant groups for HMF were identified, signaling the possibility of mapping the QTL more closely. Due to suppression of recombination from the proximity of the centromere, and a newly discovered inversion region, the associated QTL was only confined to a 25 Mb region, within which only a single recombinant was observed among ca. 9,000 BC1 individuals. Nevertheless, 79 gene models were identified within this 25 Mb region. Additionally, 10 promising candidate genes, based on RNA-seq data, are described for future evaluation, while the narrowed down genome region is accessible for straightforward introgression into elite germplasm by BC methods.

Genetic basis of maize maternal haploid induction beyond MATRILINEAL and ZmDMP.

In maize, doubled haploid (DH) lines are created in vivo through crosses with maternal haploid inducers. Their induction ability, usually expressed as haploid induction rate (HIR), is known to be under polygenic control. Although two major genes (MTL and ZmDMP) affecting this trait were recently described, many others remain unknown. To identify them, we designed and performed a SNP based (~9007) genome-wide association study using a large and diverse panel of 159 maternal haploid inducers. Our analyses identified a major gene near MTL, which is present in all inducers and necessary to disrupt haploid induction. We also found a significant quantitative trait loci (QTL) on chromosome 10 using a case-control mapping approach, in which 793 noninducers were used as controls. This QTL harbors a kokopelli ortholog, whose role in maternal haploid induction was recently described in Arabidopsis. QTL with smaller effects were identified on six of the ten maize chromosomes, confirming the polygenic nature of this trait. These QTL could be incorporated into inducer breeding programs through marker-assisted selection approaches. Further improving HIR is important to reduce the cost of DH line production.

Molecular characterization of doubled haploid lines derived from different cycles of the Iowa Stiff Stalk Synthetic (BSSS) maize population.

Molecular characterization of a given set of maize germplasm could be useful for understanding the use of the assembled germplasm for further improvement in a breeding program, such as analyzing genetic diversity, selecting a parental line, assigning heterotic groups, creating a core set of germplasm and/or performing association analysis for traits of interest. In this study, we used single nucleotide polymorphism (SNP) markers to assess the genetic variability in a set of doubled haploid (DH) lines derived from the unselected Iowa Stiff Stalk Synthetic (BSSS) maize population, denoted as C0 (BSSS(R)C0), the seventeenth cycle of reciprocal recurrent selection in BSSS (BSSS(R)C17), denoted as C17 and the cross between BSSS(R)C0 and BSSS(R)C17 denoted as C0/C17. With the aim to explore if we have potentially lost diversity from C0 to C17 derived DH lines and observe whether useful genetic variation in C0 was left behind during the selection process since C0 could be a reservoir of genetic diversity that could be untapped using DH technology. Additionally, we quantify the contribution of the BSSS progenitors in each set of DH lines. The molecular characterization analysis confirmed the apparent separation and the loss of genetic variability from C0 to C17 through the recurrent selection process. Which was observed by the degree of differentiation between the C0_DHL versus C17_DHL groups by Wright's F-statistics (FST). Similarly for the population structure based on principal component analysis (PCA) revealed a clear separation among groups of DH lines. Some of the progenitors had a higher genetic contribution in C0 compared with C0/C17 and C17 derived DH lines. Although genetic drift can explain most of the genetic structure genome-wide, phenotypic data provide evidence that selection has altered favorable allele frequencies in the BSSS maize population through the reciprocal recurrent selection program.

Self-supervised maize kernel classification and segmentation for embryo identification.

Introduction: Computer vision and deep learning (DL) techniques have succeeded in a wide range of diverse fields. Recently, these techniques have been successfully deployed in plant science applications to address food security, productivity, and environmental sustainability problems for a growing global population. However, training these DL models often necessitates the large-scale manual annotation of data which frequently becomes a tedious and time-and-resource- intensive process. Recent advances in self-supervised learning (SSL) methods have proven instrumental in overcoming these obstacles, using purely unlabeled datasets to pre-train DL models. Methods: Here, we implement the popular self-supervised contrastive learning methods of NNCLR Nearest neighbor Contrastive Learning of visual Representations) and SimCLR (Simple framework for Contrastive Learning of visual Representations) for the classification of spatial orientation and segmentation of embryos of maize kernels. Maize kernels are imaged using a commercial high-throughput imaging system. This image data is often used in multiple downstream applications across both production and breeding applications, for instance, sorting for oil content based on segmenting and quantifying the scutellum's size and for classifying haploid and diploid kernels. Results and discussion: We show that in both classification and segmentation problems, SSL techniques outperform their purely supervised transfer learning-based counterparts and are significantly more annotation efficient. Additionally, we show that a single SSL pre-trained model can be efficiently finetuned for both classification and segmentation, indicating good transferability across multiple downstream applications. Segmentation models with SSL-pretrained backbones produce DICE similarity coefficients of 0.81, higher than the 0.78 and 0.73 of those with ImageNet-pretrained and randomly initialized backbones, respectively. We observe that finetuning classification and segmentation models on as little as 1% annotation produces competitive results. These results show SSL provides a meaningful step forward in data efficiency with agricultural deep learning and computer vision.

A Comparison between Inbred and Hybrid Maize Haploid Inducers.

The effectiveness of haploid induction systems is regarded not only for high haploid induction rate (HIR) but also resource savings. Isolation fields are proposed for hybrid induction. However, efficient haploid production depends on inducer traits such as high HIR, abundant pollen production, and tall plants. Seven hybrid inducers and their respective parents were evaluated over three years for HIR, seeds set in cross-pollinations, plant and ear height, tassel size, and tassel branching. Mid-parent heterosis was estimated to quantify how much inducer traits improve in hybrids in comparison to their parents. Heterosis benefits hybrid inducers for plant height, ear height, and tassel size. Two hybrid inducers, BH201/LH82-Ped126 and BH201/LH82-Ped128, are promising for haploid induction in isolation fields. Hybrid inducers offer convenience and resource-effectiveness for haploid induction by means of improving plant vigor without compromising HIR.

Genome-wide association analysis of plant architecture traits using doubled haploid lines derived from different cycles of the Iowa Stiff Stalk Synthetic maize population.

Selection in the Iowa Stiff Stalk Synthetic (BSSS) maize population for high yield, grain moisture, and root and stalk lodging has indirectly modified plant architecture traits that are important for adaptation to high plant density. In this study, we developed doubled haploid (DH) lines from the BSSS maize population in the earliest cycle of recurrent selection (BSSS), cycle 17 of reciprocal recurrent selection, [BSSS(R)17] and the cross between the two cycles [BSSS/BSSS(R)C17]. We aimed to determine the phenotypic variation and changes in agronomic traits that have occurred through the recurrent selection program in this population and to identify genes or regions in the genome associated with the plant architecture changes observed in the different cycles of selection. We conducted a per se evaluation of DH lines focusing on high heritability traits important for adaptation to high planting density and grain yield. Trends for reducing flowering time, anthesis-silking interval, ear height, and the number of primary tassel branches in BSSS(R)17 DH lines compared to BSSS and BSSS/BSSS(R)C17 DH lines were observed. Additionally, the BSSS(R)C17 DH lines showed more upright flag leaf angles. Using the entire panel of DH lines increased the number of SNP markers identified within candidate genes associated with plant architecture traits. The genomic regions identified for plant architecture traits in this study may help to elucidate the genetic basis of these traits and facilitate future work about marker-assisted selection or map-based cloning in maize breeding programs.

Development of doubled haploid inducer lines facilitates selection of superior haploid inducers in maize.

Haploid inducers are key components of doubled haploid (DH) technology in maize. Robust agronomic performance and better haploid induction ability of inducers are persistently sought through genetic improvement. We herein developed C1-I inducers enabling large-scale in vivo haploid induction of inducers and discovered superior inducers from the DH progenies. The haploid induction rate (HIR) of C1-I inducers ranged between 5.8% and 12.0%. Overall, the success rate of DH production was 13% on average across the 23 different inducer crosses. The anthesis-silking interval and days to flowering of inducer F1s are significantly correlated with the success rate of DH production (r = -0.48 and 0.47, respectively). Transgressive segregants in DH inducers (DHIs) were found for the traits (days to flowering, HIR, plant height, and total primary branch length). Moreover, the best HIR in DHIs exceeded 23%. Parental genome contributions to DHI progenies ranged between 0.40 and 0.55, respectively, in 25 and 75 percentage quantiles, and the mean and median were 0.48. The allele frequency of the four traits from inducer parents to DHI progenies did not correspond with the phenotypic difference between superior and inferior individuals in the DH populations by genome-wide Fst analysis. This study demonstrated that the recombinant DHIs can be accessed on a large scale and used as materials to facilitate the genetic improvement of maternal haploid inducers by in vivo DH technology.

Investigating the Effect of the Interaction of Maize Inducer and Donor Backgrounds on Haploid Induction Rates.

Doubled haploid technology is a feasible, fast, and cost-efficient way of producing completely homozygous lines in maize. Many factors contribute to the success of this system including the haploid induction rate (HIR) of inducer lines, the inducibility of donor background, and environmental conditions. Sixteen inducer lines were tested on eight different genetic backgrounds of five categories in different environments for the HIR to determine possible interaction specificity. The HIR was assessed using the R1-nj phenotype and corrected using the red root marker or using a gold-standard test that uses plant traits. RWS and Mo-17-derived inducers showed higher average induction rates and the commercial dent hybrid background showed higher inducibility. In contrast, sweet corn and flint backgrounds had a relatively lower inducibility, while non-stiff stalk and stiff stalk backgrounds showed intermediate inducibility. For the poor-performing donors (sweet corn and flint), there was no difference in the HIR among the inducers. Anthocyanin inhibitor genes in such donors were assumed to have increased the misclassification rate in the F1 fraction and, hence, result in a lower HIR.

QTL Mapping for Haploid Inducibility Using Genotyping by Sequencing in Maize.

Doubled haploid (DH) technology in maize takes advantage of in vivo haploid induction (HI) triggered by pollination of donors of interest with inducer genotypes. However, the ability of different donors to be induced-inducibility (IND), varies among germplasm and the underlying molecular mechanisms are still unclear. In this study, the phenotypic variation for IND in a mapping population of temperate inbred lines was evaluated to identify regions in the maize genome associated with IND. A total of 247 F2:3 families derived from a biparental cross of two elite inbred lines, A427 and CR1Ht, were grown in three different locations and Inclusive Composite Interval Mapping (ICIM) was used to identify quantitative trait loci (QTL) for IND. In total, four QTL were detected, explaining 37.4% of the phenotypic variance. No stable QTL was found across locations. The joint analysis revealed QTL × location interactions, suggesting minor QTL control IND, which are affected by the environment.

Protocols for In Vivo Doubled Haploid (DH) Technology in Maize Breeding: From Haploid Inducer Development to Haploid Genome Doubling.

Doubled haploid (DH) technology reduces the time required to obtain homozygous genotypes and accelerates plant breeding among other advantages. It is established in major crop species such as wheat, barley, maize, and canola. DH lines can be produced by both in vitro and in vivo methods and the latter is focused here. The major steps involved in in vivo DH technology are haploid induction, haploid selection/identification, and haploid genome doubling. Herein, we elaborate on the various steps of DH technology in maize breeding from haploid induction to haploid genome doubling to produce DH lines. Detailed protocols on the following topics are discussed: in vivo haploid inducer line development, haploid selection using seed and root color markers and automated seed sorting based on embryo oil content using QSorter, artificial genome doubling, and the identification of genotypes with spontaneous haploid genome doubling (SHGD) ability.

Usefulness of temperate-adapted maize lines developed by doubled haploid and single-seed descent methods.

KEY MESSAGE: Spontaneous haploid genome doubling is not associated with undesirable linkage drag effects. The presence of spontaneous doubling genes allows maximum exploitation of variability from the temperate-adapted BS39 population Tropical non-elite maize (Zea mays L.) germplasm, such as BS39, provides a unique opportunity for broadening the genetic base of U.S. Corn Belt germplasm. In vivo doubled haploid (DH) technology has been used to efficiently exploit non-elite germplasm. It can help to purge deleterious recessive alleles. The objectives of this study were to determine the usefulness of BS39-derived inbred lines using both SSD and DH methods, to determine the impact of spontaneous as compared with artificial haploid genome doubling on genetic variance among BS39-derived DH lines, and to identify SNP markers associated with agronomic traits among BS39 inbreds monitored at testcross level. We developed two sets of inbred lines directly from BS39 by DH and SSD methods, named BS39_DH and BS39_SSD. Additionally, two sets were derived from a cross between BS39 and A427 (SHGD donor) by DH and SSD methods, named BS39 × A427_DH and BS39 × A427_SSD, respectively. Grain yield, moisture, plant height, ear height, stalk lodging, and root lodging were measured to estimate genetic parameters. For genome-wide association analysis, inbred lines were genotyped using genotype-by-sequencing and Diversity Array Technology Sequencing (DArTSeq). Some BS39-derived inbred lines performed better than elite germplasm inbreds and all sets showed significant genetic variance. The presence of spontaneous haploid genome doubling genes did not affect performance of inbred lines. Five SNPs were significant and three of them located within genes related to plant development or abiotic stresses. These results demonstrate the potential of BS39 to add novel alleles to temperate elite germplasm.

Major locus for spontaneous haploid genome doubling detected by a case-control GWAS in exotic maize germplasm.

KEY MESSAGE: A major locus for spontaneous haploid genome doubling was detected by a case-control GWAS in an exotic maize germplasm. The combination of double haploid breeding method with this locus leads to segregation distortion on genomic regions of chromosome five. Temperate maize (Zea mays L.) breeding programs often rely on limited genetic diversity, which can be expanded by incorporating exotic germplasm. The aims of this study were to perform characterization of inbred lines derived from the tropical BS39 population using different breeding methods, to identify genomic regions showing segregation distortion in lines derived by the DH process using spontaneous haploid genome doubling (SHGD), and use case-control association mapping to identify loci controlling SHGD. Four different sets were used: BS39_DH and BS39_SSD were derived from the BS39 population by DH and single-seed descendent (SSD) methods, and BS39 × A427_DH and BS39 × A427_SSD from the cross between BS39 and A427. A total of 663 inbred lines were genotyped. The analyses of gene diversity and genetic differentiation for the DH sets provided evidence of the presence of a SHGD locus near the centromere of chromosome 5. The case-control GWAS for the DH set also pinpointed this locus. Haplotype sharing analysis showed almost 100% exclusive contribution of the A427 genome in the same region on chromosome 5 of BS39 × A427_DH, presumably due to an allele in this region affecting SHGD. This locus enables DH line production in exotic populations without colchicine or other artificial haploid genome doubling.

Genomic prediction of maternal haploid induction rate in maize.

Genomic prediction (GP) might be an efficient way to improve haploid induction rate (HIR) and to reduce the laborious and time-consuming task of phenotypic selection for HIR in maize (Zea mays L.). In this study, we evaluated GP accuracies for HIR and other agronomic traits of importance to inducers by independent and cross-validation. We propose the use of GP for cross prediction and parental selection in the development of new inducer breeding populations. A panel of 159 inducers from Iowa State University (ISU set) was genotyped and phenotyped for HIR and several agronomic traits. The data of an independent set of 53 inducers evaluated by the University of Hohenheim (UOH set) was used for independent validation. The HIR ranged from 0.61 to 20.74% and exhibited high heritability (0.90). High cross-validation prediction accuracy was observed for HIR (r = 0.82), whereas for other traits it ranged from 0.36 (self-induction rate) to 0.74 (days to anthesis). Prediction accuracies across different sets were higher when the larger panel (ISU set) was used as a training population (r = 0.54). The average HIR of the 12,561 superior predicted progenies (µSP ) ranged from 1.00-18.36% and was closely related to the corresponding midparent genomic estimated breeding value (GEBV). A predicted genetic variance (VG ) of reduced magnitude was observed in the twenty crosses with highest midparent GEBV or µSP for HIR. Our results indicate that although GP is a useful tool for parental selection, decisions about which cross combinations should be pursued need to be based on optimal trade-offs between maximizing both µSP and VG .

MYO, a Candidate Gene for Haploid Induction in Maize Causes Male Sterility.

Doubled haploid technology is highly successful in maize breeding programs and is contingent on the ability of maize inducers to efficiently produce haploids. Knowledge of the genes involved in haploid induction is important for not only developing better maize inducers, but also to create inducers in other crops. The main quantitative trait loci involved in maize haploid induction are qhir1 and qhir8. The gene underlying qhir1 has been discovered and validated by independent research groups. Prior to initiation of this study, the gene associated with qhir8 had yet to be recognized. Therefore, this research focused on characterizing positional candidate genes underlying qhir8. Pursuing this goal, a strong candidate for qhir8, GRMZM2G435294 (MYO), was silenced by RNAi. Analysis of crosses with these heterozygous RNAi-transgenic lines for haploid induction rate revealed that the silencing of MYO significantly enhanced haploid induction rate by an average of 0.6% in the presence of qhir1. Recently, GRMZM2G465053 (ZmDMP) was identified by map-based gene isolation and shown to be responsible for qhir8. While our results suggest that MYO may contribute to haploid induction rate, results were inconsistent and only showing minor increases in haploid induction rate compared to ZmDMP. Instead, reciprocal crosses clearly revealed that the silencing of MYO causes male sterility.

Breeding Maize Maternal Haploid Inducers.

Maize doubled haploid (DH) lines are usually created in vivo, through crosses with maternal haploid inducers. These inducers have the inherent ability of generating seeds with haploid embryos when used to pollinate other genotypes. The resulting haploid plants are treated with a doubling agent and self-pollinated, producing completely homozygous seeds. This rapid method of inbred line production reduces the length of breeding cycles and, consequently, increases genetic gain. Such advantages explain the wide adoption of this technique by large, well-established maize breeding programs. However, a slower rate of adoption was observed in medium to small-scale breeding programs. The high price and/or lack of environmental adaptation of inducers available for licensing, or the poor performance of those free of cost, might explain why smaller operations did not take full advantage of this technique. The lack of adapted inducers is especially felt in tropical countries, where inducer breeding efforts are more recent. Therefore, defining optimal breeding approaches for inducer development could benefit many breeding programs which are in the process of adopting the DH technique. In this manuscript, we review traits important to maize maternal haploid inducers, explain their genetic basis, listing known genes and quantitative trait loci (QTL), and discuss different breeding approaches for inducer development. The performance of haploid inducers has an important impact on the cost of DH line production.

QTL mapping of spontaneous haploid genome doubling using genotyping-by-sequencing in maize (Zea mays L.).

KEY MESSAGE: A major QTL for SHGD was identified on chromosome 5 with stable expression across environments. The introgression this QTL can overcome the need of colchicine in DH lines development. Genome doubling of haploids is one of the major constraints of large-scale doubled haploid (DH) technology. Improving spontaneous haploid genome doubling (SHGD) is an alternative to overcome this limitation. In this study, we aimed to construct a high-density linkage map based on genotyping by sequencing of single nucleotide polymorphism, to detect QTL and QTL by environment (Q by E) interactions affecting SHGD and to identify the best trait for mapping and selection of haploid male fertility (HMF). To this end, a biparental population of 220 F2:3 families was developed from a cross between A427 (high HMF) and CR1Ht (moderate HMF) to be used as donor. A high-density linkage map was constructed containing 4171 SNP markers distributed over 10 chromosomes with an average distance between adjacent markers of 0.51 cM. QTL mapping for haploid fertile anther emergence, pollen production, tassel size, and HMF, identified 27 QTL across three environments, and Q by E interactions were significant. A major QTL was identified on chromosome 5. This QTL explained over 45% of the observed variance for all traits across all environments. The introgression of this major QTL, using marker-assisted backcrossing, has great potential to overcome the need of using colchicine in DH line development.

Impact of Spontaneous Haploid Genome Doubling in Maize Breeding.

Doubled haploid (DH) technology has changed the maize-breeding landscape in recent years. Traditionally, DH production requires the use of chemical doubling agents to induce haploid genome doubling and, subsequently, male fertility. These chemicals can be harmful to humans and the plants themselves, and typically result in a doubling rate of 10%-30%. Spontaneous genome doubling and male fertility of maize haploids, without using chemical doubling agents, have been observed to a limited extent, for nearly 70 years. Rates of spontaneous haploid genome doubling (SHGD) have ranged from less than 5% to greater than 50%. Recently, there has been increased interest to forgo chemical treatment and instead utilize this natural method of doubling. Genetic-mapping studies comprising worldwide germplasm have been conducted. Of particular interest has been the detection of large-effect quantitative trait loci (QTL) affecting SHGD. Having a single large-effect QTL with an additive nature provides flexibility for the method of introgression, such as marker-assisted backcrossing, marker-assisted gene pyramiding, and systematic design. Moreover, it allows implementation of new methodologies, such as haploid-inducer mediated genome editing (HI-edit) and promotion of alleles by genome editing. We believe the use of SHGD can further enhance the impact of DH technology in maize.

Mapping of QTL and identification of candidate genes conferring spontaneous haploid genome doubling in maize (Zea mays L.).

In vivo doubled haploid (DH) technology is widely used in commercial maize (Zea mays L.) breeding. Haploid genome doubling is a critical step in DH breeding. In this study, inbred lines GF1 (0.65), GF3(0.29), and GF5 (0) with high, moderate, and poor spontaneous haploid genome doubling (SHGD), respectively, were selected to develop mapping populations for SHGD. Three QTL, qshgd1, qshgd2, and qshgd3, related to SHGD were identified by selective genotyping. With the exception of qshgd3, the source of haploid genome doubling alleles were derived from GF1. Furthermore, RNA-Seq was conducted to identify putative candidate genes between GF1 and GF5 within the qshgd1 region. A differentially expressed formin-like protein 5 transcript was identified within the qshgd1 region.

Fine mapping a self-fertility locus in perennial ryegrass.

KEY MESSAGE: A self-fertility locus was fine mapped to a 1.6 cM region on linkage group 5 in a perennial ryegrass population. This locus was the main determinant of pollen self-compatibility. In grasses, self-incompatibility (SI) is characterized by a two-loci gametophytic (S and Z) mechanism acting together in the recognition and inhibition of self-pollen. Mutations affecting the expression of SI have been reported in a few grass species. In perennial ryegrass (Lolium perenne L.), a mutation independent from S and Z, and mapping on linkage group 5 (LG 5), was previously reported to produce self-fertile plants. Here, we describe fine mapping of the self-fertility (SF) gene in a perennial ryegrass population and determine whether there is any effect of other genomic regions on the pollen compatibility. The phenotypic segregation of SF showed a bimodal distribution with one mean at 49% pollen compatibility and the other at 91%. Marker-trait association analysis showed that only markers on LG 5 were significantly associated with the trait. A single gene model explained 82% of the observed variability and no effects of the other regions were detected. Using segregation and linkage analysis, the SF locus was located to a 1.6 cM region on LG 5. The flanking marker sequences were aligned to rice and Brachypodium distachyon reference genomes to estimate the physical distance. We provide markers tightly linked to SF that can be used for introgression of this trait into advanced breeding germplasm. Moreover, our results represent a further step towards the identification of the SF gene in LG 5.