The genetic architecture of soybean photothermal adaptation to high latitudes.

Soybean is a major plant protein source for both human food and animal feed, but to meet global demands as well as a trend towards regional production, soybean cultivation needs to be expanded to higher latitudes. In this study, we developed a large diversity panel consisting of 1503 early-maturing soybean lines and used genome-wide association mapping to dissect the genetic architecture underlying two crucial adaptation traits, flowering time and maturity. This revealed several known maturity loci, E1, E2, E3, and E4, and the growth habit locus Dt2 as causal candidate loci, and also a novel putative causal locus, GmFRL1, encoding a homolog of the vernalization pathway gene FRIGIDA-like 1. In addition, the scan for quantitative trait locus (QTL)-by-environment interactions identified GmAPETALA1d as a candidate gene for a QTL with environment-dependent reversed allelic effects. The polymorphisms of these candidate genes were identified using whole-genome resequencing data of 338 soybeans, which also revealed a novel E4 variant, e4-par, carried by 11 lines, with nine of them originating from Central Europe. Collectively, our results illustrate how combinations of QTL and their interactions with the environment facilitate the photothermal adaptation of soybean to regions far beyond its center of origin.

Do We Need to Breed for Regional Adaptation in Soybean?-Evaluation of Genotype-by-Location Interaction and Trait Stability of Soybean in Germany.

Soybean is a crop in high demand, in particular as a crucial source of plant protein. As a short-day plant, soybean is sensitive to the latitude of the growing site. Consequently, varieties that are well adapted to higher latitudes are required to expand the cultivation. In this study, we employed 50 soybean genotypes to perform a multi-location trial at seven locations across Germany in 2021. Two environmental target regions were determined following the latitude of the locations. Adaptation and trait stability of seed yield and protein content across all locations were evaluated using Genotype plus Genotype-by-Environment (GGE) biplots and Shukla's stability variance. We found a moderate level of crossing-over type genotype-by-location interaction across all locations. Within the environmental target regions, the genotype-by-location interaction could be minimised. Despite the positive correlation (R = 0.59) of seed yield between the environmental target regions and the same best-performing genotype, the genotype rankings differed in part substantially. In conclusion, we found that soybean can be grown at a wide range of latitudes across Germany. However, the performance of genotypes differed between the northern and southern locations, with an 18.8% higher mean yield in the south. This in combination with the observed rank changes of high-performing genotypes between both environmental target regions suggests that selection targeted towards environments in northern Germany could improve soybean breeding for those higher latitude regions.

Unraveling the potential of phenomic selection within and among diverse breeding material of maize (Zea mays L.).

Genomic selection is a well-investigated approach that facilitates and supports selection decisions for complex traits and has meanwhile become a standard tool in modern plant breeding. Phenomic selection has only recently been suggested and uses the same statistical procedures to predict the targeted traits but replaces marker data with near-infrared spectroscopy data. It may represent an attractive low-cost, high-throughput alternative but has not been sufficiently studied until now. Here, we used 400 genotypes of maize (Zea mays L.) comprising elite lines of the Flint and Dent heterotic pools as well as 6 Flint landraces, which were phenotyped in multienvironment trials for anthesis-silking-interval, early vigor, final plant height, grain dry matter content, grain yield, and phosphorus concentration in the maize kernels, to compare the predictive abilities of genomic as well as phenomic prediction under different scenarios. We found that both approaches generally achieved comparable predictive abilities within material groups. However, phenomic prediction was less affected by population structure and performed better than its genomic counterpart for predictions among diverse groups of breeding material. We therefore conclude that phenomic prediction is a promising tool for practical breeding, for instance when working with unknown and rather diverse germplasm. Moreover, it may make the highly monopolized sector of plant breeding more accessible also for low-tech institutions by combining well established, widely available, and cost-efficient spectral phenotyping with the statistical procedures elaborated for genomic prediction - while achieving similar or even better results than with marker data.

Can we abandon phosphorus starter fertilizer in maize? Results from a diverse panel of elite and doubled haploid landrace lines of maize (Zea mays L.).

The importance of phosphorus (P) in agriculture contrasts with the negative environmental impact and the limited resources worldwide. Reducing P fertilizer application by utilizing more efficient genotypes is a promising way to address these issues. To approach this, a large panel of maize (Zea mays L.) comprising each 100 Flint and Dent elite lines and 199 doubled haploid lines from six landraces was assessed in multi-environment field trials with and without the application of P starter fertilizer. The treatment comparison showed that omitting the starter fertilizer can significantly affect traits in early plant development but had no effect on grain yield. Young maize plants provided with additional P showed an increased biomass, faster growth and superior vigor, which, however, was only the case under environmental conditions considered stressful for maize cultivation. Importantly, though the genotype-by-treatment interaction variance was comparably small, there is genotypic variation for this response that can be utilized in breeding. The comparison of elite and doubled haploid landrace lines revealed a superior agronomic performance of elite material but also potentially valuable variation for early traits in the landrace doubled haploid lines. In conclusion, our results illustrate that breeding for P efficient maize cultivars is possible towards a reduction of P fertilizer in a more sustainable agriculture.

The performance of phenomic selection depends on the genetic architecture of the target trait.

KEY MESSAGE: The phenomic predictive ability depends on the genetic architecture of the target trait, being high for complex traits and low for traits with major QTL. Genomic selection is a powerful tool to assist breeding of complex traits, but a limitation is the costs required for genotyping. Recently, phenomic selection has been suggested, which uses spectral data instead of molecular markers as predictors. It was shown to be competitive with genomic prediction, as it achieved predictive abilities as high or even higher than its genomic counterpart. The objective of this study was to evaluate the performance of phenomic prediction for triticale and the dependency of the predictive ability on the genetic architecture of the target trait. We found that for traits with a complex genetic architecture, like grain yield, phenomic prediction with NIRS data as predictors achieved high predictive abilities and performed better than genomic prediction. By contrast, for mono- or oligogenic traits, for example, yellow rust, marker-based approaches achieved high predictive abilities, while those of phenomic prediction were very low. Compared with molecular markers, the predictive ability obtained using NIRS data was more robust to varying degrees of genetic relatedness between the training and prediction set. Moreover, for grain yield, smaller training sets were required to achieve a similar predictive ability for phenomic prediction than for genomic prediction. In addition, our results illustrate the potential of using field-based spectral data for phenomic prediction. Overall, our result confirmed phenomic prediction as an efficient approach to improve the selection gain for complex traits in plant breeding.

High-resolution association mapping with libraries of immortalized lines from ancestral landraces.

KEY MESSAGE: Association mapping with immortalized lines of landraces offers several advantages including a high mapping resolution, as demonstrated here in maize by identifying the causal variants underlying QTL for oil content and the metabolite allantoin. Landraces are traditional varieties of crops that present a valuable yet largely untapped reservoir of genetic variation to meet future challenges of agriculture. Here, we performed association mapping in a panel comprising 358 immortalized maize lines from six European Flint landraces. Linkage disequilibrium decayed much faster in the landraces than in the elite lines included for comparison, permitting a high mapping resolution. We demonstrate this by fine-mapping a quantitative trait locus (QTL) for oil content down to the phenylalanine insertion F469 in DGAT1-2 as the causal variant. For the metabolite allantoin, related to abiotic stress response, we identified promoter polymorphisms and differential expression of an allantoinase as putative cause of variation. Our results demonstrate the power of this approach to dissect QTL potentially down to the causal variants, toward the utilization of natural or engineered alleles in breeding. Moreover, we provide guidelines for studies using ancestral landraces for crop genetic research and breeding.

Genetic Dissection of Phosphorus Use Efficiency in a Maize Association Population under Two P Levels in the Field.

Phosphorus (P) deficiency is an important challenge the world faces while having to increase crop yields. It is therefore necessary to select maize (Zea may L.) genotypes with high phosphorus use efficiency (PUE). Here, we extensively analyzed the biomass, grain yield, and PUE-related traits of 359 maize inbred lines grown under both low-P and normal-P conditions. A significant decrease in grain yield per plant and biomass, an increase in PUE under low-P condition, as well as significant correlations between the two treatments were observed. In a genome-wide association study, 49, 53, and 48 candidate genes were identified for eleven traits under low-P, normal-P conditions, and in low-P tolerance index (phenotype under low-P divided by phenotype under normal-P condition) datasets, respectively. Several gene ontology pathways were enriched for the genes identified under low-P condition. In addition, seven key genes related to phosphate transporter or stress response were molecularly characterized. Further analyses uncovered the favorable haplotype for several core genes, which is less prevalent in modern lines but often enriched in a specific subpopulation. Collectively, our research provides progress in the genetic dissection and molecular characterization of PUE in maize.

Training set design in genomic prediction with multiple biparental families.

Genomic selection is a powerful tool to reduce the cycle length and enhance the genetic gain of complex traits in plant breeding. However, questions remain about the optimum design and composition of the training set. In this study, we used 944 soybean [Glycine max (L.) Merr.] recombinant inbred lines from eight families derived through a partial-diallel mating design among five parental lines. The cross-validated prediction accuracies for the six traits seed yield, 1,000-seed weight, protein yield, plant height, protein content, and oil content were high, ranging from 0.79 to 0.87. We investigated among-family predictions, making use of the special mating design with different degrees of relatedness among families. Generally, the prediction accuracy decreased from full-sibs to half-sib families to unrelated families. However, half-sib and unrelated families also showed substantial variation in their prediction accuracy for a given family, which appeared to be caused at least in part by the shared segregation of quantitative trait loci in both the training and prediction sets. Combining several half-sib families in composite training sets generally led to an increase in the prediction accuracy compared with the best family alone. The prediction accuracy increased with the size of the training set, but for comparable prediction accuracy, substantially more half-sibs were required than full-sibs. Collectively, our results highlight the potential of genomic selection for soybean breeding and, in a broader context, illustrate the importance of the targeted design of the training set.

Optimizing the P balance: How do modern maize hybrids react to different starter fertilizers?

Phosphorus (P) is an essential macronutrient for plants, but also a limited resource worldwide. Strict regulations for fertilizer applications in the European Union are a consequence of the negative environmental effects in case of improper use. Maize is typically grown with the application of P starter fertilizer, which, however, might be reduced or even omitted if suitable varieties were available. This study was performed with the 20 commercially most important maize hybrids in Germany evaluated in multi-location field trials with the aim to investigate the potential to breed for high-performing maize hybrids under reduced P starter fertilizer. At the core location, three starter fertilizers with either phosphate (triple superphosphate, TSP), ammonium nitrate (calcium ammonium nitrate, CAN), or a combination of ammonium and phosphate (diammonium phosphate, DAP) were evaluated relative to a control and traits from youth development to grain yield were assessed. Significant differences were mainly observed for the DAP starter fertilizer, which was also reflected in a yield increase of on average +0.67 t/ha (+5.34%) compared to the control. Correlations among the investigated traits varied with starter fertilizer, but the general trends remained. As expected, grain yield was negatively correlated with grain P concentration, likely due to a dilution effect. Importantly, the genotype-by-starter fertilizer interaction was always non-significant in the multi-location analysis. This indicates that best performing genotypes can be identified irrespective of the starter fertilizer. Taken together, our results provide valuable insights regarding the potential to reduce starter fertilizers in maize cultivation as well as for breeding maize for P efficiency under well-supplied conditions.

Genetic Architecture of Cereal Leaf Beetle Resistance in Wheat.

Wheat production can be severely damaged by endemic and invasive insect pests. Here, we investigated resistance to cereal leaf beetle in a panel of 876 winter wheat cultivars, and dissected the genetic architecture underlying this insect resistance by association mapping. We observed an effect of heading date on cereal leaf beetle infestation, with earlier heading cultivars being more heavily infested. Flag leaf glaucousness was also found to be correlated with resistance. In line with the strong effect of heading time, we identified Ppd-D1 as a major quantitative trait locus (QTL), explaining 35% of the genotypic variance of cereal leaf beetle resistance. The other identified putative QTL explained much less of the genotypic variance, suggesting a genetic architecture with many small-effect QTL, which was corroborated by a genomic prediction approach. Collectively, our results add to our understanding of the genetic control underlying insect resistances in small-grain cereals.

Speed breeding short-day crops by LED-controlled light schemes.

KEY MESSAGE: A simple and rapid speed breeding system was developed for short-day crops that enables up to five generations per year using LED lighting systems that allow very specific adjustments regarding light intensity and quality. Plant breeding is a key element for future agricultural production that needs to cope with a growing human population and climate change. However, the process of developing suitable cultivars is time-consuming, not least because of the long generation times of crops. Recently, speed breeding has been introduced for long-day crops, but a similar protocol for short-day crops is lacking to date. In this study, we present a speed breeding protocol based on light-emitting diodes (LEDs) that allow to modify light quality, and exemplarily demonstrate its effectiveness for the short-day crops soybean (Glycine max), rice (Oryza sativa) and amaranth (Amaranthus spp.). Adjusting the photoperiod to 10 h and using a blue-light enriched, far-red-deprived light spectrum facilitated the growth of short and sturdy soybean plants that flowered ~ 23 days after sowing and matured within 77 days, thus allowing up to five generations per year. In rice and amaranth, flowering was achieved ~ 60 and ~ 35 days after sowing, respectively. Interestingly, the use of far-red light advanced flowering by 10 and 20 days in some amaranth and rice genotypes, respectively, but had no impact on flowering in soybeans, highlighting the importance of light quality for speed breeding protocols. Taken together, our short-day crops' speed breeding protocol enables several generations per year using crop-specific LED-based lighting regimes, without the need of tissue culture tools such as embryo rescue. Moreover, this approach can be readily applied to a multi-storey 96-cell tray-based system to integrate speed breeding with genomics, toward a higher improvement rate in breeding.

Misexpression of a transcriptional repressor candidate provides a molecular mechanism for the suppression of awns by Tipped 1 in wheat.

Awns are bristle-like structures formed at the tip of the lemma on the florets of some cereal grasses. Wild-type wheat is awned, but awnletted and awnless variants have been selected and nowadays all forms are cultivated. In this study, we dissected the genetic control underlying variation of this characteristic feature by association mapping in a large panel of 1110 winter wheat cultivars of worldwide origin. We identified the B1 (Tipped 1) locus on chromosome 5A as the major determinant of awnlessness globally. Using a combination of fine-mapping and expression analysis, we identified a putative C2H2 zinc finger protein with an EAR domain, characteristic of transcriptional repressors, as a likely candidate for Tipped 1. This gene was found to be up-regulated in awnless B1 compared with awned b1 plants, indicating that misexpression of this transcriptional regulator may contribute to the reduction of awn length in B1 plants. Taken together, our study provides an entry point towards a better molecular understanding of the evolution of morphological features in cereals through selection and breeding.

Refining the genetic architecture of flag leaf glaucousness in wheat.

KEY MESSAGE: The cuticle is the plant's barrier against abiotic and biotic stresses, and the deposition of epicuticular wax crystals results in the scattering of light, an effect termed glaucousness. Here, we dissect the genetic architecture of flag leaf glaucousness in wheat toward a future targeted design of the cuticle. The cuticle serves as a barrier that protects plants against abiotic and biotic stresses. Differences in cuticle composition can be detected by the scattering of light on epicuticular wax crystals, which causes a phenotype termed glaucousness. In this study, we dissected the genetic architecture of flag leaf glaucousness in a panel of 1106 wheat cultivars of global origin. We observed a large genotypic variation, but the geographic pattern suggests that other wax layer characteristics besides glaucousness may be important in conferring tolerance to abiotic stresses such as heat and drought. Genome-wide association mapping identified two major quantitative trait loci (QTL) on chromosomes 3A and 2B. The latter corresponds to the W1 locus, but further characterization revealed that it is likely to contain additional QTL. The same holds true for the major QTL on 3A, which was also found to show an epistatic interaction with another locus located a few centiMorgan distal to it. Genome-wide prediction and the identification of a few additional putative QTL revealed that small-effect QTL also contribute to the trait. Collectively, our results illustrate the complexity of the genetic control of flag leaf glaucousness, with additive effects and epistasis, and lay the foundation for the cloning of the underlying genes toward a more targeted design of the cuticle by plant breeding.

Copy number variation of Ppd-B1 is the major determinant of heading time in durum wheat.

BACKGROUND: Heading time is an important adaptive trait in durum wheat. In hexaploid wheat, Photoperiod-1 (Ppd) loci are essential regulators of heading time, with Ppd-B1 conferring photoperiod insensitivity through copy number variations (CNV). In tetraploid wheat, the D-genome Ppd-D1 locus is absent and generally, our knowledge on the genetic architecture underlying heading time lacks behind that of bread wheat. RESULTS: In this study, we employed a panel of 328 diverse European durum genotypes that were evaluated for heading time at five environments. Genome-wide association mapping identified six putative QTL, with a major QTL on chromosome 2B explaining 26.2% of the genotypic variance. This QTL was shown to correspond to copy number variation at Ppd-B1, for which two copy number variants appear to be present. The higher copy number confers earlier heading and was more frequent in the heat and drought prone countries of lower latitude. In addition, two other QTL, corresponding to Vrn-B3 (TaFT) and Ppd-A1, were found to explain 9.5 and 5.3% of the genotypic variance, respectively. CONCLUSIONS: Our results revealed the yet unknown role of copy number variation of Ppd-B1 as the major source underlying the variation in heading time in European durum wheat. The observed geographic patterns underline the adaptive value of this polymorphism and suggest that it is already used in durum breeding to tailor cultivars to specific target environments. In a broader context our findings provide further support for a more widespread role of copy number variation in mediating abiotic and biotic stress tolerance in plants.

Evaluation of the genetic architecture and the potential of genomics-assisted breeding of quality traits in two large panels of durum wheat.

KEY MESSAGE: New QTL for important quality traits in durum were identified, but for most QTL their effect varies depending on the investigated germplasm. Most of the global durum wheat (Triticum turgidum ssp. durum) production is used for human consumption via pasta and to a lower extent couscous and bulgur. Therefore, durum wheat varieties have to fulfill high demands regarding quality traits. In this study, we evaluated the quality traits protein content, sedimentation volume, falling number, vitreousity and thousand kernel weight in a Central European (CP) and a Southern and Western European panel (SP) with 183 and 159 durum lines, respectively, and investigated their genetic architecture by genome-wide association mapping. Except for protein content, we identified QTL explaining a large proportion of the genotypic variance for different traits. However, most of them were identified only in one panel. Nevertheless, for sedimentation volume a genomic region on chromosome 1B appeared important in both durum panels and a BLAST search against the emmer and bread wheat reference genomes points toward the candidate gene Glu-B3. This was further supported by the protein subunit banding pattern via SDS-PAGE gel electrophoresis. For vitreousity, genomic regions on chromosome 7A explained a larger proportion of the genotypic variance in both panels, whereas one QTL, possibly related to the Pinb-2 locus, also slightly influenced the protein content. Within each panel, high prediction abilities for genomic selection were obtained, which, however, dropped considerably when predicting across both panels. Nevertheless, the across-panel prediction ability was still larger than 0.4 for protein content and sedimentation volume, underlining the potential for genomics-aided durum breeding, if laboratory and logistical facilities are available.

The soybean experiment '1000 Gardens': a case study of citizen science for research, education, and beyond.

KEY MESSAGE: Citizen science, an approach that includes normal citizens in scientific research, holds great potential also for plant sciences and breeding and can be a powerful research tool to complement traditional approaches. Citizen science is an approach that includes normal citizens in scientific research, but has so far not been exploited by the various disciplines in plant sciences. Moreover, global threats challenge human well-being and science can provide solutions, but needs to leave the ivory tower in the mind of the broader public. In 2016, we performed the '1000 Gardens-the soybean experiment' citizen science project, that aimed at finding citizens in Germany who would grow soybean lines in their own gardens and evaluate them for a range of traits related to adaptation and agronomic performance. Here, we describe details of this project, i.e. the recruitment, performance, and compliance of the citizen scientists. A total of 2492 citizen scientists volunteered for the project, but through the high media coverage a much broader audience than just the participants was reached. Our 1000 Gardens project was successful in collecting a scientifically unique data set with heritabilities ranging up to 0.60 for maturity date or 0.69 for plant height. Our results suggest that the citizen science approach holds great potential also for plant sciences and can be a powerful research tool to complement traditional approaches. Our project was also successful in raising public awareness about the importance of plant breeding and in communicating key messages on the manifold benefits of legumes for a sustainable agriculture to a broader public. Thus, citizen science appears as a promising avenue to demonstrate the value of breeding and science to the general public by including normal citizens in scientific research.

QTL mapping and validation of fertility restoration in West African sorghum A1 cytoplasm and identification of a potential causative mutation for Rf2.

Key message Major A 1 cytoplasm fertility restoration loci, Rf 2 and Rf 5 , were found in the West African sorghum. A potential causative mutation for Rf 2 was identified. KASP markers were validated on independent material. To accelerate the identification and development of hybrid parental lines in West African (WA) sorghum, this study aimed to understand the genetics underlying the fertility restoration (Rf) in WA A1 cytoplasmic male sterility system and to develop markers for a routine use in WA breeding programs. We genotyped by sequencing three F2 populations to map the Rf quantitative trait loci (QTL), validated the molecular KASP markers developed from those QTL in two F2:3 populations, and assessed the most promising markers on a set of 95 R- and B-lines from WA breeding programs. Seven QTL were found across the three F2 populations. On chromosome SBI-05, we found a major fertility restorer locus (Rf5) for two populations with the same male parent, explaining 19 and 14% of the phenotypic variation in either population. Minor QTL were detected in these two populations on chromosomes SBI-02, SBI-03, SBI-04 and SBI-10. In the third population, we identified one major fertility restorer locus on chromosome SBI-02, Rf2, explaining 31% of the phenotypic variation. Pentatricopeptide repeat genes in the Rf2 QTL region were sequenced, and we detected in Sobic.002G057050 a missense mutation in the first exon, explaining 81% of the phenotypic variation in a F2:3 population and clearly separating B- from R-lines. The KASP marker developed from this mutation stands as a promising tool for routine use in WA breeding programs.

Assessing the variation and genetic architecture of asparagine content in wheat: What can plant breeding contribute to a reduction in the acrylamide precursor?

KEY MESSAGE: A large genetic variation, moderately high heritability, and promising prediction ability for genomic selection show that wheat breeding can substantially reduce the acrylamide forming potential in bread wheat by a reduction in its precursor asparagine. Acrylamide is a potentially carcinogenic substance that is formed in baked products of wheat via the Maillard reaction from carbonyl sources and asparagine. In bread, the acrylamide content increases almost linearly with the asparagine content of the wheat grains. Our objective was, therefore, to investigate the potential of wheat breeding to contribute to a reduction in acrylamide by decreasing the asparagine content in wheat grains. To this end, we evaluated 149 wheat varieties from Central Europe at three locations for asparagine content, as well as for sulfur content, and five important quality traits regularly assessed in bread wheat breeding. The mean asparagine content ranged from 143.25 to 392.75 mg/kg for the different wheat varieties, thus underlining the possibility to reduce the acrylamide content of baked wheat products considerably by selecting appropriate varieties. Furthermore, a moderately high heritability of 0.65 and no negative correlations with quality traits like protein content, sedimentation volume and falling number show that breeding of quality wheat with low asparagine content is feasible. Genome-wide association mapping identified few QTL for asparagine content, the largest explaining 18% of the genotypic variance. Combining these QTL with a genome-wide prediction approach yielded a mean cross-validated prediction ability of 0.62. As we observed a high genotype-by-environment interaction for asparagine content, we recommend the costly and slow laboratory analysis only for late breeding generations, while selection in early generations could be based on marker-assisted or genomic selection.

Phenotypic and genetic analysis of spike and kernel characteristics in wheat reveals long-term genetic trends of grain yield components.

KEY MESSAGE: Phenotypic and genetic analysis of six spike and kernel characteristics in wheat revealed geographic patterns as well as long-term trends arising from breeding progress, particularly in regard to spikelet fertility, i.e. the number of kernels per spikelet, a grain yield component that appears to underlie the increase in the number of kernels per spike. Wheat is a staple crop of global relevance that faces continuous demands for improved grain yield. In this study, we evaluated a panel of 407 winter wheat cultivars for six characteristics of spike and kernel development. All traits showed a large genotypic variation and had high heritabilities. We observed geographic patterns for some traits in addition to long-term trends showing a continuous increase in the number of kernels per spike. This breeding progress is likely due to the increase in spikelet fertility, i.e. the number of kernels per spikelet. While the number of kernels per spike and spikelet fertility were significantly positively correlated, both traits showed a significant negative correlation with thousand-kernel weight. Genome-wide association mapping identified only small- and moderate-effect QTL and an effect of the phenology loci Rht-D1 and Ppd-D1 on some of the traits. The allele frequencies of some QTL matched the observed geographic patterns. The quantitative inheritance of all traits with contributions of additional small-effect QTL was substantiated by genomic prediction. Taken together, our results suggest that some of the examined traits were already the basis of grain yield progress in wheat in the past decades. A more targeted exploitation of the available variation, potentially coupled with genomic approaches, may assist wheat breeding in continuing to increase yield levels globally.

Mapping a male-fertility restoration locus for the A4 cytoplasmic-genic male-sterility system in pearl millet using a genotyping-by-sequencing-based linkage map.

BACKGROUND: Pearl millet (Pennisetum glaucum (L.) R. Br., syn. Cenchrus americanus (L.) R. Br) is an important cereal and fodder crop in hot and arid environments. There is great potential to improve pearl millet production through hybrid breeding. Cytoplasmic male sterility (CMS) and the corresponding nuclear fertility restoration / sterility maintenance genes (Rfs) are essential tools for economic hybrid seed production in pearl millet. Mapping the Rf genes of the A4 CMS system in pearl millet would enable more efficient introgression of both dominant male-fertility restoration alleles (Rf) and their recessive male-sterility maintenance counterparts (rf). RESULTS: A high density linkage map based on single nucleotide polymorphism (SNP) markers was generated using an F2 mapping population and genotyping-by-sequencing (GBS). The parents of this cross were 'ICMA 02777' and 'ICMR 08888', which segregate for the A4 Rf locus. The linkage map consists of 460 SNP markers distributed mostly evenly and has a total length of 462 cM. The segregation ratio of male-fertile and male-sterile plants (3:1) based on pollen production (presence/absence) indicated monogenic dominant inheritance of male-fertility restoration. Correspondingly, a major quantitative trait locus (QTL) for pollen production was found on linkage group 2, with cross-validation showing a very high QTL occurrence (97%). The major QTL was confirmed using selfed seed set as phenotypic trait, though with a lower precision. However, these QTL explained only 14.5% and 9.9% of the phenotypic variance of pollen production and selfed seed set, respectively, which was below expectation. Two functional KASP markers were developed for the identified locus. CONCLUSION: This study identified a major QTL for male-fertility restoration using a GBS-based linkage map and developed KASP markers which support high-throughput screening of the haploblock. This is a first step toward marker-assisted selection of A4 male-fertility restoration and male-sterility maintenance in pearl millet.