Tasselyzer, a machine learning method to quantify anther extrusion in maize, based on PlantCV

Male fertility in maize is controlled by development and genetic programming and is directly impacted by environmental factors such as light, temperature, water, and nutrient availability; the control of this trait has substantial agronomic utility. Maize anthers emerge from male florets, which are clustered to form the tassel at the top of the plant separated from the female ear. Quantification of anther extrusion is one important aspect in the determination of male fertility. To address the lack of an automated method to measure anther extrusion on a large scale, we developed 'Tasselyzer', a quantitative, image-based color trait analysis pipeline for tassel image segmentation, based on the existing PlantCV platform, and we applied it to determine the proportion of anther extrusion. We evaluated Tasselyzer in maize during the seven-day period of pollen shedding as well as in the temperature-sensitive male sterile mutant dcl5. With tassel images obtained with a smart phone camera, we show that the anther scores positively correlate with anther extrusion, and such methods can be used to measure environmental impacts on the dcl5 mutant. Altogether, this work establishes an automated and inexpensive method to quantify anther extrusion in maize, which would be useful for research and breeding.

Evaluation of Genetic Variability, Heritability and Genetic Advance in Advanced Breeding Lines of Wheat (Triticum aestivum L)

The Present investigation was carried out using 522 advanced breeding lines of wheat genotypes at BISA Farm, Jabalpur during Rabi, 2018-2019 in randomized block design with two replications. Analysis of variance found the significant difference between breeding lines for all the characters were studied. Appropriate variability was obtainable in the advance breeding lines under study for all the characters indicating sufficient genetic variability among the genotypes. Phenotypic coefficient of variation (PCV) was marginally higher than the respective genotypic coefficient of variation (GCV) for all the characters indicating thereby that there is negligible influence on the characters by the environment. GCV was high for grain yield per plant, grain weight per spike, biological yield per plant, number of grains per spike and number of effective tillers per plant. High PCV was observed for grain weight per spike followed by grain weight per plant, biological yield per plant, number of grains per spike, number of effective tillers per plant and length of main spike. High broad sense heritability along with high genetic advance as percentage of mean were obtained for number of non extruded anther per spike, anther extrusion %, visual score of anther extrusion, biomass per plot and number of productive tillers per plant.

Phenotyping Anther Extrusion of Wheat Using Image Analysis

Phenotyping wheat (Triticum aestivum L.) is time-consuming and new methods are necessary to decrease labor. To develop a heterotic pool of male wheat lines for hybrid breeding, there must be an efficient way to measure both anther extrusion and the size of anthers. Five hundred and ninety-four soft red winter wheat lines in two replications of randomized complete block design were phenotyped for anther extrusion, a key trait for hybrid wheat production. A device was constructed to capture images using a mobile device. Four heads were sampled per line when anthesis was evident for half the heads in the plot. The extruded anthers were scraped onto a surface, their image was captured, and the area of the anthers was taken via ImageJ. The number of anthers extruded was estimated by counting the number of anthers per image and dividing by the number of heads sampled. The area per anther was taken by dividing the area of anthers per spike by the number of anthers per spike. A significant correlation (R=0.9, p<0.0001) was observed between the area of anthers per spike and the number of anthers per spike. This method is proposed as a substitute for field ratings of anther extrusion and to quantitatively measure the size of anthers.

Genome-Wide Association and Prediction of Male and Female Floral Hybrid Potential Traits in Elite Spring Bread Wheat Genotypes

Hybrid wheat breeding is one of the most promising technologies for further sustainable yield increases. However, the cleistogamous nature of wheat displays a major bottleneck for a successful hybrid breeding program. Thus, an optimized breeding strategy by developing appropriate parental lines with favorable floral trait combinations is the best way to enhance the outcrossing ability. This study, therefore, aimed to dissect the genetic basis of various floral traits using genome-wide association study (GWAS) and to assess the potential of genome-wide prediction (GP) for anther extrusion (AE), visual anther extrusion (VAE), pollen mass (PM), pollen shedding (PSH), pollen viability (PV), anther length (AL), openness of the flower (OPF), duration of floret opening (DFO) and stigma length. To this end, we employed 196 ICARDA spring bread wheat lines evaluated for three years and genotyped with 10,477 polymorphic SNP. In total, 70 significant markers were identified associated to the various assessed traits at FDR ≤ 0.05 contributing a minor to large proportion of the phenotypic variance (8–26.9%), affecting the traits either positively or negatively. GWAS revealed multi-marker-based associations among AE, VAE, PM, OPF and DFO, most likely linked markers, suggesting a potential genomic region controlling the genetic association of these complex traits. Of these markers, Kukri_rep_c103359_233 and wsnp_Ex_rep_c107911_91350930 deserve particular attention. The consistently significant markers with large effect could be useful for marker-assisted selection. Genomic selection revealed medium to high prediction accuracy ranging between 52% and 92% for the assessed traits with the least and maximum value observed for stigma length and visual anther extrusion, respectively. This indicates the feasibility to implement genomic selection to predict the performance of hybrid floral traits with high reliability.

Spikelet movements, anther extrusion and pollen production in wheat cultivars with contrasting tendencies to cleistogamy

Background Cleistogamic flowers are a main barrier in pollen dispersal for cross-pollination necessary in wheat hybrid breeding. The aim of our study was to gain new knowledge on the biology of wheat flowering, in particular on the differences between the cleisto- and chasmogamic forms which has certainly cognitive significance, but it can also be used in practice when seeking a female and male ideotypes for cross hybridization. Results We characterized the most significant features defining the flowering specificity in two wheat cultivars with contrasting tendency to cleistogamy: Piko (chasmogamous) and Dacanto (cleistogamous). In the field observations we assessed diurnal pattern of anther extrusion and anther extrusion capacity. For the first time we adapted the time lapse method for measuring kinetics of the spikelet movement and 3-D image correlation technique for the non-invasive measurements of potential deformations of the spikelet lemmas. We found that the two cultivars differ in the potential of pollen dispersion for-cross-pollination and in the spikelet kinetics. We also described some anatomical traits that can have potential functional role in floret opening. None of the cultivars showed any symptoms of lemma surface deformation. Conclusions The cleistogamic and chasmogamic wheat cultivars differ significantly in the potential for pollen dispersion for cross-pollination, which is mainly related to anther extrusion capacity. Although none of these features differentiated the cultivars clearly, we assume, based on spikelet kinetics and the lack of lemmas surface deformation, that the water transport and turgor of cells is essential for the floret opening and anther extrusion in wheat. The search for parental ideotype should be supported by marker assisted selection, e.g. based of polymorphisms in genes related to aquaporin biosynthesis.

Genome-Wide Association Mapping and Genomic Prediction of Anther Extrusion in CIMMYT Hybrid Wheat Breeding Program via Modeling Pedigree, Genomic Relationship, and Interaction With the Environment

Anther extrusion (AE) is the most important male floral trait for hybrid wheat seed production. AE is a complex quantitative trait that is difficult to phenotype reliably in field experiments not only due to high genotype-by-environment effects but also due to the short expression window in the field condition. In this study, we conducted a genome-wide association scan (GWAS) and explored the possibility of applying genomic prediction (GP) for AE in the CIMMYT hybrid wheat breeding program. An elite set of male lines (n = 603) were phenotype for anther count (AC) and anther visual score (VS) across three field experiments in 2017–2019 and genotyped with the 20K Infinitum is elect SNP array. GWAS produced five marker trait associations with small effects. For GP, the main effects of lines (L), environment (E), genomic (G) and pedigree relationships (A), and their interaction effects with environments were used to develop seven statistical models of incremental complexity. The base model used only L and E, whereas the most complex model included L, E, G, A, and G × E and A × E. These models were evaluated in three cross-validation scenarios (CV0, CV1, and CV2). In cross-validation CV0, data from two environments were used to predict an untested environment; in random cross-validation CV1, the test set was never evaluated in any environment; and in CV2, the genotypes in the test set were evaluated in only a subset of environments. The prediction accuracies ranged from −0.03 to 0.74 for AC and −0.01 to 0.54 for VS across different models and CV schemes. For both traits, the highest prediction accuracies with low variance were observed in CV2, and inclusion of the interaction effects increased prediction accuracy for AC only. In CV0, the prediction accuracy was 0.73 and 0.45 for AC and VS, respectively, indicating the high reliability of across environment prediction. Genomic prediction appears to be a very reliable tool for AE in hybrid wheat breeding. Moreover, high prediction accuracy in CV0 demonstrates the possibility of implementing genomic selection across breeding cycles in related germplasm, aiding the rapid breeding cycle.

Molecular Mapping of QTL for Fusarium Head Blight Resistance in a Doubled Haploid Population of Chinese Bread Wheat

Fusarium head blight (FHB) is a destructive disease of wheat worldwide, particularly in China. To map genetic loci underlying FHB resistance, a doubled haploid (DH) population consisting of 174 lines was developed from a cross between widely grown Chinese cultivars Yangmai 16 and Zhongmai 895. The DH population and parents were evaluated in field nurseries at Wuhan in 2016–2017 and 2017–2018 with both spray inoculation and natural infection, and at Jingzhou in 2017–2018 with grain-spawn inoculation. The DH lines were genotyped with a wheat 660K SNP array. FHB index, plant height, anther extrusion, and days to anthesis were recorded and used for QTL analysis. Seven QTL for FHB resistance were mapped to chromosome arms 3BL, 4AS, 4BS, 4DS, 5AL, 6AL, and 6BS, in at least two environments. QFhb.caas-4BS and QFhb.caas-4DS co-located with semi-dwarfing alleles Rht-B1b and Rht-D1b, respectively, and were also associated with anther extrusion. The other five QTL were genetically independent of the agronomic traits, indicating their potential value in breeding for FHB resistance. Based on correlations between FHB indices and agronomic traits in this population, we concluded that increasing plant height to some extent would enhance FHB resistance, anther extrusion played a more important role in environments with less severe FHB, and days to anthesis was independent of FHB response when viewed across years. PCR-based markers were developed for the 3BL and 5AL QTL, which were detected in more than three environments. The InDel marker InDel_AX-89588684 for QFhb.caas-5AL was also validated on a wheat panel, confirming its effectiveness for marker-assisted breeding for improvement of FHB resistance.

Variation in Anther Extrusion and Its Impact on Fusarium Head Blight and Deoxynivalenol Content in Oat (Avena sativa L.)

Variation and inheritance of anther extrusion and its effects on Fusarium head blight were studied. On a 0 to 9 scale, variation ranged from 1 to 6 in a North American oat panel and from 0 to 8 in a Nordic population. The inheritance was studied in two recombinant inbred line populations (Fiia × Stormogul and Svea × Stormogul). Fiia and Svea are recent white-seeded cultivars with low to medium anther extrusion, while Stormogul is an old black-seeded cultivar with high anther extrusion. Highly significant transgressive segregations and high heritabilities were observed (h2 = 0.91 in Fiia × Stormogul and h2 = 0.83 in Svea × Stormogul). Another extrusion was negatively correlated with Fusarium head blight and deoxynivalenol in spawn-inoculated field experiments, but significantly only in Fiia × Stormogul where the range in resistance was widest. Correlations were reversed in spray-inoculated greenhouse experiments, apparently spraying open florets defeated the avoidance mechanism. Anther extrusion may help oat avoid Fusarium infection in the field, but the genetic variance is inadequate and high anther extrusion is rare in modern genepools.

Anther Extrusion and Its Association with Fusarium Head Blight in CIMMYT Wheat Germplasm

Pronounced anther extrusion (AE) is associated with field resistance to Fusarium head blight (FHB), one of the most devastating diseases of wheat globally. In this study, two recombinant inbred line (RIL) populations were used to map quantitative trait loci (QTL) for AE and field FHB resistance and to investigate the association of both traits at the genetic level. Furthermore, two panels of International Maize and Wheat Improvement Center (CIMMYT) wheat breeding lines were evaluated to describe the phenotypic association between the two traits in detail. Highly significant negative correlation was identified between AE and FHB severity in the two populations and the two panels, with r-values ranging from 0.55 to 0.74. QTL analysis in the two RIL populations identified 12 QTL for AE and nine for FHB resistance, of which five QTL located on chromosomes 3BL, 4BS, 4DS, 5AL, and 5BL were associated with both AE and FHB, collectively explaining over 50% of phenotypic variation for FHB. The QTL on chromosomes 4BS, 4DS, 5AL, and 5BL were closely linked to Rht-B1, Rht-D1, Vrn-A1, and Vrn-B1 genes, respectively. In conclusion, AE is closely related to field FHB resistance and could be used as a morphological marker in wheat breeding for field FHB resistance.