Using Genotyping by Sequencing to Map Two Novel Anthracnose Resistance Loci in Sorghum bicolor

Abstract

Colletotrichum sublineola is an aggressive fungal pathogen that causes anthracnose in sorghum [Sorghum bicolor (L.) Moench]. The obvious symptoms of anthracnose are leaf blight and stem rot. Sorghum, the fifth most widely grown cereal crop in the world, can be highly susceptible to the disease, most notably in hot and humid environments. In the southeastern United States the acreage of sorghum has been increasing steadily in recent years, spurred by growing interest in producing biofuels, bio-based products, and animal feed. Resistance to anthracnose is, therefore, of paramount importance for successful sorghum production in this region. To identify anthracnose resistance loci present in the highly resistant cultivar 'Bk7', a biparental mapping population of F3:4 and F4:5 sorghum lines was generated by crossing 'Bk7' with the susceptible inbred 'Early Hegari-Sart'. Lines were phenotyped in three environments and in two different years following natural infection. The population was genotyped by sequencing. Following a stringent custom filtering protocol, totals of 5186 and 2759 informative SNP markers were identified in the two populations. Segregation data and association analysis identified resistance loci on chromosomes 7 and 9, with the resistance alleles derived from 'Bk7'. Both loci contain multiple classes of defense-related genes based on sequence similarity and gene ontologies. Genetic analysis following an independent selection experiment of lines derived from a cross between 'Bk7' and sweet sorghum 'Mer81-4' narrowed the resistance locus on chromosome 9 substantially, validating this QTL. As observed in other species, sorghum appears to have regions of clustered resistance genes. Further characterization of these regions will facilitate the development of novel germplasm with resistance to anthracnose and other diseases.

Keywords: Colletotrichum sublineola; GBS; anthracnose; sorghum.

Figure 1

Disease phenotype under field conditions. (A) Cultivar ‘Bk7’ and (B) cultivar ‘Early Hegari-Sart’ in a University of Florida breeding nursery in Marianna, FL, where anthracnose was endemic. Both genotypes were of the same age. The brown appearance of Early Hegari-Sart is due to the presence of many lesions on stem and leaves. (C) Close-up of an Early Hegari-Sart leaf with anthracnose symptoms.

Figure 2

Histograms of the averaged phenotypic disease score. Histograms were generated for each location, inbred generation (F₄ and F₅) and year.

Figure 3

Bivariate comparison between the marker orders according to the reference genome and JoinMap for chromosome 1 following custom filtering. The x-axis represents the order according to the linkage map generated by JoinMap, the y-axis represents the order according to the reference genome v2.4 (GBS ordering). When the slope of the data points is equal to one, the two methods are in agreement on the marker order. Points that do not fall on the line reflect markers that differ in order depending on the method used. The red points represent the centromere and the blue points mark an area of high marker density.

Figure 4

Association map of anthracnose resistance in the mapping population phenotyped in three environments. The graph shows the degree of association between the markers and the disease phenotype. The x-axis represents the genomic position, and the y-axis represents the –log₁₀(p-value) obtained from Fisher’s exact test. A higher y-value indicates a stronger association between the marker at that position and disease resistance. The blue, red, and green lines represent the environments of Live Oak 2013, Citra 2013, and Live Oak 2015, respectively. The vertical black lines separate the 10 chromosomes, while the horizontal lines mark the FDR significance thresholds of 5%, with the colors corresponding to the environments; the FDR-lines for the two 2013 environments are indistinguishable from one another in this figure.

Figure 5

Genotypic markers within the two QTL for individual lines organized by phenotype. The vertical columns represent individual lines, and the color within the columns indicate the genotypic state of the markers: blue represents ‘Early Hegari-Sart’, red represents ‘Bk7’, green represents heterozygosity, and brown represents missing data. The scale on the y-axis is proportional to the number of markers. The reduced QTL region is indicated by the dashed black lines, while the solid black line indicates the position of the centromere on chromosome 7. Lines with ambiguous phenotypic data were excluded for clarity.

Figure 6

Genomic positions of candidate resistance genes within the two resistance loci. The bars represent the starting location for the annotated transcript of the candidate genes on (A) the locus on chromosome 7, and (B) the locus on chromosome 9. A red bar indicates an R-gene while a blue bar indicates a defense-related gene that is not an R-gene. The thick vertical black lines on the chromosome 7 locus indicate the size reduction of this QTL based on Figure 5. The x-axis represents the genomic position within each chromosome. The x-axes in A and B differ in scale.