Fine Mapping for Identification of Citrus Alternaria Brown Spot Candidate Resistance Genes and Development of New SNP Markers for Marker-Assisted Selection

Abstract

Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group within a 3.3 Mb genome region near the centromere in chromosome III. This work addresses fine-linkage mapping of this region for identifying candidate resistance genes and develops new molecular markers for ABS-resistance effective marker-assisted selection (MAS). Markers closely linked to ABSr locus were used for fine mapping using a 268-segregating diploid progeny derived from a heterozygous susceptible × resistant cross. Fine mapping limited the genomic region containing the ABSr resistance gene to 366 kb, flanked by markers at 0.4 and 0.7 cM. This region contains nine genes related to pathogen resistance. Among them, eight are resistance (R) gene homologs, with two of them harboring a serine/threonine protein kinase domain. These two genes along with a gene encoding a S-adenosyl-L-methionine-dependent-methyltransferase protein, should be considered as strong candidates for ABS-resistance. Moreover, the closest SNP was genotyped in 40 citrus varieties, revealing very high association with the resistant/susceptible phenotype. This new marker is currently used in our citrus breeding program for ABS-resistant parent and cultivar selection, at diploid, triploid and tetraploid level.

Keywords: Alternaria alternata; fungal disease resistance; gene mapping; mandarin; molecular markers.

Figure 1

Symptoms observed 48 h after in vitro inoculation of young detached leaves from control genotypes. (A) Susceptible cultivar “Fortune.” (B) Resistant cultivar “CXS0-1.”

Figure 2

Sequence alignment of four genotypes revealing the SNP08. (A) “Fortune”; (B) “CxS0-1”; (C) “Clemenules”; (D) “Minneola.”

Figure 3

Physical maps of the ABSr locus from Cuenca et al. (2013) (A) compared with the fine genetic mapping of the region from the present study (B). Shadowed area in the physical map corresponds to the identified region flanked by the closest markers. Arrows indicate the physical position of each marker used in the fine mapping. Units are Mbases and centiMorgan in the physical and the genetic maps, respectively.

Figure 4

Physical position of candidate genes for ABS resistance found in the identified mapped within the chromosome 3, between 25.49 and 25.86 Kb. Genes in red box are NBS-LRR genes; Genes remarked in double box present LRR receptor-like and serine/threonine protein kinase domains; Gene in blue box belongs to the S-adenosyl-L-methionine-dependent methyltransferase family.

Figure 5

KASPar genotyping results for the SNP08 for the set of genotypes selected for this study. Resistant cultivars are represented by red points (TT); susceptible cultivars are represented by blue points (GT and GG).