Characterization of the Maize Chitinase Genes and Their Effect on Aspergillus flavus and Aflatoxin Accumulation Resistance

Abstract

Maize (Zea mays L.) is a crop of global importance, but prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need for intervention by farmers. Chitinolytic enzymes respond to attack by potential pathogens and have been demonstrated to increase insect and fungal resistance in plants. Here, all chitinase genes in the maize genome were characterized via sequence diversity and expression patterns. Recent evolution within this gene family was noted. Markers from within each gene were developed and used to map the phenotypic effect on resistance of each gene in up to four QTL mapping populations and one association panel. Seven chitinase genes were identified that had alleles associated with increased resistance to aflatoxin accumulation and A. flavus infection in field grown maize. The chitinase in bin 1.05 identified a new and highly significant QTL, while chitinase genes in bins 2.04 and 5.03 fell directly beneath the peaks of previously published QTL. The expression patterns of these genes corroborate possible grain resistance mechanisms. Markers from within the gene sequences or very closely linked to them are presented to aid in the use of marker assisted selection to improve this trait.

Fig 1. Dendrogram depicting relationships between gene sequences and gene transcripts within each glycoside hydrolase family (18, 19, and 20) and diagram of gene structure and protein motif for each chitinase using the PIECE database [38].

(A) GH-18 family chitinases; (B) GH-19 family chitinases; (C) GH-20 family chitinases, Intron (black); CDS (blue); untranslated region (UTR, Green); Glycoside Hydrolase protein family 18 domain (PF00704 (red)); FHIPEP protein family for type III secretions (PF00771 (purple)). Glycoside Hydrolase protein family 19 (PF00182 (yellow)); Carbohydrate-binding module protein family (PF00187 (brown)); Glycoside hydrolase protein family 20, domain 2 protein family (PF02838 (orange)), Glycoside Hydrolase protein family 20 PF00728 (pink). Blue triangle denotes large intron in GRMZM2G034598. Note change in scale.

Fig 2. The position of single nucleotide and insertion/ deletion polymorphisms studied within the chitinase gene sequences or within 500 bp of the gene position (Adapted from gramene.org [29]). Schematics of each gene are drawn from information from the MaizeGDB [28]. Other genes in the study not shown here were mapped using further up-or down-stream markers.

Footnotes: (A) GRMZM2G134251; (B) GRMZM2G103668; (C) GRMZM2G162505; (D) GRMZM2G051943; (E) GRMZM2G064360; (F) GRMZM2G145518; (G) GRMZM2G062974; (H) GRMZM2G117405. Orange section is the gene of interest. Red stars and/or arrows indicate position of SNP used. (1) S1_27303546, (2) S1_85545046, (3) S1_240766861, (4) S1_240766882, (5) S2_33534181, (6) S1_63229609, (7) S1_63229636, (8) S6_82813940, (9) S8_88812804, (10) S8_164558329; (11) S8_165558375; (12) S8_164558387. Blue Triangle indicates position of insertion/ deletion polymorphism ChiAMpVa. SNP positions provided as (Maize B73 RefGen_V2) adjusted to reflect correct position on image (Maize B73 RefGen_V3).

Fig 3. Linkage map from the F_2:3 mapping population of Mp715 x T173 (n = 192) showing the position of a new QTL on chromosome 1 centered over the SNP within the sequence of GRMZM2G103668 in bin 1.05, a GH19 protein with chitinase activity.

The mapping population was phenotyped and genotyped originally according to [25].

Fig 4. Manhattan graph generated from the association analysis of SNPs extracted from the genetic sequence of 30 chitinase genes via TASSEL.

Each graph depicts the untransformed aflatoxin means in one environment, or averaged over all environments (Ave). Star09 = Starkville, 2009; Star10 = Starkville 2010; StRa10 = Starkville, Raymond site, 2010; Lubb09 = Lubbock, 2009; Lubb10 = Lubbock, 2010; CSta09 = College Station 2009; CSta10 = College Station 2010.

Fig 5. Dot matrix view of paired alignments of maize chitinase exon sequences with similar gene structures, based on the dendogram in Fig 1.

1 = GRMZM2G051943 (Exon 1) vs. GRMZM2G052175 (exons 1 and 2) (86% identical); 2 = GRMZM2G051943 (Exon 1) vs. GRMZM2G051921 (exon 1) (77% identical); 3 = GRMZM2G051943 (Exon 2) vs. GRMZM2G052175 (exon 3) (85% identical); 4 = GRMZM2G051943 (Exon 2) vs. GRMZM2G051921 (exon 2) (87% identical); 5 = Exons 1 and 2 GRMZM2G412577 vs. GRMZM2G400497 (95% identical); 6 = Exons 4 and 5 of GRMZM2G099454 vs. exons 1–3 of GRMZM2G103668 (78% identical); 7 = GRMZM2G057093 VS GRMZM2G162505 (88% identical); 8 = GRMZM2G162359 VS GRMZM2G328171 (76% identical); 9 = GRMZM2G400999 VS GRMZM2G447795 (81% identical); 10 = GRMZM2G080547 VS GRMZM2G160265 (98% identical).