The IIIVmrMLM method uncovers new genetic variants associated with resistance to Fusarium wilt in flax

Abstract

Flax (Linum usitatissimum) is an important agricultural crop grown for fiber and oil production, playing a key role in various industries such as production of paints, linoleum, food, clothes and composite materials. Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. lini is a reason of significant economic damage in flax cultivation. The spores of the fungus can persist in the soil for a long time, so obtaining resistant varieties is important. Here we used data on the resistance of 297 flax accessions from the collection of the Federal Center for Bast Crops in Torzhok (Russian Federation) to infection by a highly virulent isolate of the fungus MI39 in 2019-2021. Genotype resistance to infection was assessed by calculating the DSI index, a normalized proportion of genotypes with the same disease symptoms. The IIIVmrMLM program in Single_env mode was used to search for regions of the flax genome associated with resistance. The IIIVmrMLM model was designed to address methodological shortcomings in identifying all types of interactions between alleles, genes and environment, and to unbiasedly estimate their genetic effects. Being a multilocus MLM model, it estimates the effects of all genes as well as the effects of all interactions simultaneously. A total of 111 QTNs were found, of which 34 fell within the body of a known gene or were located in flanking regions within 1,000 bp. The genes into which the detected variants fell were associated with resistance to abiotic and biotic stresses, root, shoot and flower growth and development. Ten of the QTNs found mapped to regions of previously identified QTLs controlling the synthesis of palmitic, oleic, and other fatty acids. QTN Chr1_1706865/Chr1_1706872 and QTN Chr8_22542741 mark regions identified previously in an association search by the GAPIT program. The allelic effect was confirmed for all the QTNs found: a Mann-Whitney test was performed, which confirmed significant differences between the DSI index value in carriers of the reference and alternative allele. An increase in the number of alleles with negative effects in the genotype leads to a statistically significant decrease in the DSI value for all three years of testing. The groups of varieties with a large number of alleles reducing the DSI index had the best resistance. A total of 5 varieties were selected from the collection for which the number of alleles reducing the DSI index value did not exceed the number of alleles with the opposite effect for all three years. These varieties can be used further in breeding programs.

Keywords: Fusarium oxysporum f. sp. lini; Fusarium wilt; GWAS; Linum usitatissimum; flax.

Table 1.. Average temperature of the growing seasons 2019–2021 (according to Torzhok meteorological station)

Table 2.. Dispersion analysis

Fig. 1.. Location of QTNs associated with fusarium wilt relative to chromosomes in flax.

a –c – Manhattan plots of GWAS results using the IIIVmrMLM package; black shows QTNs that fell in the QTL or were located near genes along with their LOD score value, which is used in IIIVmrMLM to assess significance; d – distribution of QTNs found for the DSI for the three years data, by chromosome.

Table 3.. Cumulative percentage of variation in each year’s data explained by QTNs

Notе. QTN names are formed as ChrX_N, where X is the chromosome number and N is the position in the chromosome.

Table 4.. Co-localized QTNs across the years

Notе. The corresponding lines show data for different years separated by “/”; bp – base pairs; REF – reference allele; ALT – alternative allele.

Table 5.. QTNs located within protein-coding genes and their 1-kb flanking regions

Table 5end.. Table 5end.

Notе. REF – reference allele; ALT – alternative allele. * The largest of the mean DSI values in carriers of the reference or alternative allele; ** QTNs localized both in the gene body and known QTL.

Table 6.. QTNs located within previously identified QTLs

Notе. REF – carriers of the reference allele, ALT – carriers of the alternative allele. * The largest of the mean DSI values in carriers of the reference or alternative allele. Abbreviations of trait names from (You, Cloutier, 2020): PAL (Palmitic %) – palmitic acid content; OLE (Oleic %) – oleic acid content; OIL (Oil content %) – oil content, PM (Powdery mildew rating) – powdery mildew rate; IOD (Iodine value) – iodine content; LIN (Linoleic %) – linoleic acid content; LIO (Linolenic %) – linolenic acid content.

Fig. 2.. Distribution of DSI values in different years for accessions containing different numbers of alleles that have a negative effect on the DSI value.

The upper part of the graphs shows the p-value of the statistical test.

Table 7.. Varieties that had the best combination of alleles with positive and negative effects in all three years

Fig. 3.. Normalized DSI values in carriers of the reference (REF) and alternative (ALT) alleles in an independent dataset of 100 samples for some QTNs common to both datasets.

The p-values of the Mann–Whitney test are shown.