Genome-wide association study of Senegalese sorghum seedlings responding to a Texas isolate of Colletotrichum sublineola

Sorghum seedlings are known to be more resistant compared to later growth stages. It has been assumed this may be because they contain the preformed cyanogenic glycoside dhurrin, which may play a role in seeding protection9. Similarly, cultivars of Johnsongrass [Sorghum halepense (L.) Pers.]a wild relative of sorghum, were reported to be more resistant in young plants compared to fully grown plants when inoculated to a sorghum isolate of C. sublineola13. As Table 1 depicts, the results followed the previously reported patterns when compared to 8-leaf stage inoculated sorghum plants8th. Majority of accessions were resistant in seeding stage, but they become more susceptible at the 8-leaf stage. In contrast, 11 cultivars showed completely opposite results; Susceptible seedlings were detected, but acervuli formation was not observable in 8-leaf stage plants in the previous study (PI514298, PI514299, PI514300, PI514372, PI514427, PI514448, PI514449, PI514455, PI514459, PI514474 and PI514478)8th. Both of these changes infer different resistance mechanisms operate in juvenile versus older plants. If dhurrin is inhibitory, these plants may simply be those with naturally low levels. The single isolate used in these studies was selected due to its pathogenicity on BTx635 at both stages of growth whereas a mix of isolates known to represent different pathotypes was used in in the previous tests. Nevertheless, a recent study showed that following inoculation with single or a mixture of two isolates of C. sublineolahad minimal to no effect, in that infection rates were more characteristic for the virulent isolate in mixed inoculum14. The use of the single isolate may contribute the decrease in number of high scores, but as FSP53 strain has a strong and broad virulence pattern to sorghum cultivars5:11, the effect is assumed to be minimal. The results of screening sorghum to C. sublineola can also be altered by environmental effects. For example, screening results in Texas and Puerto Rico differed in one study8th. Similarly, SAP lines responded differently to anthracnose based on inoculum and field locations4. PI514284, PI514293 and PI514473 were susceptible at both seeding and 8-leaf stage8th. It is speculated that change of phytoalexin levels may vary from seeding to 8-leaf stage in individual sorghum accessions.

The SNP locus S01_72868925 is located in protein kinase domain // Leucine rich repeat N-terminal domain (Sobic.001G451800). LRRs are a feature of nearly all cloned resistance genes and are widely known for roles in plant host defense8th. Although not the same SNP, GWAS analysis based on identical accessions after inoculation at the 8-leaf stage showed the SNP locus S06_60609133 as the top candidate SNP, and the SNP locus S06_60609133 tagged leucine rich repeat/protein tyrosine kinase (Sobic.006G274866)8th. In other studies, sorghum mini core collection and SAP lines, regarding anthracnose and head smut, leucine rich repeat containing proteins were commonly listed as top candidate genes4,5,6but chromosomal locations differed in each study.

The SNP locus S08_7370058 is 16202 bp away from a poly (ADP-ribose) polymerase (PARP), catalytic domain. PARP domains have also been implicated as factors in stress responses of plants15. Transgenic plants with reduced PARP levels have broad-spectrum stress-resistant phenotypes16.

The SNP locus S09_51943886 is located within the gene coding for flavonoid 3′-monooxygenase. Chalcone synthase (CHS) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway17. CHS expression causes accumulation of flavonoid and isoflavonoid phytoalexins and is involved in the salicylic acid defense pathway17.

As sorghum seedlings typically contain the preformed cyanogenic glycoside dhurrin9, it was expected to see majority of 1st leaves to be scored as 1. Sorghum pathologists often label an accession as susceptible when even a single sorghum plant among a number exposed to inoculum can be infected. An example is head smut caused by Sporisorium reilianum (Bold) Langdon & Fullerton. Similarly, a GWAS analysis based on the highest score of the 1st leaves based on the excised-leaf assay in each accession identified candidate genes associated with SNP loci supported by strong statistical power. As sorghum responses differed at the 1-leaf stage versus the 8-leaf stage, it is not surprising to identify novel SNP loci, including the SNP locus Sobic.001G451800 that are associated with seedling defense, but it will be essential to explore the response more deeply in the future. It is quite possible that different SNPs are highly associated with sorghum defense in seeding stage only or throughout the whole growth stages. The candidate genes identified in this study can be tested for further analysis such as real-time quantitative reverse transcription PCR (Real-time qRT-PCR) or RNA sequencing analysis (RNA-Seq) to confirm that different genes are highly expressed in seeding stages . Gene editing technology can also be applied to verify the defensive roles of the candidate genes. Lastly, level of cyanogenic glycosides can be measured at seedling and 8-leaf stages to find associations with phenotypes and the identified genes.

In sum, this study explored sorghum seeding responses to C. sublineola with an excised-leaf assay and verified that the excised-leaf assay can be useful to study seedlings, but compared to 8-leaf stage inoculation methods, a greater number of seedlings should be screened to determine susceptibility in each accession as the results can easily be skewed.

However, the excised-leaf assay applied to 1-leaf stage seedlings is expected to promote studies associated with sorghum seedlings and C. sublineola interactions; it can be conducted rapidly within laboratory setting.

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