Genetic identification of SNP markers and candidate genes associated with sugarcane smut resistance using BSR-Seq

Abstract

Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases worldwide. In this study, a cross was made between a smut-resistant variety YT93-159 and a smut-susceptible variety ROC22, and 312 progenies were obtained. Two bulks of progenies were then constructed, one consisted of 27 highly smut resistant progenies and the other 24 smut susceptible progenies. Total RNAs of the progenies of each bulk, were pooled and subject to bulked segregant RNA-sequence analysis (BSR-Seq). A total of 164.44 Gb clean data containing 2,341,449 SNPs and 64,999 genes were obtained, 7,295 of which were differentially expressed genes (DEGs). These DEGs were mainly enriched in stress-related metabolic pathways, including carbon metabolism, phenylalanine metabolism, plant hormone signal transduction, glutathione metabolism, and plant-pathogen interactions. Besides, 45,946 high-quality, credible SNPs, a 1.27 Mb region at Saccharum spontaneum chromosome Chr5B (68,904,827 to 70,172,982), and 129 candidate genes were identified to be associated with smut resistance. Among them, twenty-four genes, either encoding key enzymes involved in signaling pathways or being transcription factors, were found to be very closely associated with stress resistance. RT-qPCR analysis demonstrated that they played a positive role in smut resistance. Finally, a potential molecular mechanism of sugarcane and S. scitamineum interaction is depicted that activations of MAPK cascade signaling, ROS signaling, Ca²⁺ signaling, and PAL metabolic pathway and initiation of the glyoxalase system jointly promote the resistance to S. scitamineum in sugarcane. This study provides potential SNP markers and candidate gene resources for smut resistance breeding in sugarcane.

Keywords: BSR-seq; SNPs; expression pattern; key genes; molecular mechanism; smut resistance; sugarcane.

Figure 1

A graphic presentation of smut disease response by two selected extreme bulks of F₁ progenies and two parental varieties (X-axis) based on disease incidence (%) (Y-axis). F, female parent; M, male parent; HR, highly resistance; S1, susceptible 1; S2, susceptible 2; HS1, highly susceptible 1; HS2, highly susceptible 2.

Figure 2

Number of new genes functionally annotated in various databases.

Figure 3

MA plot (A) and cluster plot (B) of differentially expressed genes (DEGs) between YT93-159 (T01) and ROC22 (T02) and between the resistant bulk (T03) and the susceptible bulk (T04).

Figure 4

GO (A) and KEGG (B) enrichment analyses of DEGs between YT93-159 (T01) and ROC22 (T02) and between the resistant bulk (T03) and the susceptible bulk (T04).

Figure 5

 A Venn plot of SNP statistics for T01, T02, T03, and T04. The numbers of SNPs are shown.

Figure 6

Distribution of correlation values for ΔSNP-index (A) and Euclidean distance (ED) (B) methods on the S. spontaneum chromosomes. The colored dots represent the ΔSNP-index or ED value, the black line represents the fitted ΔSNP-index or ED value, and the red dotted line represents the significance threshold.

Figure 7

Expression patterns of key genes based on BSR-Seq database (A) in ROC22 (B), and under the stress of sugarcane smut pathogen (C).

Figure 8

Comparative expression analysis of 20 key genes between YT93-159 and ROC22 at 0 d (blue), 1 d (red), 2 d (green), and 5 d (purple) post S. scitamineum-inoculation by RT-qPCR. Different letters indicate a significant difference at 5% level (p ≤ 0.05). All data points are mean ± standard error (n = 3). (A) LRR-RLK; (B) MAPK; (C) MEK; (D) Raf-like; (E) CAT1; (F) POD; (G) GLYI10; (H) GLYII21; (I) CML42; (J) CIPK; (K) PHD-ZFP; (L) BED-ZFP; (M) eIF2B; (N) MYB; (O) WRKY53; (P) LOX; (Q) KUP; (R) RNK; (S) SPI; (T) PAP.

Figure 9

 A potential molecular mechanism of sugarcane and S. scitamineum interaction. (A) A heat map of the key genes identified by BSR-Seq and WGCNA. Values indicate log ratios of relative expression levels of the key genes by RT-qPCR in YT93-159 and ROC22 at 0 d, 1 d, 2 d, and 5 d post S. scitamineum-inoculation. (B) The potential molecular mechanism diagram. a, ROS signaling pathway; b, MAPK cascade signaling pathway; c, Ca²⁺ signaling pathway; d, PAL metabolic pathway; e, the glyoxalase system; PAMPs, pathogen-associated molecular proteins; RLKs, receptor-like kinases; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; CIPK, calcineurin B-Like interacting protein kinase; CDPK, calcium-dependent protein kinase; CML, calmodulin like; NOS, nitric-oxide synthesis; PAL, phenylpropanoid; MG, methylglyoxal; GSH, glutathione; GLYI, glyoxalase I; GLYII, glyoxalase II; SD-lactoylglutathione (S-LG).