A male-sterile mutant with necrosis-like dark spots on anthers was generated in cotton

Abstract

Although conventional hybrid breeding has paved the way for improving cotton production and other properties, it is undoubtedly time and labor consuming, while the cultivation of male sterile line can fix the problem. Here, we induced male sterile mutants by simultaneously editing three cotton EXCESS MICROSPOROCYTES1 (GhEMS1) genes by CRISPR/Cas9. Notably, the GhEMS1 genes are homologous to AtEMS1 genes, which inhibit the production of middle layer and tapetum cells as well, leading to male sterility in cotton. Interestingly, there are necrosis-like dark spots on the surface of the anthers of GhEMS1s mutants, which is different from AtEMS1 mutant whose anther surface is clean and smooth, suggesting that the function of EMS1 gene has not been uncovered yet. Moreover, we have detected mutations in GhEMS1 genes from T₀ to T₃ mutant plants, which had necrosis-like dark spots as well, indicating that the mutation of the three GhEMS1 genes could be stably inherited. Dynamic transcriptomes showed plant hormone pathway and anther development genetic network were differential expression in mutant and wild-type anthers. And the lower level of IAA content in the mutant anthers than that in the wild type at four anther developmental stages may be the reason for the male sterility. This study not only facilitates the exploration of the basic research of cotton male sterile lines, but also provides germplasms for accelerating the hybrid breeding in cotton.

Keywords: CRISPR/Cas9; GhEMS1s; cotton; male-sterile line; necrosis-like dark spots.

Figure 1

Creation of GhEMS1 genes mutants by using CRISPR/Cas9. (A) A phylogenetic tree for EMS1 genes in Gossypium hirsutum and AtEMS1 was constructed using the neighbor-joining method in MEGA 10.1.8 followed by bootstrapping with 1,000 replicates; (B) Expression profiles of GhEMS1 genes in flower buds of different lengths (3~5 mm, 5~6 mm, 6~7 mm, 7~8 mm, 8~9 mm, 9~10 mm, 10~11 mm, 11~12 mm, 12~13 mm, 13~14 mm, 14~16 mm, 16~19 mm, 19~24 mm)in G. hirsutum H05 determined by transcriptome sequencing (Zhang et al., 2022); (C) Creation of a male-sterile line pool by CRISPR/Cas9. Two sgRNAs were serially connected to each vector to increase the knockout efficiency; (D) Two pairs of sgRNAs on exons were designed and vectors containing polycistronic tRNA–gRNA genes (PTG). Ghir_A08G010860 (GhEMS_A08), Ghir_D08G010810 (GhEMS_D08), and Ghir_A09G018830 (GhEMS_A09) were knocked out by sgRNA1 and sgRNA2 (PTG1). GhEMS_A08 and GhEMS_D08 (PTG2) were knocked out by sgRNA3 and sgRNA4 (PTG2).

Figure 2

Phenotypes of GhEMS1s mutants. (A) The phenotypes of the WT and KO1 - KO5 transgenic lines, three genes Ghir_A08G010860 (GhEMS_A08), Ghir_D08G010810 (GhEMS_D08), and Ghir_A09G018830 (GhEMS_A09) of PTG1 in KO1- KO3 sterile plants. GhEMS_A08 and GhEMS_D08 (PTG2) were knocked out in KO4 and KO5 plants. Scale bars: 2 mm; (B) Partial enlarged view of anther in picture (a); (C) TTC (2,3,5-triphenyl tetrazolium chloride) was used to detect the pollen viability. No pollen was observed in the anthers of KO1 and KO2 plants. Scale bars: 100 µm. (D) Statistics for pollen fertility in the GhEMS1 mutants. During the phenotypic investigation, we divided plant fertility into six levels: Grade 1 indicates that anthers with necrosis-like dark spots and all anthers without pollen; Grade 2 indicates that < 25% of the anthers have a few inactive pollen grains without dehiscence; Grade 3, 4, and 5 indicate that 25%, 50%, and 75% of the anthers spread pollen, respectively; Grade 6 indicates that all anthers dehiscence and release active pollen. The phenotype of every flower was recorded, and the fertility of different plants was counted. (E) Analysis of gene editing efficiency in different GhEMS mutant lines by Sanger sequencing.

Figure 3

The sterile phenotype of surface necrosis of T3. (A) wild type, KO1 T3 plants, and enlarged images. (B) Changes in the anthers with different degrees of necrosis-like spots. Compared with those of the WT, yellow spots appeared on the anthers of sterile KO1 plants at stage 7, these spots gradually deepened at stage 10 and stage 14 to form necrosis-like dark spots eventually. KO1, GhEMS 3-genes simultaneous mutant. The white arrows indicate necrotic spots.

Figure 4

KO1 editing types were inherited in T0 to T3. (A, B) GhEMS_A/D08 were edited in sgRNA1 and sgRNA2, respectively. (C, D) GhEMS_A09 were edited in sgRNA1 and sgRNA2, respectively. (E) Sequencing peak photos of GhEMS_A/D08 with 5 bp deletions at the sgRNA1 site. (F) Sequencing peak photos of GhEMS_A/D08 with 2 bp deletions at the sgRNA2 site. (G) Sequencing peak photos of GhEMS_A09 with 6 bp deletions at the sgRNA1 site. (H) Sequencing peak photos of GhEMS_A09 with 1 bp deletions at the sgRNA2 site.

Figure 5

Comparison of the histological characteristics of the WT and KO1 sterile line T3 anthers. (A) Stage 6-11 histological characteristics of the WT and KO1, Scale bars: 100 µm (B, C) Analysis of DNA damage in anthers of WT and KO1 male-sterile plants. The degree of DNA fragmentation of anthers was detected by TUNEL. Scale bars: 100 µm. DMs: degenerated microspores; E, epidermis; En, endothecium; Msc, microsporocyte; Mp, mature pollen; ML, middle layer; Msp, microspore; T, tapetum; Tds, tetrads; WT, wild type.

Figure 6

Transcriptome analysis of four developmental stages. (A) Difference gene statistics of wild type and mutant in four periods; (B) Intersection of differential genes at different stages; (C) WGCNA co-expression module, a total of 8 different modules; (D) Analysis of the co-expression network of the red module. The shades of color represent correlations; (E) Gene expression of the regulatory network of pollen development; (F) IAA content at four developmental stages; (G) H₂O₂ content at four developmental stages. W, wild type; M, mutant; AG, AGAMOUS; AMS, ABORTED MICROSPORE; DYT1, DYSFUNCTIONAL TAPETUM 1; EMS1, EXCESS MICROSPOROCYTES 1; MS1, MALE STERILITY 1; NZZ/SPL, SPOROCYTELESS/NOZZLE. Data are presented as means ± SE from five biologically independent experiments. Asterisks indicate statistically significant differences (***, P <0.001; *, P <0.05); by Student’s t-test.