Characterization of epistatic interaction of QTLs LH8 and EH3 controlling heading date in rice

Abstract

Heading date is a critical trait for adaptation of rice to different cultivation areas and cropping seasons. We evaluated the heading dates of 1,123 chromosome segments substitution lines (CSSLs) in the genetic background of an elite rice variety Huajingxian74 (HJX74). A CSSL with the substituted segments from Zihui100 exhibited late heading under both natural long-day (NLD) and natural short-day (NSD) conditions, and the late heading phenotype was controlled by two novel epistatic loci on chromosome 8 and chromosome 3, respectively, termed LH8 and EH3. The function of EH3 was dependent on the LH8 genotype through epistatic interaction between EH3(Zihui100) and LH8(Zihui100) alleles. Genetic and molecular characterization revealed LH8 encodes a CCAAT-box-binding transcription factor with Heading date1 (Hd1)-binding activity and may delay flowering by repressing the expression of Early heading date1 (Ehd1). Our work provides a solid foundation for further study on gene interaction in heading date and has application in breeding rice with greater adaptability.

Figure 1. Flowchart of the development of CSSLs and QTL analysis.

MAS: marker-assisted selection.

Figure 2. The heading dates of HJX74, CSSL5 and their F₂ population.

(a). Phenotypes of HJX74 (left) and CSSL5 (right). Yellow bar = 15 cm. (b). Days-to-heading in HJX74 and CSSL5 in different photoperiodic conditions. NLD, natural long-day conditions; NSD, natural short-day conditions. Each column represents mean ± s.d. (n = 30). (c). Frenquency distributions of days-to-heading in an F₂ population from HJX74 x CSSL5 under NLD conditions. Black arrows indicate the mean DTH of HJX74 and CSSL5 (n = 30), respectively.

Figure 3. Physical map of CSSL5 based on the whole-genome resequencing data.

Red areas indicated the substituted chromosome segments from Zihui100, while the blue areas indicated chromosome regions of HJX74. The tiny yellow area on chromosome 9 indicated a heterozygous segment between HJX74 and Zihui100.

Figure 4. Epistatic interaction between LH8 and EH3.

Differences in days-to-heading for nine genotype classes for combinations of LH8 and EH3 under NLD (a) and NSD (b) conditions in the F₂ populations. Genotypes were determined using the closely linked markers Id83-Id82 (LH8) and Id32-Id33 (EH3). Z, Hetero and H indicate homozygous for Zihui100 allele, heterozygous and homozygous for the HJX74 allele, respectively. P_AA, P_AD, P_DA and P_DD are significant probalities for AA, AD, DA and DD interactions between LH8 and EH3, respectively.

Figure 5. Map-based cloning of the LH8 gene in rice.

(a and b). Frenquency distributions of days-to-heading in an F₃ population from HJX74 x CSSL5 under NLD (a) and NSD (b) conditions. Black arrows indicate the mean DTH of HJX74 and NIL-LH8 (n = 30), respectively. (c). High-resolution mapping of LH8. The genetic map of LH8 was based on recombinant events among 2,159 F₃ plants from HJX74 x CSSL5. The number of recombinants between adjacent markers is shown above the bar. White, shadow and black regions shown in the recombinants indicate homozygous regions for HJX74 allele, heterozygous and homozygous regions for the Zihui100 allele, respectively.

Figure 6. Sequence comparision of LH8 from HJX74 and CSSL5.

(a). Structure of LH8 in HJX74 and CSSL5. Black and gray regions represent the ORF and UTR regions of LH8, respectively. The dotted line represents the 1,116 bp deletion in HJX74. (b). Protein alignment of LH8 from the predicted protein of HJX74 and CSSL5 using the EMBL software ClustalW2 multiple sequence alignment tool.

Figure 7. Characterization of the LH8 gene in rice.

(a). Expression of LH8, Hd1, OsMADS50, Ehd1, Hd3a and RFT1 in HJX74 (white pillars), CSSL5 (gray pillars) and NIL-LH8 (black pillars) at dawn (for LH8, OsMADS50, Ehd1, Hd3a and RFT1) and dusk (for Hd1) under NSD conditions. The analysis was performed in two independent experiments. The mean values of the relative expression levels of genes in CSSL5 and NIL-LH8 compared with HJX74 are shown by cloumns, and standard deviations are indicated by the error bars. (b). Interaction of LH8 and Hd1 by yeast-two-hybrid assay. The yeast cells were grown on SD/-Leu/-Trp/-His/-Ade medium. The interaction between SE and HYL1 was served as positive control, while the combination of empty prey pGADT7, and that of the empty bait vector pGBKT7 with/without LH8 from Zihui100 were served as negative controls.

Figure 8. Schematic representation of the LH8 and EH3 mediated flowering pathway under natural growth conditions.

(a). Epistatic model of rice heading controlled by LH8 and EH3. (b). A proposed model for the flowering pathway controlled by LH8 and EH3 in rice under natural growth conditions. LH8 could down-regulate Ehd1, which could act on Hd3a and RFT1, thus delaying flowering in rice under natural growth conditions. EH3 might function as a modifier of LH8 to shorten heading date only when specific LH8 background was present. The interaction between LH8 and Hd1 depended on different alleles presented in different genetic backgrounds, possibly resulting in varied functions in flowering control.