A retrotransposon insertion in MUTL-HOMOLOG 1 affects wild rice seed set and cultivated rice crossover rate

Abstract

Wild rice (Oryza rufipogon) has a lower panicle seed setting rate (PSSR) and gamete fertility than domesticated rice (Oryza sativa), but the genetic mechanisms of this phenomenon remain unknown. Here, we cloned a null allele of OsMLH1, an ortholog of MutL-homolog 1 to yeast and mammals, from wild rice O. rufipogon W1943 and revealed a 5.4-kb retrotransposon insertion in OsMLH1 is responsible for the low PSSR in wild rice. In contrast to the wild-type, a near isogenic line NIL-mlh1 exhibits defective crossover (CO) formation during meiosis, resulting in reduced pollen viability, partial embryo lethality, and low PSSR. Except for the mutant of mismatch repair gene postmeiotic segregation 1 (Ospms1), all other MutL mutants from O. sativa indica subspecies displayed male and female semi-sterility similar to NIL-mlh1, but less severe than those from O. sativa japonica subspecies. MLH1 and MLH3 did not contribute in an additive fashion to fertility. Two types of MutL heterodimers, MLH1-PMS1 and MLH1-MLH3, were identified in rice, but only the latter functions in promoting meiotic CO formation. Compared to japonica varieties, indica cultivars had greater numbers of CO events per meiosis. Our results suggest that low fertility in wild rice may be caused by different gene defects, and indica and japonica subspecies have substantially different CO rates responsible for the discrepancy between the fertility of mlh1 and mlh3 mutants.

Figure 1

Mapping and characterization of OsMLH1. A, QTL mapping for PSSR. B, The positional cloning of MLH1. The candidate region was mapped to a ∼49-kb genomic DNA region lying between markers XG10 and XG12 and co-segregated with XG11 and XG3 in a large population (n = 1,536). In total, seven genes were predicted within the 49-kb region. Rec, number of recombinants. C, The relative expression levels of the seven candidate genes in panicles of GLA4 and NIL-mlh1 like plants. Values are means ± sem, n = 4. Significance was assessed by Student’s t test. D, Comparison of a GLA4 plant and a NIL-mlh1 plant. Bar = 10 cm. E–G, Comparison of panicles of GLA4 and NIL-mlh1 (E). Bar = 5 cm. Histograms of PSSR (F) and grain number per panicle (G) are shown beside. Values are means ± sem, n = 30. Significance was assessed by Student’s t test. H, Comparison of a GLA4 and a NIL-mlh1 spikelet after removing the lemma and palea. Bar = 2 mm. I, KI-I₂ staining of GLA4 pollen and NIL-mlh1 pollen. Bar = 100 μm. J, Quantifications of the pollen fertility in GLA4 and NIL-mlh1. Values are means ± sem, n = 20. Significance was assessed by Student’s t test. K, Comparison of GLA4 mature embryo sac and NIL-mlh1 abnormal mature embryo sac spikelet. Bar = 50 μm. L, Quantifications of the fertility of embryo sac in GLA4 and NIL-mlh1.

Figure 2

Cytological Analysis of GLA4 and NIL-mlh1. A, Meiotic chromosome behaviors in GLA4 and NIL-mlh1. Some unpaired chromosomes in NIL-mlh1 are indicated with white arrows. Bar = 5  μm. B, Quantification of the number of bivalents in GLA4 and NIL-mlh1.

Figure 3

Association Mapping. A, Schematic of the gene structure and allelic variation on OsMLH1 between GLA4 and NIL-mlh1 indicated by vertical lines at bottom. B, Haplotype analysis of the OsMLH1 gene region from 112 rice cultivars and NIL-mlh1. C, The PSSR of the five haplotypes. Values are means ± sem. The presence of the same lower letter denotes a nonsignificant difference among them (P > 0.01). P-values were calculated by one-way analysis of variance (ANOVA). D, LUC reporter gene transient assay of promoter activity in rice protoplasts. Left, three constructs, one of which is a site-directed mutation at one SNP in the promoter region. Right, the relative expression was quantified according to LUC/REN values. Values are means ± sem, n = 4. The presence of the same lower letter denotes a nonsignificant difference among them (P > 0.01). P-values were calculated by one-way ANOVA.

Figure 4

Protein sub-cellular localization and protein–protein interactions. A, Sub-cellular localization of OsMLH1-GFP fusion protein in rice protoplasts. Bar = 5 μm. B, The illustration of the conserved domains of three MutL proteins and OsSWIB in rice. C, Yeast two-hybrid assay to examine interactions among MutL proteins and chromatin remodeling complex subunit SWIB. Transformed yeast cells were grown on a synthetic medium lacking Trp and Leu (DDO) or Trp, Leu, His, and Ade (QDO). AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. D, BiFC analysis of the interaction between MLH1 and each of PMS1, MLH3, and SWIB. Bar = 20 μm. E, LUC complementation assay between MLH1 and each of PMS1, MLH3, and SWIB.

Figure 5

Targeted mutation of MutL genes in japonica (Dongjing). A, Gross morphology of mature plants (upper parts) and pollen grains with KI-I₂ solution (lower parts) of WT and mutants. Bar = 10 cm (upper). Bar = 100 μm (lower). B, Histograms of PSSR. Values are means ± sem, n = 40. The presence of the same lower letter denotes a nonsignificant difference among them (P > 0.01). P-values were calculated by one-way ANOVA. C, Histograms of pollen fertility. Values are means ± sem, n = 20. The presence of the same lower letter denotes a nonsignificant difference among them (P > 0.01). P-values were calculated by one-way ANOVA. D, Representative images of metaphase I cells are shown. Bar = 5 μm. E, Quantification of the number of bivalents in WT and mutants. Bivalents, as a readout for CO formation, were assessed in chromosome spreads of the indicated mutant lines.

Figure 6

Targeted mutation of MutL genes in indica (GLA4). A, Gross morphology of mature plants (upper) and pollen grains with KI-I₂ solution (lower) of WT and mutants. Bar = 10 cm (upper). Bar = 100 μm (lower). B, Histograms of PSSR. Values are means ± sem, n = 40. The presence of the same lower letter denotes a nonsignificant difference among them (P > 0.01). P-values were calculated by one-way ANOVA. C, Histograms of pollen fertility. Values are means ±  sem, n = 20. The presence of the same lower letter denotes a nonsignificant difference among them (P > 0.01). P-values were calculated by one-way ANOVA. D, Representative images of metaphase I cells are shown in WT and mutants of indica. Bar = 5 μm. E, Quantification of the number of bivalents in WT and mutants of indica. Bivalents, as a readout for CO formation, were assessed in chromosome spreads of the indicated mutant lines.

Figure 7

Comparison of fertility between indica and japonica with lack of MLH1/3. A, Averages of the number of bivalents per meiocyte in all other mutants of indica and japonica, exclusive of Ospms1. Values are means ± sem, n = 5. B, Averages of the embryo sac fertility in all other mutants of indica and japonica, exclusive of Ospms1. Middle horizontal lines in the box represent the means. Significance was assessed by Student’s t test.

Figure 8

CO frequencies in indica and japonica. A, Histogram of CO number per F₂ individual in indica and japonica populations. Vertical dash lines indicate mean values. B, CO s per chromosome per F₂ compared with chromosome length in indica and japonica.