Molecular characteristics of S-RNase alleles as the determinant of self-incompatibility in the style of Fragaria viridis

Abstract

Strawberry (Fragaria spp.) is a member of the Rosoideae subfamily in the family Rosaceae. The self-incompatibility (SI) of some diploid species is a key agronomic trait that acts as a basic pollination barrier; however, the genetic mechanism underlying SI control in strawberry remains unclear. Two candidate S-RNases (S_a- and S_b-RNase) identified in the transcriptome of the styles of the self-incompatible Fragaria viridis 42 were confirmed to be SI determinants at the S locus following genotype identification and intraspecific hybridization using selfing progenies. Whole-genome collinearity and RNase T2 family analysis revealed that only an S locus exists in Fragaria; however, none of the compatible species contained S-RNase. Although the results of interspecific hybridization experiments showed that F. viridis (SI) styles could accept pollen from F. mandshurica (self-compatible), the reciprocal cross was incompatible. S_a and S_b-RNase contain large introns, and their noncoding sequences (promotors and introns) can be transcribed into long noncoding RNAs (lncRNAs). Overall, the genus Fragaria exhibits S-RNase-based gametophytic SI, and S-RNase loss occurs at the S locus of compatible germplasms. In addition, a type of SI-independent unilateral incompatibility exists between compatible and incompatible Fragaria species. Furthermore, the large introns and neighboring lncRNAs in S-RNase in Fragaria could offer clues about S-RNase expression strategies.

Fig. 1. The phylogenetic tree of the RNase T2 gene family and the conserved gene sequence of candidate S-RNases in the genus Fragaria.

A The S-RNase genes with red, orange, and light blue backgrounds are from the tribe Amygdaleae, the family Solanaceae, and the tribe Maleae, respectively. The red circles represent Malus, and the yellow five-pointed stars represent Pyrus. The genes in red and green fonts are from F. viridis and F. vesca, respectively. Groups 1, 2, and 3 represent the three groups clustered together, which are marked with red, yellow, and green circular bands, respectively. Bootstrapping was performed with 1000 replicates. B S_a and S_b-RNase are closely related to the genus Prunus; therefore, 41 Prunus S-RNases (Supplementary Table S2) were used to compare and analyze the conserved S-RNase structure in F. viridis. Based on the S-RNase characteristics of Rosaceae^,,, the conserved S-RNase structure in F. viridis was divided further into C1–C3, RC4, and C5 as conserved regions and RHV as a hypervariable region. These regions are marked with thick lines

Fig. 2. The structural pattern and potential neighboring lncRNAs of S-RNase.

A S_aI-1 and S_aI-2 represent the first and second introns of S_a-RNase, respectively; S_bI-1 and S_bI-2 represent the first and second introns of S_b-RNase, respectively. The purified PCR product was used for DNA agarose gel electrophoresis. A 15-kb marker is provided on the left as a reference. B The area between the back slashes on the gene represents introns. The first intron of S_b-RNase is magnified by a grid-filled arrow and highlighted with a blue background. The thick black line with arrow indicates the position and transcription direction of lncRNA. The analysis of the transcription direction of lncRNAs according to the characteristic structure of intron boundaries is also shown. The transcription direction could not be determined because there was no intron structure in lncRNA1-4, and the thick gray lines with arrows represent only the transcription positions

Fig. 3. Analysis of S-RNase expression by RT and qRT-PCR.

Lanes 1, 2, 3, 4, 5, 6, 7, 8, and 9 in A–D represent the flower balls, styles, ovaries, receptacles, pedicels, calyxes, petals, leaves, and anthers of F. viridis 42, respectively. Lanes 10, 11, 12, and 13 represent the flower balls of F. vesca 41, F. mandshurica 43, F. nilgerrensis 45, and F.× ananassa “Benihoppe”, respectively. A, B represent the expression of S_a and S_b-RNase based on specific primers. C Shows the expression results obtained using degenerate primers for S_a and S_b-RNase. D Represents the detection results of all cDNA samples with primers for a reference gene, EF-1α. E Illustrates the expression trends of S-RNase at different periods in styles after pollination. The abscissa represents the time after pollination, and the ordinate represents the level of expression

Fig. 4. Analysis of the collinear region between the S loci of Rosa (rose) and Prunus (almond) and F. vesca.

Fve_v4_6 represents chromosome 6 of F. vesca, and its enlarged region represents the area that is highly collinear with the S loci of the two rose genomes (Rosa_v1_3, from 38160380 to 43142292, and Rosa_v2_3, from 3037968 to 7988569) and the almond genome (Prunus_v2_6, from 24640828 to 29556668). The genomes of F. vesca and Prunus (almond) are represented by Fve_v4 and Prunus_v2, and the versions analyzed were the F. vesca genome v4.0.a1 and the P. dulcis “Texas” genome v2.0, respectively. Rosa_v1 and Rosa_v2 are rose genomes, and the versions analyzed were Rosa chinensis genome v1.0 and Rosa chinensis “Old Blush” homozygous genome v2.0, respectively. The numbers after the first and second “_” represent the genome version and the serial number of the chromosome, respectively. The red text on the right side of each chromosome represents the RNase T2 family genes, the black text represents the F-box family genes, the green text represents other types of genes, and the genes connected by red lines are those that were randomly selected for collinearity analysis in the enlarged area

Fig. 5. Identification of the S-RNase genotype by polyacrylamide gel electrophoresis.

Each band indicated by a red arrow with the S_a tag is from S_a-RNase, and each band indicated by the a arrow with the S_b tag is from S_b-RNase. M represents the reference band for 300 bp and 400 bp markers. A1–101, B1–101, and D57–68 are the selfing progeny lines of “S1-02-S2-76-S3-09” (S_aS_b), and C1–45 and C71–94 are the selfing progeny lines of “S1-02-S2-30-S3-09” (S_aS_a). B46-70 are the selfing progeny lines of “S1-02-S2-76-S3-11” (S_bS_b). D1–56 are the hybrid progenies between “S1-02-S2-30-S3-09” (S_aS_a) and “S1-02-S2-76-S3-11” (S_bS_b). D69–97 are the selfing progeny lines of F. viridis 42 (0–3 generations). The serial number and order (from left to right and top to bottom) of the selfing lines are the same as those in Table 2

Fig. 6. Fruit development following hybridization of different genotype lines from F. viridis 42.

The S genotype lines were selected as shown in Table 2. The S-RNase genotype is shown in brackets

Fig. 7. Growth status of compatible and incompatible pollen tubes in the styles of F. viridis lines 48 h after pollination.

Scale bar = 100 μm. The left side shows pollen tube growth in the incompatible state, that is, the hybridizations that are not highlighted in “Fig. 6”. The right side shows pollen tube growth in the compatible state; that is, the hybridizations that are shown in boxes in “Fig. 6”

Fig. 8. Fruit development and pollen tube extension in the style after interspecific cross-pollination.

F. viridis 42 is a self-incompatible species, and F. vesca 41, F. mandshurica 43, and F. nilgerrensis 45 are self-compatible species. All are wild germplasm resources. Pictures were obtained 15–25 days after pollination, and the pollen tube was selected for observation 48 h after pollination

Fig. 9. Hybridization of four wild strawberry diploid germplasms.

The arrows in the diagram point to the female parents. √ denotes compatibility, × denotes incompatibility, and √* denotes hybridizations that were considered compatible in this study. In fact, the √* hybridizations are different from the other completely compatible interspecific hybridizations (that is, the receptacle develops normally, the number of achenes is high, and most germinated pollen tubes can reach the bases of styles), although a certain compatibility was observed based on receptacle development and achene level. In the √* interspecific hybridizations, most pollen tubes were inhibited at 1/3–1/2 the length of the style, and only some of the styles facilitated pollen tubes without restriction