Identification of Substitution Segments and Phenotypic Analysis of Z414

In this study, we used eight polymorphic simple sequence repeat (SSR) markers in the substitution segments of Z414, and 233 polymorphic SSR markers outside the substitution segments of this line to examine the molecular background of Z414. The substitution segments of 10 Z414 plants were consistent, and no other residual segments from Huhan 3 were detected. Z414 contains four substitution segments from Huhan 3, which are on chromosomes 3, 5, and 11. The total estimated length of the substitution segments was 12.17 Mb, and the average length was 3.04 Mb (Fig. 1).

Fig. 1
figure 1

Chromosome substitution segments of Z414. Physical distances (Mb) and mapped QTL are indicated on the left; markers and substitution segment lengths are shown on the right. The black sections on each chromosome are substitution segments. PL, panicle length; GW, grain width; GL, grain length; RLW, ratio of length to width; GWT, 1000-grain weight; BRR, brown rice rate

Z414 displayed a similar plant type and different grain size to Xihui 18 (Fig. 2a–c). There were significant increases in grain width (+ 23.5%; Fig. 2d), 1000-grain weight (+ 16.4%; Fig. 2e), brown rice rate (+ 8.8%; Fig. 2f), and degree of chalkiness (+ 26.1%; Fig. 2g). By contrast, Z414 showed significant decreases in panicle length (− 17.5%; Fig. 2h), grain length (− 7.7%; Fig. 2i), and the ratio of length to width (− 25.1%; Fig. 2j). There were no significant differences in other traits, such as plant height, panicle number per plant, spikelet number per panicle, grain number per panicle, seed-setting rate, yield per plant, head rice rate, chalky rice rate, and gel consistency.

Fig. 2
figure 2

Phenotypes of Xihui 18 and Z414. a Plant types of Xihui 18 and Z414. b Panicles of Xihui 18 and Z414. c Grains, brown rice, and polished rice of Xihui 18 and Z414. Bars represent 20 cm in a, 5 cm in b, and 5 mm in c. dj Statistical analysis of the differences in seven traits between Z414 and Xihui 18, in order as grain width (d), 1000-grain weight (e), brown rice rate (f), chalkiness degree (g), panicle length (h), grain length (i), ratio of grain length to width (j). * and ** indicate significant differences at the 0.05 and 0.01 level, respectively

Cytological Analysis of Z414 and Xihui 18 Glumes

Since the grain lengths and widths differed between Xihui 18 and Z414 (Fig. 3a, b), we used scanning electron microscopy (SEM) to analyze the cell morphology of Xihui 18 and Z414 glumes at the heading stage (Fig. 3c–f). The cell width in the inner epidermis of the glumes was significantly higher in Z414 than in Xihui 18 (+ 22.23%; Fig. 3g). There was no significant difference between Z414 and Xihui 18 in cell length of the inner glume epidermis (Fig. 3h). The total cell number in the outer epidermis of the glume along the longitudinal axis was significantly lower in Z414 than in Xihui 18 (− 13.52%; Fig. 3i). These results indicate that the short, wide grains of Z414 are mainly attributed to a decrease in glume cell number and an increase in glume cell width.

Fig. 3
figure 3

Scanning electron microscopy of Xihui 18 and Z414 glumes. af Scanning electron micrographs of the lemma (a, b), inner epidermis (c, d) and outer epidermis (e, f) of Xihui 18 (a, c, e) and Z414 (b, d, f) glumes. g, h, i show cell width, cell length, and total number of cells in the outer epidermis of the lemma at 200× magnification, respectively. ** and * indicate significant differences at the 0.01 and 0.05 level between Xihui 18 and Z414, respectively. Bars represent 10 mm in a and b, 500 µm in c and d, and 500 µm in e and f

Identification of QTL Using a Secondary F2 Population from Xihui 18/Z414

Seven QTL were identified for six traits that differed between Z414 and Xihui 18, which explained 7.00–42.50% of the phenotypic variation (Table 1). According to the setting of mapQTL, the positive genetic effect of each QTL indicated that the allele of Huhan 3 substitution segment of Z414 increased the phenotypic value, whereas the negative effect indicated that the allele of Xihui18 in substitution interval increased the phenotypic value. Thus, the additive effect of the allele qGW5 from a Huhan 3 substitution segment of Z414 increased grain width by 0.16 mm, and the dominant effect of qGW5/qgw5 was 0.0004, explaining 42.50% of the phenotypic variance. The additive effect of the qGWT5 allele from the Huhan 3 substitution segment of Z414 increased the 1000-grain weight by 1.25 g, and the dominant effect of qGWT5/qgwt5 was 0.15. Furthermore, qGW5, qRLW5, and qGWT5 were all linked to the same marker, RM5874. The additive of the allele qGL11 from Xihui 18 in the substitution interval increased grain length by 0.09 mm, and the dominant effect of qGL11/qgl11 was 0.012, explaining 9.61% of the phenotypic variance in grain length. Similarly, qGL11, qRLW11 and qBRR11 were all linked to the same marker, RM1812. However, the additive effects of alleles qGL11 and qRLW11 from Xihui 18 in the substitution interval increased the values of the corresponding traits, whereas the qBRR11 allele from the Huhan 3 substitution segment of Z414 increased the phenotypic value (Fig. 1, Table 1). In addition, the additive effect of the qPL3 allele from Xihui18 in the substitution interval increased panicle length of Z414 by 1.17 cm per panicle, and the dominant effect of qPL3/qpl3 was 0.59 (Table 1).

Table 1 QTL for agronomic and quality traits identified in substitution segments of Z414

Verification and Pyramiding of QTL Using the Newly Developed SSSLs and DSSLs

Based on the results of primary QTL mapping, we developed six SSSLs (S1–S6) and two DSSLs (D1 and D2) in the F3 population by marker-assisted selection (MAS). Among these, S3, S4, and S5 are SSSLs with overlapping substitution segments (Fig. 4a).

Fig. 4
figure 4

Additive and epistatic effects of QTL for related traits in the SSSLs and DSSLs. a Diagram of the locations of substitution segments and QTL in S1–S6 and D1 and D2. bf Parameters of QTL in different SSSLs and DSSLs, including grain length (b), degree of chalkiness (c), panicle length (d), grain width (e), 1000-grain weight (f), and ratio of grain length to width (g). Different lowercase letters indicate a significant difference (P < 0.05), as determined by Duncan’s multiple comparison. μ: the average value of each line; ai: additive effect for each QTL controlling the trait, whose positive value shows allele from substitution segment increasing phenotypic value, while negative value decreasing one. I: epistatic effect between QTL. P < 0.05in SSSL indicates that a QTL existed in the substitution segment of the SSSL, as determined by one-way ANOVA and LSD multiple comparison with Xihui 18; P < 0.05 in DSSL indicates that an epistatic effect of Q1 × Q2 existed in DSSL, as detected by two-way ANOVA. S1 (Chr. 3 RM3417 (6.1 Mb)–RM3766 (7.4 Mb)-RM14809 (11.7 Mb)–RM7425 (13.0 Mb)); S2 (Chr. 3 RM5813 (35.2 Mb)–RM3346 (37.6 Mb)–RM1221 (40.1 Mb)); S3 (Chr. 5 RM2010 (1.2 Mb)–RM405 (3.3 Mb)–RM5874 (3.7 Mb)); S4 (Chr. 5 RM2010 (1.2 Mb)–RM405( 3.3 Mb)-RM5874 (3.7 Mb)-RM3322 (4.4 Mb)–RM3328 (5.6 Mb)); S5 (Chr. 5 RM405 (3.3 Mb)–RM5874 (3.7 Mb)-RM3322 (4.4 Mb)–RM3328 (5.6 Mb)); S6 (Chr. 11 RM26038 (1.3 Mb)–RM26045 (1.6 Mb)-RM1812 (2.2 Mb)-RM26114 (2.8 Mb)–RM6085 (3.0 Mb)); D1 (Chr. 3 RM3417 (6.1 Mb)–RM3766 (7.4 Mb)-RM14809 (11.7 Mb)–RM7425 (13.0 Mb), Chr.3 RM5813 (35.2 Mb)–RM3346 (37.6 Mb)–RM1221 (40.1 Mb)); D2 (Chr. 3 RM3417 (6.1 Mb)–RM3766 (7.4 Mb)-RM14809 (11.7 Mb)–RM7425 (13.0 Mb), Chr. 11 RM26038 (1.3 Mb)–RM26045 (1.6 Mb)-RM1812 (2.2 Mb)-RM26114 (2.8 Mb)–RM6085 (3.0 Mb)); the internal markers connected with hyphens indicate the substitution segment from the donor, whereas the markers at each end of the substitution segment linked with ‘–’ indicate that segment recombination might have occurred

Six QTL (qPL3, qGW5, qGL11, qRLW5, qRLW11, and qGWT5) were identified in the SSSLs (Fig. 4a–f), indicating that these QTL are inherited stably. qBRR11 was not detected in S6, suggesting that the genetic effect of some minor QTL might be influenced by the environment; this QTL contributed only 7.00%. In addition, four QTL (qGL3, qGL5, qCD3, and qCD5) for grain length and degree of chalkiness were detected in S1 and S5 (Fig. 4b, c; Additional file 1) but were not detected in the secondary F2 population (Table 1). The reason for this might be genetic noise from the other three substitution segments in F2 individuals, which was cancelled out by SSSLs, meaning that QTL were detected more efficiently in SSSLs than in F2 plants.

In this section, the positive or negative additive effect (a) of a QTL detected in substitution line indicates increasing or decreasing phenotypic value for the allele from Huhan3 substitition segment compared with recipient Xihui18.

The grain lengths (9.66 and 9.55 mm, respectively) of S5, carrying qGL5 (a = − 0.26), and S6, carrying qGL11 (a = − 0.37), were significantly shorter than that of Xihui 18 (9.92 mm). By contrast, the grain length (10.35 mm) of S1, carrying qGL3 (a = 0.43), was significantly longer than that of Xihui 18 (9.92 mm), and the grain lengths of S2–S4, which lack QTL for grain length, were not significantly different from that of Xihui 18 (Fig. 4b; Additional file 1).

The degree of chalkiness (19.76%) of S1, harboring qCD3 (a = − 1.17), was significantly lower than that of Xihui 18 (20.93%), while the degree of chalkiness (24.02%) of S5, carrying qCD5 (a = 3.09), was significantly higher than that of Xihui 18 (20.93%). The degree of chalkiness of the other SSSLs (S2–S4 and S6) without QTL for this trait were not significantly different from that of Xihui 18 (Fig. 4c; Additional file 1).

The panicle length (25.49 cm) of S1 carrying qPL3 (a = − 1.53) was significantly shorter than that of Xihui 18 (27.03 cm), whereas the panicle lengths of S2–S6, which lacked QTL for this trait, were not significantly different from that of Xihui 18 (Fig. 4d; Additional file 1).

The grain widths (3.59 and 3.52 mm, respectively) of S4 and S5, harboring qGW5 (a = 0.47 and a = 0.40, respectively), were significantly larger than that of Xihui 18 (3.12 mm), while the grain widths of S1–S3, and S6, which lack QTL for grain width, were not significantly different from that of Xihui 18. qGW5 was localized to the same substitution interval (RM405–RM5874-RM3322–RM3328) of chromosome 5, based on substitution mapping (Fig. 4e; Additional file 1).

The 1000-grain weights (34.40 and 33.43 g, respectively) of S4 and S5, containing qGWT5 (a = 3.64 and a = 2.68, respectively), were significantly larger than that of Xihui 18 (30.75 g), whereas the 1000-grain weights of the other SSSLs (S1–S3 and S6), which lack QTL for this trait, were not significantly different from that of Xihui 18. qGWT5 was localized to the same substitution interval of chromosome 5 as qGW5 (Fig. 4f; Additional file 1).

The ratios of length to width (2.75, 2.75, and 2.99, respectively) of S4 and S5, carrying qRLW5 (both a = − 0.43), and S6, harboring qRLW11 (a = − 0.20), were significantly lower than that of Xihui 18 (3.19). By contrast, the ratios of length to width of S1–S3, which lack a QTL for this trait, were not significantly different from that of Xihui 18 (Fig. 4g; Additional file 1).

Pyramiding of qGL3 (a = 0.43) and qGL11 (a = − 0.37) yielded an epistatic effect of − 0.62, which resulted in a 0.56 mm reduction in grain length in D2. Pyramiding of qGL3 and qGL11 resulted in shorter grains than S6 (containing qGL11) (Table 2, Fig. 4b; Additional file 2), indicating that qGL11 is epistatic to qGL3 (Table 2). However, qGL3 (a = 0.43) and a substitution locus without a QTL for grain length on chromosome 3 in D1 showed independent inheritance. The grain length of D1 (10.25 mm) was not significantly different from that of S1 (10.35 mm), whereas these grains were significantly longer than those of Xihui 18 and S2 (Table 2, Fig. 4b, Additional file 2). Pyramiding two substitution loci without QTL for 1000-grain weight on chromosomes 3 and 11 in D2 produced an epistatic effect of − 4.88, resulting in a 4.88 g decrease in 1000-grain weight in D2. Thus, the 1000-grain weight of D2 (28.99 g) was significantly lower than that of S1, S6, and Xihui 18 (32.65, 31.98, and 30.75 g, respectively) (Fig. 4f; Additional file 2). All the other QTL in D1 and D2 were independently inherited (Table 2; Fig. 4c, d, e, g; Additional file 2).

Table 2 Epistasis between QTLs in DSSL detection by two-way ANOVA

Substitution Mapping and Candidate Gene Analysis of qGL11

Based on the above results, we further analyzed qGL11 using S6, whose estimated and maximum substitution lengths were 1.42 Mb and 1.66 Mb, respectively (Fig. 5a). For fine mapping of qGL11, we developed five novel secondary SSSLs (S7–S11) by crossing Xihui 18 with S6. Based on substitution mapping, qGL11 was delimited to an estimated substitution interval of 405 kb and a maximum length of 810 kb (Fig. 5a). Ninety genes were identified in this interval of 810 kb, including 40 genes with specific functional descriptions and 50 genes with poorly elucidated functions, such as expressed protein, or retrotransposon protein. Among 40 genes, LOC_Os11g05850 (CycT1;3) was selected as the candidate gene for qGL11 based on the signaling pathway regulating grain size (Li and Li 2016) and observations of the cell morphology of Z414 glumes. A comparison of the DNA sequences of Xihui 18 with S6 revealed six single nucleotide polymorphisms (SNPs) and a 25-base pair insertion in the 5′ untranslated region (UTR), one SNP in the 3′ UTR, and one SNP in the coding sequence that did not cause an amino acid change (Fig. 5b). Furthermore, the protein structure of CycT1;3 was predicted by SWISS-MODEL (https:/swissmodel.expasy.org/) (Arnold et al, 2006). There was no difference between S6 and Xihui 18 (Fig. 5c). However, the expression levels of LOC_Os11g05850 were significantly higher in the sheaths and panicles of S6 than in Xihui 18 (Fig. 5d). Thus, this suggests that LOC_Os11g05850 (CycT1;3) might be the gene responsible for qGL11.

Fig. 5
figure 5

Substitution mapping, sequence analysis, and relative expression level of qGL11 in Xihui 18 versus S6. a Substitution mapping of qGL11. Black regions indicate the estimated length of the substitution segment. b DNA sequence of CycT1;3 in S6 compared with Xihui 18. In the candidate gene sequence, the red box represents the coding sequence, the white boxes represent the 5′ UTR and 3′ UTR, the solid red lines represent introns, the black line in the gene sequence represents the mutation site, and the arrow represents a sequence change from Xihui18 to S6. c Protein structure of CycT1;3 predicted by SWISS-MODEL. d Relative expression levels of the candidate gene CycT1;3 in root, stem, leaf, sheath, and panicle tissue of Xihui 18 versus S6

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