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Wheat hybrids have been widely demonstrated to have remarkable heterosis or hybrid vigor in increasing yield potential and stability since the 1960s. Two-line hybrid wheat can achieve higher yields than local varieties, especially in marginal environments. However, the commercial application of hybrid wheat is hindered by higher seed costs, primarily due to lower yields in hybrid seed production. Stigma exsertion has been verified as a decisive factor in increasing rice's hybrid seed yield, but more investigation is needed in hybrid wheat breeding and production. In this study, four thermo-photo-sensitive genic male sterile lines, including K41S, K64S, K66S, and K68S, with different stigma exsertion rates (SERs) were used to compare the differences in floral architecture relative to stigma exsertion over two growing seasons. The results revealed that the K41S and K64S exhibited a relatively higher SER at 21.87% and 22.81%, respectively. No exserted stigma was observed in K66S, and K68S had an SER of only 0.82%. This study found that the stigma length, glume width and the length–width ratio of the glume were significantly correlated with the SER, with correlation coefficients of 0.46, −0.46 and 0.60, respectively. Other stigma features such as the branch angle, stretch width and hairbrush length, as well as the glume length, also had a weakly positive correlation with SER (r = 0.09–0.27). For K41S and K64S, the SER was significantly affected by the differences in the stigma branch angle and stigma stretch width among florets. A cross-pollination survey showed that the out-crossing ability of florets with an exserted stigma was about three times as high as that of florets with a non-exserted stigma. As a result, the stigma-exserted florets that accounted for 21.87% and 22.81% of the total florets in K41S and K64S produced 46.80% and 48.53% of the total cross-pollinated seeds in both sterile lines. These findings suggest that a longer stigma combined with a slender glume appears to be the essential floral feature of stigma exsertion in sterile wheat lines. It is expected that breeding and utilizing sterile lines with a higher SER would be a promising solution to cost-effective hybrid wheat seed production. [ABSTRACT FROM AUTHOR]

The stigma exsertion rate (SER) is one of the important factors affecting the seed production of hybrid rice. In the genus Oryza, wild rice species usually show higher SERs than cultivars. We previously identified one novel SER-QTL, qSERg-1b, from the single-segment substitution line (SSSL) SG22 of O. glumaepatula. In this study, qSERg-1b was fine-mapped by developing the secondary SSSLs (s-SSSLs) of SG22 and substitution mapping among the s-SSSLs. A total of 11 s-SSSLs were developed and used for qSERg-1b fine-mapping. As a result, qSERg-1b was narrowed down to the 122.59 kb physical region between InDel markers M01 and M49. There were 19 genes annotated in both O. glumaepatula and the HJX74 genomes at the qSERg-1b interval. Based on the genome re-sequencing of SG22 and HJX74, and the expression levels of the annotated functional genes, multiple nucleotide variations were found in LOC_Os01g53630, LOC_Os01g53680, and LOC_Os01g53710, including base substitutions and insertions/deletions of the genes, and they also showed significant differences in expression levels between SG22 and HJX74. Therefore, LOC_Os01g53630, LOC_Os01g53680, and LOC_Os01g53710 were selected as the most possible candidate genes of qSERg-1b for further validation. The above results laid an important foundation for qSERg-1b cloning and provided valuable resources for molecular breeding of SER in rice. [ABSTRACT FROM AUTHOR]

Wheat hybrids have been widely demonstrated to have remarkable heterosis or hybrid vigor in increasing yield potential and stability since the 1960s. Two-line hybrid wheat can achieve higher yields than local varieties, especially in marginal environments. However, the commercial application of hybrid wheat is hindered by higher seed costs, primarily due to lower yields in hybrid seed production. Stigma exsertion has been verified as a decisive factor in increasing rice’s hybrid seed yield, but more investigation is needed in hybrid wheat breeding and production. In this study, four thermo-photo-sensitive genic male sterile lines, including K41S, K64S, K66S, and K68S, with different stigma exsertion rates (SERs) were used to compare the differences in floral architecture relative to stigma exsertion over two growing seasons. The results revealed that the K41S and K64S exhibited a relatively higher SER at 21.87% and 22.81%, respectively. No exserted stigma was observed in K66S, and K68S had an SER of only 0.82%. This study found that the stigma length, glume width and the length–width ratio of the glume were significantly correlated with the SER, with correlation coefficients of 0.46, −0.46 and 0.60, respectively. Other stigma features such as the branch angle, stretch width and hairbrush length, as well as the glume length, also had a weakly positive correlation with SER (r = 0.09–0.27). For K41S and K64S, the SER was significantly affected by the differences in the stigma branch angle and stigma stretch width among florets. A cross-pollination survey showed that the out-crossing ability of florets with an exserted stigma was about three times as high as that of florets with a non-exserted stigma. As a result, the stigma-exserted florets that accounted for 21.87% and 22.81% of the total florets in K41S and K64S produced 46.80% and 48.53% of the total cross-pollinated seeds in both sterile lines. These findings suggest that a longer stigma combined with a slender glume appears to be the essential floral feature of stigma exsertion in sterile wheat lines. It is expected that breeding and utilizing sterile lines with a higher SER would be a promising solution to cost-effective hybrid wheat seed production.

The stigma exsertion rate (SER) is one of the important factors affecting the seed production of hybrid rice. In the genus Oryza, wild rice species usually show higher SERs than cultivars. We previously identified one novel SER-QTL, qSERg-1b, from the single-segment substitution line (SSSL) SG22 of O. glumaepatula. In this study, qSERg-1b was fine-mapped by developing the secondary SSSLs (s-SSSLs) of SG22 and substitution mapping among the s-SSSLs. A total of 11 s-SSSLs were developed and used for qSERg-1b fine-mapping. As a result, qSERg-1b was narrowed down to the 122.59 kb physical region between InDel markers M01 and M49. There were 19 genes annotated in both O. glumaepatula and the HJX74 genomes at the qSERg-1b interval. Based on the genome re-sequencing of SG22 and HJX74, and the expression levels of the annotated functional genes, multiple nucleotide variations were found in LOC_Os01g53630, LOC_Os01g53680, and LOC_Os01g53710, including base substitutions and insertions/deletions of the genes, and they also showed significant differences in expression levels between SG22 and HJX74. Therefore, LOC_Os01g53630, LOC_Os01g53680, and LOC_Os01g53710 were selected as the most possible candidate genes of qSERg-1b for further validation. The above results laid an important foundation for qSERg-1b cloning and provided valuable resources for molecular breeding of SER in rice.

[Objectives]Stigma exsertion rate is an important determinant of outcrossing in hybrid rice seed production. The purpose of this study was to detect quantitative trait loci (QTL)controlling stigma exsertion rate and fine mapping related genes in rice. [Methods]In this study, an F2 population derived from japonica rice variety ‘02428' with low stigma exsertion rate and parent ZH464 with high stigma exsertion rate was used for preliminary detection of QTL. Molecular markers were used to screen heterozygous individual plants in the target region to obtain F2:3 and F3:4 populations by continuous selfing for multi-year stable QTL mapping. At the same time, BSA-seq technology was used to analyze gene mapping and candidate gene sequence of the target region. [Results] The results of correlation analysis showed that there was a very significant correlation among the three traits of single stigma exsertion rate, double stigma exsertion rate and total stigma exsertion rate. In this study, the total stigma exsertion rate was used as phenotypic data, and the constructed genome-wide molecular linkage genetic map was used for QTL mapping. Two loci, qTSE2 and qTSE4, were detected on chromosomes 2 and 4, respectively. qTSE4 with high contribution rate was selected as the target locus for follow-up study. This locus was repeatedly detected in the F2:3 and F3:4 planting population. Based on BSA-seq result, two genes with differences were screened in the qTSE4 locus interval. [Conclusions] qTSE4 locus as the major QTL related to stigma exsertion rate, can be used for subsequent fine-mapping. The two screened candidate genes need to be further verified by transgenic experiment. [ABSTRACT FROM AUTHOR]

Summary: Stigma exsertion rate (SER) of the male sterile line is a key limiting factor for hybrid seed production in rice. Although a large number of quantitative trait loci associated with SER have been reported, few genes have been molecularly cloned and functionally characterized, severely hindering the genetic improvement of SER of the male sterile line and the breeding efficiency of hybrid rice. In this study, we identified three grain shape regulatory genes, GS3, GW8 and GS9, as potential candidate genes for targeted manipulation of grain shape and SER. We show that simultaneously knocking out these three genes could effectively increase SER by increasing the ratio of spikelet length/spikelet width and length of stigma and style, without negative impacts on other agronomic traits. Cellular examination and transcriptomic analyses revealed a role of these genes in coordinated regulation of transverse and longitudinal cell division in the pistils. Moreover, we demonstrate that targeted manipulation of these grain shape genes could significantly improve the outcrossing rate in both the ZH11 (a japonica variety) and Zhu6S (an indica male sterile line) backgrounds. Our results provide new insights into the mechanisms of rice SER regulation and develop an effective strategy to improve SER and out‐crossing rate in rice, thus facilitating hybrid rice production. [ABSTRACT FROM AUTHOR]

The stigma exsertion rate (SER) is a complex agronomy phenotype controlled by multiple genes and climate and a key trait affecting the efficiency of hybrid rice seed production. Using a japonica two-line male sterile line (DaS) with a high SER as the donor and a tropical japonica rice (D50) with a low SER as the acceptor to construct a near-isogenic line [NIL (qSE4DaS)]. Populations were segregated into 2,143 individuals of BC3F2 and BC4F2, and the stigma exsertion quantitative trait locus (QTL) qSE4 was determined to be located within 410.4 Kb between markers RM17157 and RM17227 on chromosome 4. Bioinformatic analysis revealed 13 candidate genes in this region. Sequencing and haplotype analysis indicated that the promoter region of LOC_Os04g43910 (ARF10) had a one-base substitution between the two parents. Further Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis showed that the expression level of ARF10 in DaS was significantly higher than in D50. After knocking out ARF10 in the DaS background, it was found that the SER of arf10 (the total SER of the arf10- 1 and the arf10-2 were 62.54 and 66.68%, respectively) was significantly lower than that of the wild type (the total SER was 80.97%). Transcriptome and hormone assay analysis showed that arf10 had significantly higher auxin synthesis genes and contents than the wild type and the expression of auxin signaling-related genes was significantly different, Similar results were observed for abscisic acid and jasmonic acid. These results indicate that LOC_Os04g43910 is mostly likely the target gene of qSE4, and the study of its gene function is of great significance for understanding the molecular mechanisms of SER and improving the efficiency of hybrid seed production. [ABSTRACT FROM AUTHOR]

Abstract Background Stigma exsertion rate (SER) is a key determinant of outcrossing in hybrid rice seed production. A quantitative trait locus (QTL) for stigma exsertion rate in rice, qSER-7, has previously been detected on chromosome 7 by using a F2 population derived from two indica cytoplasmic male sterility (CMS) maintainers, Huhan 1B and II-32B. Results The chromosomal location of qSER-7 was precisely delimited by fine-scale mapping. Near-isogenic lines (NILs) were established, one of which isolated the locus in the qSER-7 II-32B line, which contains an introgressed segment of II-32B in the Huhan 1B genetic background, and exhibits a significantly higher stigma exsertion rate than that of the recurrent parent. Using 3192 individuals from the BC4F2 segregation population, the QTL qSER-7 was narrowed down to a 28.4-kb region between the markers RM3859 and Indel4373 on chromosome 7. According to the rice genome annotation database, three genes were predicted within the target region. Real-time PCR analysis showed significantly higher expression levels of LOC_Os07g15370 and LOC_Os07g15390 in II-32B than in Huhan 1B. LOC_Os07g15370(OsNRAMP5) was a previously reported gene for Mn and Cd transporter. The stigma exertion rates of OsNRAMP5-overexpressing plants were significantly higher than that of wild type plants, in contrast, a T-DNA insertion mutant osnramp5 showed a lower stigma exertion rate. Conclusions In the present study, the QTL qSER-7 was isolated to a region between the markers RM3859 and Indel4373. Two candidate genes were selected based on the expression difference between the two parents, which can facilitate the further cloning of the gene underlying the quantitative trait associated with qSER-7 as well as the marker-assisted transfer of desirable genes for stigma exsertion rate improvement in rice.

The efficiency of hybrid rice seed production can be improved by increasing the percentage of exserted stigmas. To identify quantitative trait loci (QTLs) for this trait, we conducted QTL mapping using 75 chromosome segment substitution lines (CSSLs) developed from a cross between the donor parent, Xieqingzao B (XQZB), a maintainer line which has high stigma exsertion and the recurrent parent, Zhonghui 9308 (ZH9308), a restorer line which has low stigma exsertion. A total of nine QTLs (qSSE5, qSSE10, qSSE11, qDSE10, qDSE11, qTSE5, qTSE6, qTSE10, and qTSE11) for single stigma exsertion (SSE), dual stigma exsertion (DSE) and total stigma exsertion (TSE) were assessed in two environments (Hainan and Zhejiang). Six of these QTLs (qSSE10, qSSE11, qDSE10, qDSE11, qTSE10, and qTSE11) were found in both environments, while one QTL (qTSE6) was found in only Hainan, and two QTLs (qSSE5 and qTSE5) were found in only Zhejiang. The qSSE10, qSSE11, qDSE10, qDSE11, qTSE6, qTSE10, and qTSE11 alleles, which are derived from the parent XQZB, exhibited a positive additive effect. In contrast, the qSSE5 and qTSE5 alleles, which are derived from the parent ZH9308, exhibited a negative additive effect. The SSE, DSE and TSE traits were significantly correlated with each other in an environmentally dependent manner. These results indicated that the lines showing higher values for SSE were more likely to exhibit increased values for DSE, which would ultimately increase TSE. To evaluate the advantage of exserted stigmas for cross-pollination, single, dual and total stigma exsertion should be considered separately in future attempts at genetic improvement to achieve increased production of rice hybrid seeds. This study also provides information for fine mapping, gene cloning and particularly marker-assisted selection (MAS), on the latter and with an emphasis the phenotypic effects and implications of the QTLs for practical use in hybrid rice breeding.

The rate of stigma exsertion (SE) is an important trait in rice breeding because the efficiency of hybrid rice seed production can be improved by increasing the percentage of stigmas that exsert. In this study, we developed a near isogenic line (NIL) from two parents, XieqingzaoB (XQZB) and Zhonghoi9308 (ZH9308), which have high and low SE rates in that order. In our previous study, we employed 75 chromosome segment substitution lines (CSSLs) and analyzed quantitative trait loci (QTLs) for their influence on SE rate. The single gene QTL (qSE11), which is located on chromosome 11, was responsible for this trait. In this study, we focused on one of the CSSLs (C65), namely, the NIL (qSE11XB). It contains an introgression segment of XQZB in the genetic background of ZH9308, and exhibits a significantly higher SE rate than that of the parents. We demonstrated that qSE11 regulated both the single and the dual SE rates. They both contribute to the total SE rate. Genetic analysis revealed that qSE11 acted as a single Mendelian factor and that the allele from XQZB increased the SE rate. The validity of our conclusions was established when C65 was used to develop secondary F2 (BC5F2) and F2:3 (BC5F2:3) populations by backcrossing to ZH9308, with subsequent selfing. We entered 3600 plants from the F2 population and 3200 from the F2:3 populations into a genetic dissection program and dissected the major QTL qSE11 to a 350.7-kb region located on chromosome 11. This study will contribute to the future isolation of candidate genes of SE and will play a vital role in future hybrid rice seed production programs.

The rate of stigma exsertion (SE) is an important trait in rice breeding because the efficiency of hybrid rice seed production can be improved by increasing the percentage of stigmas that exsert. In this study, we developed a near isogenic line (NIL) from two parents, XieqingzaoB (XQZB) and Zhonghoi9308 (ZH9308), which have high and low SE rates in that order. In our previous study, we employed 75 chromosome segment substitution lines (CSSLs) and analyzed quantitative trait loci (QTLs) for their influence on SE rate. The single gene QTL (qSE11), which is located on chromosome 11, was responsible for this trait. In this study, we focused on one of the CSSLs (C65), namely, the NIL (qSE11XB). It contains an introgression segment of XQZB in the genetic background of ZH9308, and exhibits a significantly higher SE rate than that of the parents. We demonstrated that qSE11 regulated both the single and the dual SE rates. They both contribute to the total SE rate. Genetic analysis revealed that qSE11 acted as a single Mendelian factor and that the allele from XQZB increased the SE rate. The validity of our conclusions was established when C65 was used to develop secondary F2 (BC5F2) and F2:3 (BC5F2:3) populations by backcrossing to ZH9308, with subsequent selfing. We entered 3600 plants from the F2 population and 3200 from the F2:3 populations into a genetic dissection program and dissected the major QTL qSE11 to a 350.7-kb region located on chromosome 11. This study will contribute to the future isolation of candidate genes of SE and will play a vital role in future hybrid rice seed production programs. [ABSTRACT FROM AUTHOR]

Stigma exsertion rate is a key factor which influences the efficiency of hybrid seed production in rice. To clarify the genetic basis of stigma exsertion rate, a recombinant inbred line population was developed from two superior rice cultivars, Zhongguo Xiangdao (ZX) and Chuanxiang 29B (CX29B), and a total of 11 quantitative trait loci (QTLs) for stigma exsertion rate were detected over 2 years. Two QTLs on chromosome 1 and four QTLs on chromosome 6 formed two QTL cluster regions, qSe1 and qSe6, respectively. To further validate the effect of qSe1 and qSe6, the near-isogenic lines (NILs) of the two QTL cluster regions were constructed with the genetic background of CX29B. Compared to CX29B, CX29B ( qSe1), the NIL carrying homozygous qSe1 regions from ZX, showed a dual stigma exsertion rate increased by 18.35 % and a single stigma exsertion rate increased by 16.24 %; in contrast, CX29B ( qSe6), the NIL carrying homozygous qSe6 regions from ZX, showed a dual stigma exsertion rate decreased by 16.87 % and a single stigma exsertion rate decreased by 20.47 %. Therefore, the qSe1 region from ZX and the qSe6 region from CX29B have positive effects on improvement of stigma exsertion rate and would be very useful in rice breeding. [ABSTRACT FROM AUTHOR]

The stigma exsertion rate (SER) is a complex agronomy phenotype controlled by multiple genes and climate and a key trait affecting the efficiency of hybrid rice seed production. Using a japonica two-line male sterile line (DaS) with a high SER as the donor and a tropical japonica rice (D50) with a low SER as the acceptor to construct a near-isogenic line [NIL ( qSE4 DaS )]. Populations were segregated into 2,143 individuals of BC 3 F 2 and BC 4 F 2 , and the stigma exsertion quantitative trait locus (QTL) qSE4 was determined to be located within 410.4 Kb between markers RM17157 and RM17227 on chromosome 4. Bioinformatic analysis revealed 13 candidate genes in this region. Sequencing and haplotype analysis indicated that the promoter region of LOC_Os04g43910 ( ARF10 ) had a one-base substitution between the two parents. Further Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis showed that the expression level of ARF10 in DaS was significantly higher than in D50. After knocking out ARF10 in the DaS background, it was found that the SER of arf10 (the total SER of the arf10-1 and the arf10-2 were 62.54 and 66.68%, respectively) was significantly lower than that of the wild type (the total SER was 80.97%). Transcriptome and hormone assay analysis showed that arf10 had significantly higher auxin synthesis genes and contents than the wild type and the expression of auxin signaling-related genes was significantly different, Similar results were observed for abscisic acid and jasmonic acid. These results indicate that LOC_Os04g43910 is mostly likely the target gene of qSE4 , and the study of its gene function is of great significance for understanding the molecular mechanisms of SER and improving the efficiency of hybrid seed production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guo, Wang, Chen, Tang, An, Wei, Hu, Tang, Shao, Jiao, Xie, Wang, Sheng and Hu.)