Background Rice farming faces a significant challenge from the brown planthopper (BPH), a destructive pest that threatens crop yields. Developing BPH-resistant rice varieties is critical for ensuring food security. The pyramiding of BPH resistance genes, BPH14 and BPH15, has proven effective in providing protection in elite rice strains. MicroRNAs (miRNAs) play a pivotal role in plant defense by fine-tuning resistance responses through the modulation of genes involved in various signaling pathways and metabolic processes. B1415, the pyramiding line containing BPH14 and BPH15, exhibits stronger BPH resistance compared to its recurrent parent, Wushansimiao, without affecting other important agronomic traits. However, the molecular basis underlying the resistance conferred by the BPH14/BPH15 pyramiding rice remains largely unclear, particularly with respect to the potential regulatory role of miRNAs. Studying miRNAs in resistance gene pyramiding lines like B1415 is essential for advancing rice breeding efforts, as such research can uncover regulatory networks that enhance pest resistance and identify miRNA-mRNA interactions as potential targets for genetic manipulation. Results The study investigated miRNA levels in B1415 and their recurrent parent (RP) under BPH infestation employing high-throughput sequencing and revealed 136 differentially expressed miRNAs (DEMs) among 550 known miRNAs. An integrated analysis highlighted that 587 miRNA-mRNA pairs linking 95 DEMs to 537 targeted genes were enriched in phenylpropanoid and lignin metabolism, circadian rhythms, and amino acid metabolism. The candidate DEMs, miR172d-3p, and miR396 family members were identified as negative regulators to decrease their target genes Os06g0708700 (encoding a nodulin-like protein) and Os11g0129700 (encoding an AP2 domain transcription factor), suggesting their key roles in rice against BPH. Conclusions Our investigation provides the first insights into miRNA-mediated defense mechanisms in the B1415. Identifying miRNAs and their target mRNAs in BPH resistance opens a new avenue for rice breeding programs, offering potential targets for improving pest resistance. Understanding these molecular interactions paves the way for developing more resistant rice cultivars, thereby contributing to sustainable rice production and food security.

Rice, a vital crop, faces significant threats from the brown planthopper (BPH), which impacts plant growth and yield. Pyramiding the BPH resistance genes BPH14 and BPH15 provides rice crops with reliable and lasting protection against BPH. Nonetheless, current research lacks clarity on the molecular processes responsible for BPH14/BPH15-mediated resistance to BPH. In this study, utilizing high-throughput metabolomics and integrating transcriptomic data, we investigated the metabolic adaptations of the BPH14/BPH15 pyramiding line (B1415) and its recurrent parent (RP) during early and late infestation stages. The analysis identified 1007 metabolites, mainly consisting of lipids and lipid-like molecules, together with phenylpropanoid and polyketide classes. Differentially accumulated metabolites (DAMs) displayed different patterns in B1415 and RP, particularly in flavonoid and phenylpropanoid biosynthesis pathways, which were more pronounced in the resistant B1415. Furthermore, ferulic acid (FA) was found to negatively regulate BPH resistance. These findings elucidate critical metabolic pathways involved in rice defense mechanisms and underscore the potential of B1415’s enhanced metabolic responses in conferring durable resistance against BPH.

Although rice has many pests, brown planthopper (BPH) in particular is known to cause substantial damage. The pyramiding application of BPH-resistance genes BPH14 and BPH15 has proven effective in enhancing rice defense against BPH. However, the molecular mechanisms underlying BPH14/BPH15-conferred resistance remain unexplained. In this investigation, we analyzed the transcriptomes of near isogenic lines (NILs) containing either BPH14 (B14), BPH15 (B15), or BPH14/BPH15 (B1415), as well as their recurrent parent (RP) 'Wushansimiao'. In total, we detected 14,492 differentially expressed genes (DEGs) across 12 mRNA profiles of resistant NILs and RP at different feeding stages. In the transcriptomic analysis, 531 DEGs appeared to be common among the resistant NILs compared to RP before and after BPH feeding. These common DEGs were enriched in defense response, phosphorylation, and salt stress response. In addition, 258 DEGs shared only in resistant NILs were obtained among the different feeding stages, which were enriched in oxidative stress response, karrikin response, and chloroplast organization. Considering the expression patterns and relevant research reports associated with these DEGs, 21 were chosen as BPH resistance candidates. In rice protoplasts, the candidate DEG OsPOX8.1 was confirmed to increase reactive oxygen species (ROS) accumulation by chemiluminescence measurement. Our results provide valuable information to further explore the defense mechanism of insect-resistant gene pyramiding lines and develop robust strategies for insect control.

Knowledge of rice (Oryza sativa L.) genes and various DNA markers can be used in genomic breeding programs aimed at developing improved elite rice cultivars. We used an efficient genomic breeding approach to pyramid four resistance genes (Pi2, Xa23, Bph14, and Bph15) in the popular photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice line Feng39S. We performed foreground selection for the target genes, followed by recombinant selection and background selection. This process reduced the sizes of the genomic segments harboring the target genes (566.8 kb for Pi2, 1143.9 kb for Xa23, 774.7 kb for Bph14, and 1574.9 kb for Bph15) and accelerated the recovery of the recurrent parent genome to proportions ranging from 98.77% to 99.16%, thus resulting in four near-isogenic lines. To assemble the four resistance genes in Feng39S, we performed a double-way cross combined with foreground and background selection to generate two improved lines of Feng39S (Pi2 + Xa23 + Bph14 + Bph15) with a recurrent parent genome recovery of 98.98%. The two lines showed agronomic performance, grain quality, and fertility–sterility transition characteristics similar to those of the original Feng39S line. The newly developed PTGMS lines and corresponding hybrid combinations were resistant to various field blast isolates and seven representative isolates of bacterial blight. At the seedling stage, the lines also showed resistance against brown planthopper. This study provides an efficient and accurate genomic breeding approach for introducing desirable traits into PTGMS lines.

华两优2809是孝感市农业科学院、武汉楚禾汇科技有限公司、湖北工程学院用两系不育系华1228S与恢复系R409配组育成的两系杂交中稻新组合.该组合高产稳产,对稻瘟病、白叶枯病、纹枯病抗性强,适应性广,2021年7月通过湖北省农作物品种审定委员会审定.

The number of grains per panicle is one of the most important factors of rice yield and determinated by branch and spikelet number. Genetic and molecular mechanisms responsible for panicle branching and spikelet development remain unclear in rice. Here, we report a mutant lax2-4 mainly showing lax panicle, longer grains, a little abnormal spikelet, lower setting rate and abaxial curled leaf. Genetic analysis indicated that the defective panicle phenotype of lax2-4 was controlled by single recessive gene. Further mapping and re-sequencing revealed that the fourth exon of LAX2 was lost caused by an approximately 2-Mb INV in lax2-4, thus LAX2-4 could be a new allele of LAX2. Overexpression assays showed that lax panicle was rescued in correspondence with the gradually increased expression level of wild-type LAX2-4 transcript. CRISPR/Cas9-mediated knockout of the fourth exon verified that LAX2-4 participates the regulation of sterile lemma and lemma development in rice. LAX2-4 could interact with an E-class gene, OsMADS1, and the fourth exon was necessary for its function. Collectively, we proposed that LAX2-4 plays a dual role in lateral spikelet and lemma development.

华两优2824是以荆州农业科学院为主选育的两系杂交中稻新品种,该品种具有产量高、生育期短、抗病性强的特点;栽培上要做到培育壮秧、合理密植、科学运筹肥水、合理防治病虫害.

为了提高常规籼稻品种'黄华占'和'五山丝苗'的褐飞虱抗性,采用杂交、回交和分子标记辅助选择技术,将褐飞虱抗性基因Bph14和Bph15分别导入到'黄华占'和'五山丝苗'的遗传背景中,选育出了具有'黄华占'和'五山丝苗'遗传背景,同时携带纯合Bph14和Bph15基因的新株系各2个.苗期褐飞虱抗性鉴定结果表明,选出的新株系均表现为抗褐飞虱.产量比较试验、性状考查和米质分析结果表明,新株系的产量、主要性状和稻米品质指标都与受体亲本相似,因此可以作为'黄华占'和'五山丝苗'的抗褐飞虱新品系进一步进行多点试验和区域试验.

Heterosis of grain yield is closely associated with heading date in crops. Gene combinations of the major heading date genes Ghd7, Ghd8, and Hd1 play important roles in enhancing grain yield and adaptation to ecological regions in rice. However, the predominant three-gene combinations for a specific ecological region remain unclear in both three-line and two-line hybrids. In this study, we sequenced these three genes of 50 cytoplasmic male sterile/maintainer lines, 31 photo-thermo-sensitive genic male sterile lines, and 109 restorer lines. Sequence analysis showed that hybrids carrying strong functional alleles of Ghd7 and Hd1 and non-functional Ghd8 are predominant in three-line hybrids and are recommended for rice production in the subtropics around 30°N/S. Hybrids carrying strong functional Ghd7 and Ghd8 and non-functional Hd1 are predominant in two-line hybrids and are recommended for low latitude areas around 23.5°N/S rich in photothermal resources. Hybrids carrying strong functional Ghd7 and Ghd8 and functional Hd1 were not identified in commercial hybrids in the middle and lower reaches of the Yangtze River, but they have high yield potential in tropical regions because they have the strongest photoperiod sensitivity. Based on these findings, two genic sterile lines, Xiangling 628S and C815S, whose hybrids often head very late, were diagnosed with these three genes, and Hd1 was targeted to be knocked out in Xiangling 628S and replaced with hd1 in C815S. The hybrids developed from both modified sterile lines in turn had appropriate heading dates and significantly improved grain yield. This study provides new insights for breeding design to develop hybrids for various regions.

香5是由湖北省农科院选育的优质两系杂交稻恢复系,所配组合广两优5号(广占63-4S/香5)于2013年通过了湖北省审定.利用回交和分子标记辅助选择技术,将供体亲本MD12086-1351中的抗稻瘟病基因Pi9、抗褐飞虱基因Bph14、Bph15和抗白叶枯病基因Xa23渗入到香5背景中,育成了3个同时携带Pi9、Bph14、Bph15和Xa23基因的新株系.鉴定结果表明,新株系的叶瘟抗性明显提高,穗颈瘟抗性部分提高,苗期抗褐飞虱,分蘖盛期高抗白叶枯病;产量、主要农艺性状、香味和稻米品质主要指标与香5相似.新株系所配的组合在产量、主要农艺性状上与香5所配的组合相似.表明新株系可以作为香5的替代系用于培育抗稻瘟病、抗褐飞虱和抗白叶枯病的两系杂交稻新组合.

Background Rice blast caused by Magnaporthe oryzae is one of the most widespread biotic constraints that threaten rice production. Using major resistance genes for rice blast resistance improvement is considered to be an efficient and technically feasible approach to achieve optimal grain yield. Results We report here the introgression of the broad-spectrum blast resistance gene Pi2 into the genetic background of an elite PTGMS line, Feng39S, for enhancing it and its derived hybrid blast resistance through marker-assisted backcrossing (MABC) coupled with genomics-based background selection. Two PTGMS lines, designated as DB16206–34 and DB16206–38, stacking homozygous Pi2 were selected, and their genetic background had recurrent parent genome recovery of 99.67% detected by the SNP array RICE6K. DB16206–34 and DB16206–38 had high resistance frequency, with an average of 94.7%, when infected with 57 blast isolates over 2 years, and the resistance frequency of their derived hybrids ranged from 68.2% to 95.5% under inoculation of 22 blast isolates. The evaluation of results under natural blast epidemic field conditions showed that the selected PTGMS lines and their derived hybrids were resistant against leaf and neck blast. The characterizations of the critical temperature point of fertility-sterility alternation of the selected PTGMS lines, yield, main agronomic traits, and rice quality of the selected PTGMS lines and their hybrids were identical to those of the recurrent parent and its hybrids. DB16206–34/9311 or DB16206–38/9311 can be used as a blast-resistant version to replace the popular hybrid Fengliangyou 4. Likewise, DB16206–34/FXH No.1 or DB16206–38/FXH No.1 can also be used as a blast-resistant version to replace another popular hybrid Fengliangyou Xiang 1. Conclusions Our evaluation is the first successful case to apply MABC with genomics-based background selection to improve the blast resistance of PTGMS lines for two-line hybrid rice breeding.

Background Thousand grain weight is a key component of grain yield in rice, and a trait closely related to grain length (GL) and grain width (GW) that are important traits for grain quality. Causal genes for 16 quantitative trait loci (QTL) affecting these traits have been cloned, but more QTL remain to be characterized for establishing a genetic regulating network. A QTL controlling grain size in rice, qGS10 , was previously mapped in the interval RM6100–RM228 on chromosome 10. This study aimed to delimitate this QTL to a more precise location. Method A total of 12 populations were used. The ZC9 population comprised 203 S 1:2 families derived from a residual heterozygous (RH) plant in the F 9 generation of the indica rice cross Teqing (TQ)/IRBB52, segregating the upper region of RM6100–RM228 and three more regions on chromosomes 1, 9, and 11. The Ti52-1 population comprised 171 S 1 plants derived from one RH plant in F 7 of TQ/IRBB52, segregating a single interval that was in the lower portion of RM6100–RM228. The other ten populations were all derived from Ti52-1, including five S 1 populations with sequential segregating regions covering the target region and five near isogenic line (NIL) populations maintaining the same segregating pattern. QTL analysis for 1,000-grain weight, GL, and GW was performed using QTL IciMapping and SAS procedure GLM. Result Three QTL were separated in the original qGS10 region. The qGL10.1 was located in the upper region RM6704–RM3773, shown to affect GL only. The qGS10.1 was located within a 207.1-kb interval flanked by InDel markers Te20811 and Te21018, having a stable and relatively high effect on all the three traits analyzed. The qGS10.2 was located within a 1.2-Mb interval flanked by simple sequence repeat markers RM3123 and RM6673. This QTL also affected all the three traits but the effect was inconsistent across different experiments. QTL for grain size were also detected in all the other three segregating regions. Conclusion Three QTL for grain size that were tightly linked on the long arm of chromosome 10 of rice were separated using NIL populations with sequential segregating regions. One of them, qGS10.1 , had a stable and relatively high effect on grain weight, GL, and GW, providing a good candidate for gene cloning. Another QTL, qGS10.2 , had a significant effect on all the three traits but the effect was inconsistent across different experiments, providing an example of genotype-by-environmental interaction.

Though inter-subspecific hybrids from indica and japonica rice have gained great success for yield increases over the recent decade, there are severe sterility problems in such hybrids. To overcome hybrid sterility, we previously obtained the line PL-(S5-n + f5-n) in the genetic background of an elite indica restorer line 9311, via breeding strategy for construction of wide compatibility line, which was proven to be an effective strategy in improving the seed setting rate of indica-japonica hybrid rice. Here, a novel strategy for developing japonica-compatible indica lines was employed by introducing the japonica allele pf12-j into 9311. The obtained NIL-(pf12-j) could significantly improve pollen and embryo-sac fertility by 29.7% and 28.6% in indica-japonica hybrids, thus leading to 20.9% improvement of spikelet fertility. In addition, we achieved the pyramiding line PL-(S5-n + f5-n + pf12-j) by a combinational strategy using both wide compatibility line and japonica-compatible indica line. Compared to PL-(S5-n + f5-n) with 38.9% increase of spikelet fertility, the pyramiding line PL-(S5-n + f5-n + pf12-j) showed 49.7% increase of spikelet fertility, suggesting cumulative effect of wide compatibility alleles S5-n + f5-n and japonica allele pf12-j to shape normal fertility of inter-subspecies hybrid. Interestingly, these lines also showed compatibility to indica. Hence, our results demonstrate that the two strategies could be simultaneously applied for indica-japonica hybrid breeding, and S5-n + f5-n + pf12-j are the optional allelic combination for overcoming hybrid sterility. This finding will greatly enhance our understanding for breeding indica-japonica hybrid rice by molecular-assisted selection strategy.

R1813是湖南隆平种业有限公司选育的高产、抗倒、迟熟优良籼型两系恢复系,以R1813为父本,已经选配出一批组合通过审定.该研究利用分子标记辅助选择和回交转育技术,将MD12086-41携带的抗稻瘟病基因Pi9、抗白叶枯病基因Xa23、抗褐飞虱基因Bph14和Bph15渗入到R1813的背景中,选出3个携带Pi9、Xa23、Bph14和Bph15基因的近等基因系ST15005-1-106-9、ST15005-1-318-4和ST15005-1-318-14.抗性鉴定结果表明,这3个改良株系均表现抗稻瘟病、高抗白叶枯病和中抗褐飞虱,与R1813相比,3种病虫害的抗性都明显提高.田间试验和品质分析表明,3个改良株系的产量、主要农艺性状和稻米品质主要指标都与R1813相似.对少量测配组合的抗性鉴定、田间试验和品质分析表明,除了抗性有明显提高以外,产量、主要农艺性状、稻米品质主要指标都与R1813所配组合相似.

Rice source- and sink-associated traits are important for grain yield and are sensitive to environmental conditions. The continuing increase of CO2 concentrations in the atmosphere will become a major challenge for rice growth and development in the future due to changes in our climate such as extremes in temperature. To guarantee food safety, novel genetic loci need to be identified for source- and sink-associated traits that are specifically expressed under elevated CO2 conditions. Eighty chromosome segment substitution lines carrying japonica (Nipponbare) chromosome segments in the indica (9311) background were used in this study. QTL analysis was conducted for source- and sink-related traits, including flag leaf length, flag leaf width, flag leaf fresh weight, flag leaf dry weight, primary branch number, secondary branch number, grain number per panicle, panicle weight per plant, chlorophyll a, chlorophyll b, and carotenoid contents, under ambient CO2 concentrations and free-air CO2 enrichment. A total of 49 QTLs for these traits were detected on chromosomes 1, 3, 5, 6, 7, 9, and 12 under the two conditions; the variance explained by these QTLs varied from 6.22 to 38.15%. Among these QTLs, 19 of them were detected under the natural field conditions and 30 were detected in the elevated CO2 conditions. In addition, 2 and 13 QTLs were specifically expressed in the natural and CO2-enriched conditions, respectively. Our findings have important implications on the utilization of germplasm resources for ensuring food security under elevated CO2 levels, especially for QTLs that were specifically detected under the elevated CO2 condition.

Grain weight is one of the most important determinants of grain yield in rice. In this study, QTL analysis for grain weight, grain length, and grain width was performed using populations derived from crosses between major parental lines of three-line indica hybrid rice. A total of 27 QTL for grain weight were detected using three recombinant inbred line populations derived from the crosses Teqing/IRBB lines, Zhenshan 97/Milyang 46, and Xieqingzao/Milyang 46. Of these, 10 were found in only a single population and the other 17 in two or all three populations. Nine of the 17 common QTL were located in regions where no QTL associated with grain weight have been cloned and one was selected for fine-mapping. Eight populations segregating in an isogenic background were derived from one F7 residual heterozygote of Teqing/IRBB52. The target QTL,q TGW10-20.8 controlling grain weight, grain length, and grain width, was localized to a 70.7-kb region flanked by In Del markers Te20811 and Te20882 on the long arm of chromosome 10. The QTL region contains seven annotated genes, of which six encode proteins with known functional domains and one encodes a hypothetical protein. One of the genes, Os10 g0536100 encoding the MIKC-type MADS-box protein Os MADS56, is the most likely candidate for q TGW10-20.8. These results provide a basis for cloning q TGW10-20.8, which has an important contribution to grain weight variation in rice.

Brown planthopper (BPH) is a devastating pest that threatens the food security of rice-producing countries. At present, most cultivars planted in farmers’ paddies lack effective BPH resistance, which constitutes a potential threat to rice yield. Moreover, developing BPH-resistant rice varieties using traditional breeding approaches is time-consuming, labor-intensive, and unpredictable. In this study, we successfully enhanced BPH resistance of the elite rice cultivar Wushansimiao by introgressing the resistance genes BPH14 and BPH15 through positive selection, negative selection, and whole genome background selection. Through backcrossing, the introgression fragments were reduced to 428.3 kb for BPH14 and 413.1 kb for BPH15. Except for these two fragments, the residual genetic background of the selected near-isogenic lines (NILs) was nearly identical to that of the recurrent parent, with a genetic background recovery rate of 99.78%. As a result, the selected NILs exhibited much stronger BPH resistance at the seedling and adult stages compared to the recurrent parent. Moreover, field tests showed that grain yield, major agronomic traits, and grain quality of the five selected NILs were statistically indistinguishable from those of the recurrent parent. Our results provide an effective approach for directionally upgrading the target traits and will inform and facilitate rice breeding.

Background: Breeding two-line hybrid rice with disease resistance is an effective approach to stabilize rice yield in commercial rice production of China. Results: We improved the blast and bacterial blight resistance of Guangzhan63-4S, an elite photoperiod-and thermo-sensitive male sterile (P/TGMS) line widely used in two-line hybrid rice, by introducing the R genes Pi2 and Xa7 conferring resistance to rice blast and bacterial blight, respectively. Through the backcrossing and gene pyramiding breeding coupled with molecular marker-assisted selection, a new P/TGMS line Hua1228S carrying Pi2, Xa7, and tms5 was developed. Based on 200,000 SNP markers by next-generation sequencing, Hua1228S covered 87.6% of the recurrent genome, as well as 4.5% of the donor genome from VE6219 and 7.9% from YR7029-39. When infected with seven tested Xanthomonas oryzae pv. oryzae strains, Hua1228S conferred high resistance (0 level) to six bacterial blight strains. Moreover, Hua1228S showed broad-spectrum resistance to rice blast isolates with a high resistance frequency of 90.91%. High levels of resistance to leaf blast and neck blast were observed under heavy disease pressure in natural field. Importantly, Hua1228S showed identical fertility-sterility alteration pattern to Guangzhan63-4S. Thus, two hybrid combinations Hua Liangyou 2821 and Hua Liangyou 284 derived from Hua1228S exhibited enhanced resistance and higher yield compared with the control variety Feng Liangyou 4. Conclusions: These results indicate that Hua1228S has tremendous potentiality to increase and stabilize the rice yield, through the introgression of two R genes by marker-assisted selection strategy.

Grain size is a key determinant of grain weight and a trait having critical influence on grain quality in rice. While increasing evidences are shown for the importance of minor-effect QTL in controlling complex traits, the attention has not been given to grain size until recently. In previous studies, five QTL having small effects for grain size were resolved on the long arm of chromosome 1 using populations derived from indica rice cross Zhenshan 97///Zhenshan 97//Zhenshan 97/Milyang 46. One of them, qTGW1.2c that was located in a 2.1-Mb region, was targeted for fine-mapping in the present study.

Hua 1201S is a medium indica photo-thermo-sensitive genic male sterile (PTGMS) line in rice.It was developed from the cross between Guangzhan 63-4S (recipient) and VE6219 (donor of Gene Pi2) through molecular marker-assisted selection (MAS) for tracking Pi2 with SSR marker RM527 and Indel marker Pi2-4,identification of phenotype and blast resistance and successive backcrossing.The results of field identification of blast resistance under natural conditions at the blast epidemic area of Lianghe Village,Enshi City,Hubei Province showed that Hua 1201S was ranked at Scale 1.3 to 2.3 in the complex resistance index of rice blast and Scale 1 in the loss rate of panicle neck blast in 2013 and 2014,which was evaluated to be resistant to blast in both years.The results of nursery identification of seedling blast resistant spectrum in Guangdong Academy of Agricultural Sciences in 2015 showed that Hua 1201S had 96.97％ of the resistance frequency while Guangzhan 63-4S had only 12.12％ of the resistance frequency among 33 inoculated blast isolates.The critical sterility-inducing temperature of fertility alteration of Hua 1201S was 23 ℃ with a stable male sterile period of 64 days at Wuhan,Hubei and a seed-setting rate of over 70％ under natural conditions in winter season in Hainan.Hua 1201S was technically identified by Hubei Crop Variety Appraisal Committee in August,2015.