Breeding of CMS maintainer lines through anther culture assisted by high-resolution melting-based markers

The integrated use of molecular marker-assisted selection (MAS) and anther culture has potential to significantly increase efficiency in plant breeding;however, reports on this kind of practical use are very limited. In the present study, we report the development of cytoplasmic male sterile (CMS) maintainers with aroma, disease resistance and red-brown hulls, as an example of integration of MAS and anther culture in rice breeding. A high-resolution melting (HRM)-based functional molecular marker was developed for the red-brown hull trait caused by a unique mutation (rbh1) in OsCAD2. Functional molecular markers for genes of rice blast resistance (Pi2), aroma (fgr) and red-brown hull (rbh1) were used for precise genotyping of individual plants in the BC1 and BC2F2 populations derived from a cross between CMS maintainers Huaxiang B (pi2–/rbh1–/fgr–) and Rong 3B (Pi2+/RBH1+/Fgr+). A total of 89 doubled haploid (DH) lines were generated from selected BC2F2 plants (Pi2+/rbh1–/fgr–) by anther culture. Seven DH lines were subsequently selected as the potential new CMS maintainers based on their overall performance and high resistance to blast. Our study demonstrated that integration of MAS and anther culture significantly accelerated the development of CMS maintainers with multiple stacked genes.

Rice gene OsUGT75A regulates seedling emergence under deep-sowing conditions

Poor seedling emergence is a challenge for direct seeding of rice under deep-sowing field conditions.Here we reveal that UDP-glucosyltransferase OsUGT75A promotes rice seedling emergence under deepsowing conditions by increasing shoot length.Expression of OsUGT75A was higher in the middle regions of the shoot and in shoots under deep-sowing conditions.Levels of free abscisic acid(ABA)and jasmonates(JA)were higher in shoots of OsUGT75A mutants than in those of wild-type plants,and OsUGT75A mutants were more sensitive to ABA and JA treatments.Reduced shoot length was attributed to higher ABA INSENSITIVE 3(OsABI3)expression and lower JASMONATE-ZIM domain protein(OsJAZ)expression in shoots.Shoot extension by OsUGT75A is achieved mainly by promotion of cell elongation.An elite haplotype of OsUGT75A associated with increased shoot length was identified among indica rice accessions.OsUGT75A acts to increase seedling emergence under deep-sowing conditions.

Identification of QTL and candidate genes involved in early seedling growth in rice via high-density genetic mapping and RNA-seq

Early seedling vigor(ESV)is a major breeding target in rice,especially under direct seeding.To identify quantitative trait locus(QTL)affecting ESV,a recombinant inbred line population derived from a cross between 02428 and YZX,two cultivars differing in vigor during early seedling growth,was used for QTL analysis.Nine traits associated with ESV were examined using a high-density map.Of 16 additive loci identified,three were detected in two generations and thus considered stable.Four epistatic interactions were detected,one of which was repeated in two generations.Further analysis of the pyramiding effect of the three stable QTL showed that the phenotypic value could be effectively improved with an increasing number of QTL.These results were combined with results from our previous QTL analysis of the germination index.The lines G58 and G182 combined all the favourable alleles of all three stable QTL for ESV and three QTL for germination speed.These two lines showed rapid germination and strong ESV.A total of 37 candidate differentially expressed genes were obtained from the regions of the three stable QTL by analysis of the dynamic transcriptomic expression profile during the seedling growth period of the two parents.The QTL are targets for ESV breeding and the candidate genes await functional validation.This study provides a theoretical basis and a genetic resource for the breeding of directseeded rice.

Development of elite restoring lines by integrating blast resistance and low amylose content using MAS

Blast resistance and grain quality are major problems in hybrid rice production in China. In this study, two resistance （R） genes, Pi46 and Pita, along with the gene Wxb, which mainly affects rice endosperm amylose content （AC）, were introgressed into an elite indica restoring line, R8166, which has little blast resistance and poor grain quality through marker-assisted selection （MAS）. Eight improved lines were found to have recurrent genome recovery ratios ranging from 88.68 to 96.23%. Two improved lines, R163 and R167, were selected for subsequent studies. R167, which has the highest recovery ratio （96.23%）, showed no significant differences in multiple agronomic traits. In contrast, R163 with the lowest recovery ratio （88.68%） exhibited significant differences in heading date and yield per plant compared with the recurrent parent. At two developmental stages, R163 and R167 had greatly enhanced resistance to blast over the recurrent parent. Similar trends were also observed for agronomic traits and blast resistance in R163- and R167-derived hybrids when compared with the counterparts from R8166. In addition, R163, R167, and their derived hybrids significantly improved the grain quality traits, including amylose content （AC）, gel consistency （GC）, chalky grain rate （CGR）, and degree of endosperm chalkiness （DEC）. It confirmed the success of efficiently developing elite restoring lines using MAS in this study.

Significant association of the novel Rf4-targeted SNP marker with the restorer for WA-CMS in different rice backgrounds and its utilization in molecular screening

In the rice cytoplasmic-genetic male sterility （CMS） system, the combination of a CMS line, maintainer line and restorer line carrying the restorer gene to restore fertility, is indispensable for the development of hybrids. However, the process of screening for the trait of fertility restoration is laborious and time-consuming. In the present study, we analyzed the nucleotide sequence of the Rf4 gene, which is the major locus controlling fertility restoration, to identify allele-specific variation. A single nucleotide polymorphism （SNP） A/C at ＋474 in the coding sequence （CDS） was found to be capable of strictly distinguishing groups of alleles Rf4 （A） and rf4 （C）. Using KASP genotyping, this valuable SNP was converted to an allele-specific PCR marker. We evaluated and validated the marker among three-line parents with different backgrounds, and the results revealed a complete correlation between SNP alleles and the fertility restoration phenotype. Molecular screening was subsequently carried out for the presence of alleles of Rf4 and Rf3 among 328 diverse rice cultivars with worldwide distribution. The results demonstrate that this SNP marker could be the optimal choice for the molecular identification of potential restorers.

Pyramiding of Pi46 and Pita to improve blast resistance and to evaluate the resistance effect of the two R genes

Utilization of R（resistance） genes to develop resistant cultivars is an effective strategy to combat against rice blast disease. In this study, R genes Pi46 and Pita in a resistant accession H4 were introgressed into an elite restorer line Hang-Hui-179（HH179） using the marker-assisted backcross breeding（MABB） procedure. As a result, three improved lines（e.g., R1791 carrying Pi46 alone, R1792 carrying Pita alone and R1793 carrying both Pi46 and Pita） were developed. The three improved lines had significant genetic similarities with the recurrent parent HH179. Thus, they and HH179 could be recognized as near isogenic lines（NILs）. The resistance spectrum of the three improved lines, which was tested at seedling stage, reached 91.1, 64.7 and 97.1%, respectively. This was markedly broader than that of HH179（23.5%）. Interestingly, R1793 showed resistance to panicle blast but neither R1791 nor R1792 exhibited resistance at two natural blast nurseries. The results implied that the stacking of Pi46 and Pita resulted in enhanced resistance, which was unachievable by either R gene alone. Further comparison indicated that the three improved lines were similar to HH179 in multiple agronomic traits; including plant height, tillers per plant, panicle length, spikelet fertility, and 1 000-grain weight. Thus, the three improved lines with different R genes can be used as new sources of resistance for developing variety. There is a complementary effect between the two R genes Pi46 and Pita.

Cloning of Rice Blast Resistance Gene Pik_2-H4 and Screening of Its Interaction Protein

Abstract The broad-spectrum rice blast resistance gene Pik2-H4 was cloned from the highly resistant line H4. The full-length sequence of Pik2-H4 was 3 066 bp, which was different from the corresponding allele sequence of Nipponbare. Pik2-H4 belonged to NBS-LRR genes and coded a protein containing NB-ARC domain and leueine-rieh repeats. To find the interaction proteins with Pik2-H4 from the rice, the yeast two-hybrid system was used and three important proteins （ LOC- Os08939300, LOCOs03g25960 and LOC-Os09929130）were identified. The results could provide some new information for the mechanism of rice blast resistance mediated by Pik2-H4.

OsBLS6.2:A rice bacterial leaf streak resistance gene identified by GWAS and RNA-seq

Bacterial leaf streak(BLS),caused by Xanthomonas oryzae pv.oryzicola(Xoc),is a bacterial disease affecting rice production in Asia and Africa,whose severity is expected to increase with climate change.Identification of new quantitative-trait loci(QTL)or resistance genes for BLS resistance is essential for developing resistant rice.A genome-wide association study to identify QTL associated with BLS resistance was conducted using phenotypic and genotypic data from 429 rice accessions.Of 47 QTL identified,45 were novel and two co-localized with previously reported QTL or genes conferring BLS resistance.qBLS6.2 on chromosome 6 explained the greatest phenotypic variation.Combined analysis of differential expression and annotations of predicted genes near qBLS6.2 based on haplotype and disease phenotype identified OsBLS6.2(LOC_Os06g02960)as a candidate gene for qBLS6.2.OsBLS6.2 knockout plants showed higher resistance to Xoc than wild-type plants.Many other candidate genes for resistance to Xoc were identified.

Rice seed germination priming by salicylic acid and the emerging role of phytohormones in anaerobic germination

Direct seeding has been increasingly adopted under both rainfed and irrigated conditions due to its low cost and convenience.Not only to suppress weed germination and reduce the cost of manual weeding and/or dependence on herbicides,but also to control pests and reduce pesticide usage,farmers usually flood the direct-seeded field,resulting in inadequate seed germination and seedling establishment.

The Class Ⅲ peroxidase gene OsPrx30,transcriptionally modulated by the AT-hook protein OsATH1, mediates rice bacterial blight-induced ROS accumulation

Class Ⅲ peroxidases(CⅢ Prxs) play critical roles in plant immunity by scavenging reactive oxygen species(ROS). However, the functions of CⅢ Prxs in rice(Oryza sativa L.) immunity are largely unexplored. Here, we report a Prx precursor, OsPrx30,that is responsive to the bacterial blight Xanthomonas oryzae pv. oryzae(Xoo). OsPrx30 was primarily expressed in rice roots, leaves, and stems,and its protein product was mainly localized at the endoplasmic reticulum. Overexpression of OsPrx30 enhanced the plant’s susceptibility to Xoo by maintaining a high level of peroxidase(POD) activity and reducing the content of H2O2, whereas depletion of OsPrx30 had the opposite effects. Furthermore, we identified an AT-hook transcription factor, Os ATH1, that is specifically bound to the OsPrx30 promoter. As observed in plants overexpressing OsPrx30, depletion of Os ATH1 enhanced susceptibility to Xoo. Finally, we demonstrated that depletion of Os ATH1 increased histone H3 acetylation at the AT-rich region of the OsPrx30 promoter.Taken together, these results reveal a mechanism underlying the POD-induced natural resistance to bacterial diseases and suggest a model for transcription regulation of Prx genes in rice.