Drought is one of the major abiotic stresses threatening rice (Oryza sativa) production worldwide. Drought resistance is controlled by multiple genes, and therefore, a multi-gene genetic engineering strategy is theoretically useful for improving drought resistance. However, the experimental evidence for such a strategy is still lacking. In this study, a few drought-responsive genes from rice were assembled by a multiple-round site-specific assembly (MISSA) system, and the constructs were introduced into the rice cultivar KY131 via Agrobacterium-mediated transformation. The transgenic lines of the multi-gene and corresponding single-gene constructs were pre-evaluated for drought resistance. We found that the co-overexpression of two genes, encoding a constitutively active form of a bZIP transcription factor (OsbZIP46CA1) and a protein kinase (SAPK6) involved in the abscisic acid (ABA) signaling pathway, showed significantly enhanced drought resistance compared with the single-gene transgenic lines and the negative transgenic plants. Single-copy lines of this bi-gene combination (named XL22) and the corresponding single-gene lines were further evaluated for drought resistance in the field using agronomical traits. The results showed that XL22 exhibited greater yield, biomass, spikelet number, and grain number under moderate drought stress conditions. The seedling survival rate of XL22 and the single-gene overexpressors after drought stress treatment also supported the drought resistance results. Furthermore, expression profiling by RNA-Seq revealed that many genes involved in the stress response were specifically up-regulated in the drought-treated XL22 lines and some of the stress-related genes activated in CA1-OE and SAPK6-OE were distinct, which could partially explain the different performances of these lines with respect to drought resistance. In addition, the XL22 seedlings showed improved tolerance to heat and cold stresses. Our results demonstrate that the multi-gene assembly in an appropriate combination may be a promising approach in the genetic improvement of drought resistance.

The development of hybrid rice is a practical approach for increasing rice production. However, the brown planthopper (BPH), Nilaparvata lugens Stål, causes severe yield loss of rice (Oryza sativa L.) and can threaten food security. Therefore, breeding hybrid rice resistant to BPH is the most effective and economical strategy to maintain high and stable production. Fortunately, numerous BPH resistance genes have been identified, and abundant linkage markers are available for molecular marker-assisted selection (MAS) in breeding programs. Hence, we pyramided two BPH resistance genes, Bph14 and Bph15, into a susceptive CMS restorer line Huahui938 and its derived hybrids using MAS to improve the BPH resistance of hybrid rice. Three near-isogenic lines (NILs) with pyramided Bph14 and Bph15 were obtained by molecular marker-assisted backcross (MAB) and phenotypic selection. The genomic components of these NILs were detected using the whole-genome SNP (Single nucleotide polymorphism) array, RICE6K, suggesting that the recurrent parent genome (RPG) recovery of the NILs was 87.88, 87.70 and 86.62 %, respectively. BPH bioassays showed that the improved NILs and their derived hybrids carrying homozygous Bph14 and Bph15 were resistant to BPH. However, the hybrids with heterozygous Bph14 and Bph15 remained susceptible to BPH. The developed NILs showed no significant differences in major agronomic traits and rice qualities compared with the recurrent parent. Moreover, the improved hybrids derived from the NILs exhibited better agronomic performance and rice quality compared with the controls under natural field conditions. This study demonstrates that it is essential to stack Bph14 and Bph15 into both the maternal and paternal parents for developing BPH-resistant hybrid rice varieties. The SNP array with abundant DNA markers is an efficient tool for analyzing the RPG recovery of progenies and can be used to monitor the donor segments in NILs, thus being extremely important for rice molecular breeding.

Background: Blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the big problems in rice production in China, especially for high yield hybrid varieties made from a two-line system in which thermo-sensitive genic male sterile (TGMS) lines are used. In this study, we report the introgression of a rice blast resistance gene Pi2 from VE6219 into C815S, an elite rice TGMS line, leading to the development of blast resistant TGMS lines through marker assisted selection (MAS) and phenotypic selection approaches. Results: Four new TGMS lines with blast resistance gene Pi2 were developed from C815S (an elite TGMS line susceptible to the blast, used as recurrent parent) and VE6219 (a blast resistant line harboring Pi2, used as donor parent). The pathogenicity assays inoculated with 53 blast prevalent isolates in glasshouse showed that the blast resistant frequency of the four TGMS lines was 94.3%-98.1% that is equivalent to blast resistant donor parent VE6219. The field evaluation of the new lines and hybrids made from them at a blast epidemic site also showed high resistant levels against the blast. The genetic background of the newly developed TGMS lines were examined using a whole-genome single nucleotide polymorphism (SNP) array (RICE6K) that turned out more than 83% of the genomic markers were derived from the recurrent parent. The critical temperature points of fertility-sterility alteration of the new TGMS lines were between 22°C and 23°C of daily mean temperature, which is similar to that of C815S. The complete male sterility under natural growth conditions at Wuhan last more than 80 days. Their agronomic and grain quality traits meet the requirement for two-line hybrid rice production. Conclusions: The broad-spectrum and durable rice blast resistant gene Pi2 was introgressed into the elite TGMS line C815S background. The newly developed TGMS lines can be practically used for two-line hybrid rice breeding and must play an important role in sustainable rice production in China.

Breeding two-line hybrid rice with disease resistance is an effective approach to stabilize rice yield in commercial rice production of China. 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. 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.