Upgrading the genome of an elite japonica rice variety Kongyu 131 for lodging resistance improvement.

Developing a new rice variety requires tremendous efforts and years of input. To improve the defect traits of the excellent varieties becomes more cost and time efficient than breeding a completely new variety. Kongyu 131 is a high-performing japonica variety with early maturity, high yield, wide adaptability and cold resistance, but the poor-lodging resistance hinders the industrial production of Kongyu 131 in the Northeastern China. In this study, we attempted to improve the lodging resistance of Kongyu 131 from perspectives of both gene and trait. On the one hand, by QTL analysis and fine mapping we discovered the candidate gene loci. The following CRISPR/Cas9 and transgenic complementation study confirmed that Sd1 dominated the lodging resistance and favourable allele was mined for precise introduction and improvement. On the other hand, the Sd1 allelic variant was identified in Kongyu 131 by sequence alignment, then introduced another excellent allelic variation by backcrossing. Then, the two new resulting Kongyu 131 went through the field evaluation under different environments, planting densities and nitrogen fertilizer conditions. The results showed that the plant height of upgraded Kongyu 131 was 17%-26% lower than Kongyu 131 without penalty in yield. This study demonstrated a precise and targeted way to update the rice genome and upgrade the elite rice varieties by improving only a few gene defects from the perspective of breeding.

Improving the blast resistance of the elite rice variety Kongyu-131 by updating the pi21 locus.

BACKGROUND: As an elite japonica rice variety, Kongyu-131 has been cultivated for over 20 years in the third accumulated temperature zone of Heilongjiang Province, China. However, the cultivated area of Kongyu-131 has decreased each year due to extensive outbreaks of rice blast. To achieve the goals of improving blast resistance and preserving other desirable traits in Kongyu-131, a genome-updating method similar to repairing a bug in a computer program was adopted in this study. A new allele of the broad-spectrum blast resistance gene pi21 in the upland rice variety GKGH was mined by genetic analysis and introgressed into the genome of Kongyu-131 to upgrade its blast resistance. RESULT: QTL analysis was performed with an F2 population derived from a cross between Kongyu-131 and GKGH, and a blast resistance QTL was detected near the pi21 locus. Parental Pi21 sequence alignment showed that the pi21 of the donor (GKGH) was a new allele. By 5 InDel or SNP markers designed based on the sequence within and around pi21, the introgressed chromosome segment was shortened to less than 634 kb to minimize linkage drag by screening recombinants in the target region. The RRPG was 99.92%, calculated according to 201 SNP markers evenly distributed on 12 chromosomes. Artificial inoculation at the seedling stage showed that the blast resistance of the new Kongyu-131 was improved significantly. Field experiments also indicated that the improved Kongyu-131 had enhanced field resistance to rice blast and grain-quality traits similar to those of the original Kongyu-131. CONCLUSIONS: It is feasible to improve resistance to rice blast and preserve other desirable traits by precisely improving the Pi21 locus of Kongyu-131. Linkage drag can be eliminated effectively via recombinant selection on both sides of the target gene.

Updating the Genome of the Elite Rice Variety Kongyu131 to Expand Its Ecological Adaptation Region.

As an elite rice variety cultivated in the third accumulative temperature belt in Heilongjiang province, China, Kongyu131 has many excellent traits, such as high quality, high stability, early maturation and cold resistance. However, as with other crop varieties, Kongyu131 has regional restrictions, exhibiting decreased yields when grown at low latitudes. To address these problems, two populations were constructed from cross between japonica and indica varieties. QTL analyses were performed with these two populations to detect regional adaptation related quantitative trait locus. Results in a BC1F6 backcross inbred line population with 168 lines derived from cross between Kongyu131 and GKMP showed a large pleiotropic QTL near 9 Mb on chromosome 7, which significantly delayed the HD of Kongyu131 and increased the plant height (PH), length of main panicle (LMP), number of primary branches (NPB) and grain number of main panicles (GNP). We also found a similar QTL in the population BC3F2 derived from Kongyu131 and GKLPL. Based on the QTL, we developed a gene module named mRA7 with 5 single-nucleotide polymorphism (SNP) markers around the QTL. Through a foreground and background selection based on 197 SNP markers evenly distributed over the 12 chromosomes, we obtained a new plant (a single point substitution line, SPSL) with a new Kongyu131 genome, carrying only a small chromosomal fragment less than 800 kb from GKLPL. The background recovery ratio of the SPSL was 99.8%. Compared with Kongyu131, the SPSL exhibited a significant HD delay of approximately 31 days and increased PH, LMP and GNP values when planted in Heilongjiang province. When cultivated in Guangdong province, HD of SPSL showed only 16 days delay, and less increase in PH, LMP and GNP than in Heilongjiang province. Phenotypic evaluation showed that the SPSL could be moved to south by more than 3 latitude units and cultivated in low-latitude regions. This study exemplifies the feasibility of expanding the regions of cultivation of elite rice varieties via similar methods.

Improving rice grain length through updating the GS3 locus of an elite variety Kongyu 131.

BACKGROUND: Traditional crop breeding has made significant achievement meet food needs worldwide. However, the way has some inevitable problems including time-consuming, laborious, low predictability and reproducibility. In this study, we updated the GRAIN SIZE 3 (GS3) locus to improve the grain length of a major cultivate variety of Kongyu 131 at Heilongjiang Province, the northernmost region of China. High-resolution melting (HRM) analysis is used for single nucleotide polymorphism (SNP) genotyping. RESULTS: The improved line introgressed about 117 kb segment including gs3 allele from donor GKBR by using five SNP markers designed within and without GS3 locus, and the background recovery ratio of the recurrent parent genome is about 99.55% that are detected by 219 SNP markers evenly distributed on the 12 chromosomes. The field trial indicates that grain length, 100-grain weight and total grain weight per plant of the improved line raised by 12.05%, 16.30% and 4.47%, respectively, compared with Kongyu 131. CONCLUSIONS: This result demonstrates that update the GS3 locus is a feasible and efficient and accurate way can be applied to improve grain size of rice.

Updating the elite rice variety Kongyu 131 by improving the Gn1a locus.

BACKGROUND: Kongyu 131 is an elite japonica rice variety of Heilongjiang Province, China. It has the characteristics of early maturity, superior quality, high yield, cold tolerance and wide adaptability. However, there is potential to improve the yield of Kongyu 131 because of the relatively few grains per panicle compared with other varieties. Hence, we rebuilt the genome of Kongyu 131 by replacing the GRAIN NUMBER1a (Gn1a) locus with a high-yielding allele from a big panicle indica rice variety, GKBR. High-resolution melting (HRM) analysis was used for single nucleotide polymorphism (SNP) genotyping. RESULTS: Quantitative trait locus (QTL) analysis of the BC3F2 population showed that the introgressed segment carrying the Gn1a allele of GKBR significantly increased the branch number and grain number per panicle. Using 5 SNP markers designed against the sequence within and around Gn1a, the introgressed chromosome segment was shortened to approximately 430 Kb to minimize the linkage drag by screening recombinants in the target region. Genomic components of the new Kongyu 131 were detected using 220 SNP markers evenly distributed across 12 chromosomes, suggesting that the recovery ratio of the recurrent parent genome (RRPG) was 99.89%. Compared with Kongyu 131, the yield per plant of the new Kongyu 131 increased by 8.3% and 11.9% at Changchun and Jiamusi, respectively. CONCLUSIONS: To achieve the high yield potential of Kongyu 131, a minute chromosome fragment carrying the favorable Gn1a allele from the donor parent was introgressed into the genome of Kongyu 131, which resulted in a larger panicle and subsequent yield increase in the new Kongyu 131. These results indicate the feasibility of improving an undesirable trait of an elite variety by replacing only a small chromosome segment carrying a favorable allele.