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.

Heterotrimeric G proteins regulate nitrogen-use efficiency in rice.

The drive toward more sustainable agriculture has raised the profile of crop plant nutrient-use efficiency. Here we show that a major rice nitrogen-use efficiency quantitative trait locus (qNGR9) is synonymous with the previously identified gene DEP1 (DENSE AND ERECT PANICLES 1). The different DEP1 alleles confer different nitrogen responses, and genetic diversity analysis suggests that DEP1 has been subjected to artificial selection during Oryza sativa spp. japonica rice domestication. The plants carrying the dominant dep1-1 allele exhibit nitrogen-insensitive vegetative growth coupled with increased nitrogen uptake and assimilation, resulting in improved harvest index and grain yield at moderate levels of nitrogen fertilization. The DEP1 protein interacts in vivo with both the Gα (RGA1) and Gß (RGB1) subunits, and reduced RGA1 or enhanced RGB1 activity inhibits nitrogen responses. We conclude that the plant G protein complex regulates nitrogen signaling and modulation of heterotrimeric G protein activity provides a strategy for environmentally sustainable increases in rice grain yield.

Control of grain size, shape and quality by OsSPL16 in rice.

Grain size and shape are important components of grain yield and quality and have been under selection since cereals were first domesticated. Here, we show that a quantitative trait locus GW8 is synonymous with OsSPL16, which encodes a protein that is a positive regulator of cell proliferation. Higher expression of this gene promotes cell division and grain filling, with positive consequences for grain width and yield in rice. Conversely, a loss-of-function mutation in Basmati rice is associated with the formation of a more slender grain and better quality of appearance. The correlation between grain size and allelic variation at the GW8 locus suggests that mutations within the promoter region were likely selected in rice breeding programs. We also show that a marker-assisted strategy targeted at elite alleles of GS3 and OsSPL16 underlying grain size and shape can be effectively used to simultaneously improve grain quality and yield.

Complete loss of photoperiodic response in the rice mutant line X61 is caused by deficiency of phytochrome chromophore biosynthesis gene.

In rice (Oryza sativa), a short-day plant, photoperiod is the most favorable external signal for floral induction because of the constant seasonal change throughout the years. Compared with Arabidopsis, however, a large part of the regulation mechanism of the photoperiodic response in rice still remains unclear due mainly to the lack of induced mutant genes. An induced mutant line X61 flowers 35 days earlier than its original variety Gimbozu under a natural photoperiod in Kyoto (35°01'N). We attempted to identify the mutant gene conferring early heading to X61. Experimental results showed that the early heading of X61 was conferred by a complete loss of photoperiodic response due to a novel single recessive mutant gene se13. This locus interacts with two crucial photoperiod sensitivity loci, Se1 and E1. Wild type alleles at these two loci do not function in coexistence with se13 in a homozygous state, suggesting that Se13 is an upstream locus of the Se1 and E1 loci. Linkage analysis showed that Se13 is located in a 110 kb region between the two markers, INDEL3735_1 and INDEL3735_3 on chromosome 1. A database search suggested that the Se13 gene is identical to AK101395 (=OsHY2), which encodes phytochromobilin synthase, a key enzyme in phytochrome chromophore biosynthesis. Subsequent sequence analysis revealed that X61 harbors a 1 bp insertion in exon 1 of OsHY2, which induces a frame-shift mutation producing a premature stop codon. It is therefore considered that the complete loss of photoperiodic response of X61 is caused by a loss of function of the Se13 (OsHY2) gene involved in phytochrome chromophore biosynthesis.

Identification of a novel gene ef7 conferring an extremely long basic vegetative growth phase in rice.

A late heading-time mutant line, HS276, which was induced by gamma-irradiation of seeds of the japonica rice (Oryza sativa L.) variety Gimbozu, exhibits an extremely long basic vegetative growth phase (BVP). A genetic analysis using the F(2) population from the cross between HS276 and Gimbozu revealed that the late heading of HS276 is governed by a single recessive mutant gene. The subsequent analysis on heading responses of HS276 and Gimbozu to four photoperiods (12, 13, 14, and 15 h) and to the photoperiodic transfer treatment from a short photoperiod to a long photoperiod revealed that the mutant gene confers an extremely long BVP and increases photoperiod sensitivity under long photoperiod (14 and 15 h). The BVP durations of HS276 and Gimbozu were estimated at 30.1 and 16.0 days, respectively; the mutant gene, compared with its wild type allele, elongates the duration of BVP by 14 days. Linkage analysis showed that the mutant gene is located in the 129 kb region between the two INDEL markers, INDELAP0399_6 and INDELAP3487_2, on the distal part of the short arm of chromosome 6. None of the other BVP genes are located in this region; therefore, we declared this a newly detected mutant gene and designated it ef7. A recently established program to breed rice suitable for low latitudes, where short photoperiodic conditions continue throughout the year, aims to develop varieties with extremely long BVPs and weak photoperiod sensitivities; the mutant gene ef7, therefore, will be quite useful in these programs because it confers an extremely long BVP and little enhances photoperiod sensitivity under short photoperiod.

Multiple alleles at Early flowering 1 locus making variation in the basic vegetative growth period in rice (Oryza sativa L.).

A recently established rice breeding program in low latitudes aims to develop varieties with extremely long basic vegetative growth (BVG) periods and weak photoperiod sensitivities. The Taiwanese japonica variety Taichung 65 (T65) harbors a recessive allele ef1 at the Ef1 (Early flowering 1) locus, thereby exhibiting an extremely long BVG period. The previous reported functional allele Ehd1 (Early heading date 1), located on chromosome 10, encodes a B-type response regulator, thereby shortening the BVG period, whereas its nonfunctional allele ehd1 greatly prolongs the BVG period. A conventional analysis using F(2) and F(3) populations and a subsequent CAPS analysis based on the amino acid sequences of Ehd1 and ehd1 showed that Ef1 and Ehd1 were at the same locus. The CAPS analysis also indicated that the Taiwanese japonica varieties with extremely long BVG periods all harbor ef1, but that ef1 does not exist among indica and japonica varieties in the low latitudes. Since ef1 has not been found in any japonica varieties outside Taiwan, this allele might have originated in Taiwan. Sequence analysis revealed that the mutant allele ef1-h, which prolongs the BVG period even more than ef1 does, harbors an mPing insertion in exon 2, which causes the complete loss of gene function. Our results indicate that both ef1 or ef1-h alleles can be used as new gene sources in developing rice varieties with extremely long BVG periods for low latitudes.