Development and characterization of GR2E Golden rice introgression lines.

Golden Rice with ß-carotene in the grain helps to address the problem of vitamin A deficiency. Prior to commercialize Golden Rice, several performance and regulatory checkpoints must be achieved. We report results of marker assisted backcross breeding of the GR2E trait into three popular rice varieties followed by a series of confined field tests of event GR2E introgression lines to assess their agronomic performance and carotenoid expression. Results from confined tests in the Philippines and Bangladesh have shown that GR2E introgression lines matched the performance of the recurrent parents for agronomic and yield performance, and the key components of grain quality. Moreover, no differences were observed in terms of pest and disease reaction. The best performing lines identified in each genetic background had significant amounts of carotenoids in the milled grains. These lines can supply 30-50% of the estimated average requirements of vitamin A.

A novel resistance gene for bacterial blight in rice, Xa43(t) identified by GWAS, confirmed by QTL mapping using a bi-parental population.

Bacterial blight (BB) caused by the Xanthomonas oryzae pv. oryzae (Xoo) pathogen is a significant disease in most rice cultivation areas. The disease is estimated to cause annual rice production losses of 20-30 percent throughout rice-growing countries in Asia. The discovery and deployment of durable resistance genes for BB is an effective and sustainable means of mitigating production losses. In this study QTL analysis and fine mapping were performed using an F2 and a BC2F2 population derived from a cross with a new R-donor having broad spectrum resistance to Korean BB races. The QTL qBB11 was identified by composite interval mapping and explained 31.25% of the phenotypic variation (R2) with LOD values of 43.44 harboring two SNP markers. The single major R-gene was designated Xa43 (t). Through dissection of the target region we were able to narrow the region to within 27.83-27.95 Mbp, a physical interval of about 119-kb designated by the two flanking markers IBb27os11_14 and S_BB11.ssr_9. Of nine ORFs in the target region two ORFs revealed significantly different expression levels of the candidate genes. From these results we developed a marker specific to this R-gene, which will have utility for future BB resistance breeding and/or R-gene pyramiding using marker assisted selection. Further characterization of the R-gene would be helpful to enhance understanding of mechanisms of BB resistance in rice.

Identification of stable QTLs causing chalk in rice grains in nine environments.

KEY MESSAGE: A novel QTL cluster for chalkiness on Chr04 was identified using single environment analysis and joint mapping across 9 environments in Asia and South American. QTL NILs showed that each had a significant effect on chalk. Chalk in rice grains leads to a significant loss in the proportion of marketable grains in a harvested crop, leading to a significant financial loss to rice farmers and traders. To identify the genetic basis of chalkiness, two sets of recombinant inbred lines (RILs) derived from reciprocal crosses between Lemont and Teqing were used to find stable QTLs for chalkiness. The RILs were grown in seven locations in Asia and Latin American and in two controlled environments in phytotrons. A total of 32 (21) and 46 (22) QTLs for DEC and PGWC, most of them explaining more than 10% of phenotypic variation, were detected based on single environment analysis in T/L (L/T) population, respectively. Seven (2) and 7 (3) QTLs for DEC and PGWC were identified in the T/L (L/T) population using joined analysis across all environments, respectively. Six major QTLs clusters were found on five chromosomes: 1, 2, 4, 5 and 11. The biggest cluster at id4007289-RM252 on Chr04 was a novelty, including 16 and 4 QTLs detected by single environment analysis and joint mapping across all environments, respectively. The detected digenic epistatic QTLs explained up to 13% of phenotypic variation, suggesting that epistasis play an important role in the genetic control of chalkiness in rice. QTL NILs showed that each QTL cluster had a significant effect on chalk. These chromosomal regions could be targets for MAS, fine mapping and map-based cloning for low chalkiness breeding.

Identification and fine-mapping of a new resistance gene, Xa40, conferring resistance to bacterial blight races in rice (Oryza sativa L.).

KEY MESSAGE: A new bacterial blight resistance gene has been identified through fine-mapping, which confers high levels of resistance to all Korean Xanthomonas oryzae pv. oryzae (Xoo) races, including the new Xoo race K3a. Rice bacterial leaf blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is a serious constraint to rice production in Asia and Africa. The japonica advanced backcross breeding lines derived from the indica line IR65482-7-216-1-2 in the background of cultivar Junam are resistant to all Korean BB races, including K3a. To identify the gene(s) involved in resistance to Korean Xoo races, the association of genotypic and phenotypic variations was examined in two F2 populations derived from the crosses between 11325 (IR83261-3-7-23-6-2-1-1-2-1-2)/Anmi and 11325/Ilpum. The segregation ratios of F2 individuals from the crosses of 11325/Anmi and 11325/Ilpum were 578 resistant:209 susceptible and 555 resistant:241 susceptible, respectively, which is consistent with the expected allelic frequency of a 3:1 ratio. Genetic analysis using graphical mapping indicated that resistance (R) was controlled by a new resistance gene linked with the flanking markers RM27320 and ID55.WA18-5 within an approximately 80-kb region between 28.14 and 28.22 Mbp on chromosome 11. The eight candidate genes functionally predicted were included in the target region. Examination of the candidate genes by RT-PCR analysis only corroborated with the significant difference in transcript levels of the WAK3 gene in the presence or absence of pathogen infection. Allelism tests performed with other known BB R-genes revealed that the allele was distinct from others having a similar chromosomal location.

SNP in starch biosynthesis genes associated with nutritional and functional properties of rice.

Starch is a major component of human diets. The relative contribution of variation in the genes of starch biosynthesis to the nutritional and functional properties of the rice was evaluated in a rice breeding population. Sequencing 18 genes involved in starch synthesis in a population of 233 rice breeding lines discovered 66 functional SNPs in exonic regions. Five genes, AGPS2b, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Association analysis found 31 of the SNP were associated with differences in pasting and cooking quality properties of the rice lines. Two genes appear to be the major loci controlling traits under human selection in rice, GBSSI (waxy gene) and SSIIa. GBSSI influenced amylose content and retrogradation. Other genes contributing to retrogradation were GPT1, SSI, BEI and SSIIIa. SSIIa explained much of the variation in cooking characteristics. Other genes had relatively small effects.

Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing.

High-throughput sequencing of pooled DNA was applied to polymorphism discovery in candidate genes involved in starch synthesis. This approach employed semi- to long-range PCR (LR-PCR) followed by next-generation sequencing technology. A total of 17 rice starch synthesis genes encoding seven classes of enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), starch debranching enzyme (DBE) and starch phosphorylase (SPHOL) and phosphate translocator (GPT1) from 233 genotypes were PCR amplified using semi- to long-range PCR. The amplification products were equimolarly pooled and sequenced using massively parallel sequencing technology (MPS). By detecting single nucleotide polymorphism (SNP)/Indels in both coding and noncoding areas of the genes, we identified genetic differences and characterized the SNP/Indel variation and distribution patterns among individual starch candidate genes. Approximately, 60.9 million reads were generated, of which 54.8 million (90%) mapped to the reference sequences. The average coverage rate ranged from 12,708 to 38,300 times for SSIIa and SSIIIb, respectively. SNPs and single/multiple-base Indels were analysed in a total assembled length of 116,403 bp. In total, 501 SNPs and 113 Indels were detected across the 17 starch-related loci. The ratio of synonymous to nonsynonymous SNPs (Ka/Ks) test indicated GBSSI and isoamylase 1 (ISA1) as the least diversified (most purified) and conservative genes as the studied populations have been through cycles of selection. This report demonstrates a useful strategy for screening germplasm by MPS to discover variants in a specific target group of genes.

Influence of imidacloprid seed treatments on rice germination and early seedling growth.

BACKGROUND: Seed treatments with the chloronicotinyl insecticide imidacloprid (Gaucho 600 FS) were evaluated to determine whether differences in concentration and exposure regime influence the germination and early growth of rice. RESULTS: Continuous exposure to imidacloprid (4 days at 2000 mg AI L(-1)) significantly (P < 0.001) reduced normal germination by an average of 18% across the 15 cultivars examined. Nine days after sowing, plants showed no adverse effects from continuous imidacloprid treatment during germination, with shoot lengths and root system dry weights equalling, or occasionally exceeding (P < 0.05), those of untreated plants. Short-term imidacloprid exposure (2 h at 2000 mg L(-1)) at initial seed wetting did not affect germination (P > 0.05), and short-term (1 h) exposure of 48 h pregerminated seed to imidacloprid (2000 mg L(-1)) similarly had no significant effect on early subsequent growth. Plants arising from 48 h pregerminated seed exposed to imidacloprid (1 h) at concentrations up to 4000 mg L(-1) immediately before sowing were not significantly different from control plants at either 9 or 25 days post-sowing. CONCLUSION: Results show that imidacloprid will have no adverse effects on plant growth if applied to pregerminated rice shortly before sowing. Continuous exposure of seed during germination had more pronounced effects, and the initial response of different cultivars was highly variable. Cultivars with high levels of sensitivity (such as IR72) require further testing before continuous exposure to imidacloprid during germination can be recommended.

Gelatinization temperature of rice explained by polymorphisms in starch synthase.

The cooking quality of rice is associated with the starch gelatinization temperature (GT). Rice genotypes with low GT have probably been selected for their cooking quality by humans during domestication. We now report polymorphisms in starch synthase IIa (SSIIa) that explain the variation in rice starch GT. Sequence analysis of the eight exons of SSIIa identified significant polymorphism in only exon 8. These single nucleotide polymorphisms (SNPs) were determined in 70 diverse genotypes of rice. Two SNPs could classify all 70 genotypes into either high GT or low GT types which differed in GT by 8 degrees C. 'A' rather than 'G' at base 2412 determined whether a methionine or valine was present at the corresponding amino acid residue in SSIIa, whilst two adjacent SNPs at bases 2543 and 2544 coded for either leucine (GC) or phenylalanine (TT). Rice varieties with high GT starch had a combination of valine and leucine at these residues. In contrast, rice varieties with low GT starch had a combination of either methionine and leucine or valine and phenylalanine at these same residues. At least two distinct polymorphisms have apparently been selected for their desirable cooking qualities in the domestication of rice.