Identification of resistant germplasm containing novel resistance genes at or tightly linked to the <Emphasis Type="Italic">Pi2/9</Emphasis> locus conferring broad-spectrum resistance against rice blast

Background

The rice Pi2/9 locus harbors multiple resistance (R) genes each controlling broad-spectrum resistance against diverse isolates of Magnaporthe oryzae, a fungal pathogen causing devastating blast disease to rice. Identification of more resistance germplasm containing novel R genes at or tightly linked to the Pi2/9 locus would promote breeding of resistance rice cultivars.

Results

In this study, we aim to identify resistant germplasm containing novel R genes at or tightly linked to the Pi2/9 locus using a molecular marker, designated as Pi2/9-RH (Pi2/9 resistant haplotype), developed from the 5′ portion of the Pi2 sequence which was conserved only in the rice lines containing functional Pi2/9 alleles. DNA analysis using Pi2/9-RH identified 24 positive lines in 55 shortlisted landraces which showed resistance to 4 rice blast isolates. Analysis of partial sequences of the full-length cDNAs of Pi2/9 homologues resulted in the clustering of these 24 lines into 5 haplotypes each containing different Pi2/9 homologues which were designated as Pi2/9-A5, ?A15, ?A42, ?A53, and -A54. Interestingly, Pi2/9-A5 and Pi2/9-A54 are identical to Piz-t and Pi2, respectively. To validate the association of other three novel Pi2/9 homologues with the blast resistance, monogenic lines at BC₃F₃ generation were generated by marker assisted backcrossing (MABC). Resistance assessment of the derived monogenic lines in both the greenhouse and the field hotspot indicated that they all controlled broad-spectrum resistance against rice blast. Moreover, genetic analysis revealed that the blast resistance of these three monogenic lines was co-segregated with Pi2/9-RH, suggesting that the Pi2/9 locus or tightly linked loci could be responsible for the resistance.

Conclusion

The newly developed marker Pi2/9-RH could be used as a potentially diagnostic marker for the quick identification of resistant donors containing functional Pi2/9 alleles or unknown linked R genes. The three new monogenic lines containing the Pi2/9 introgression segment could be used as valuable materials for disease assessment and resistance donors in breeding program.

     

Large-scale deployment of a rice 6 K SNP array for genetics and breeding applications

Background

Fixed arrays of single nucleotide polymorphism (SNP) markers have advantages over reduced representation sequencing in their ease of data analysis, consistently higher call rates, and rapid turnaround times. A 6 K SNP array represents a cost-benefit “sweet spot” for routine genetics and breeding applications in rice. Selection of informative SNPs across species and subpopulations during chip design is essential to obtain useful polymorphism rates for target germplasm groups. This paper summarizes results from large-scale deployment of an Illumina 6 K SNP array for rice.

Results

Design of the Illumina Infinium 6 K SNP chip for rice, referred to as the Cornell_6K_Array_Infinium_Rice (C6AIR), includes 4429 SNPs from re-sequencing data and 1571 SNP markers from previous BeadXpress 384-SNP sets, selected based on polymorphism rate and allele frequency within and between target germplasm groups. Of the 6000 attempted bead types, 5274 passed Illumina’s production quality control. The C6AIR was widely deployed at the International Rice Research Institute (IRRI) for genetic diversity analysis, QTL mapping, and tracking introgressions and was intensively used at Cornell University for QTL analysis and developing libraries of interspecific chromosome segment substitution lines (CSSLs) between O. sativa and diverse accessions of O. rufipogon or O. meridionalis. Collectively, the array was used to genotype over 40,000 rice samples. A set of 4606 SNP markers was used to provide high quality data for O. sativa germplasm, while a slightly expanded set of 4940 SNPs was used for O. sativa X O. rufipogon populations. Biparental polymorphism rates were generally between 1900 and 2500 well-distributed SNP markers for indica x japonica or interspecific populations and between 1300 and 1500 markers for crosses within indica, while polymorphism rates were lower for pairwise crosses within U.S. tropical japonica germplasm. Recently, a second-generation array containing ~7000 SNP markers, referred to as the C7AIR, was designed by removing poor-performing SNPs from the C6AIR and adding markers selected to increase the utility of the array for elite tropical japonica material.

Conclusions

The C6AIR has been successfully used to generate rapid and high-quality genotype data for diverse genetics and breeding applications in rice, and provides the basis for an optimized design in the C7AIR.

     

Microalgal paste production of the diatom <Emphasis Type="Italic">Chaetoceros calcitrans</Emphasis> using electrolytic flocculation method at optimum culture conditions

The optimum culture conditions of the local strain Chaetoceros calcitrans were determined to improve biomass and reduce cost of production. Under outdoor culture conditions, higher cell density was attained when the cultures were enriched with Tungkang Marine Research Laboratory (TMRL) medium composed of cheap technical grade reagents and cultured at 25 g L^?1 salinity. The cultures were lighted with two 40 W cool-white GE fluorescent tubes (24–35 μmol photon m^?2 s^?1). Using semi-continuous culture system under established optimum culture conditions, C. calcitrans can be re-cultured thrice and concentrated at each culture cycle using electrolytic flocculation method to produce 4.6 kg m^?3 of diatom paste. The viability of concentrated C. calcitrans after 3 months of storage was comparable to live diatom cells. Simple preservation technique by low-temperature storage is convenient for storing algal concentrates for use as starter cultures and for feeding invertebrates. The paste costs USD 8.24 kg^?1 inclusive of the assets and flocculation materials for culturing and harvesting the diatom, respectively. This study established the suitable conditions for mass culture of C. calcitrans and produced concentrated diatoms in paste form that is readily available for aquaculture hatcheries at a lower cost.     

New trends in the control of reproduction in the bovine

The new trends in the control of animal reproduction have been theoretically well-known for 25 years, and some of these methods have now been found to have a practical value.A short-term treatment (10 days) of a progestagen implant (SC 21009) begun by oestradiol valerate administration and followed by PMSG injection, induces oestrus and ovulation in nursing cows. The synchronisation of oestrus is adequate for artificial insemination at a predetermined time, with 68% of cows ultimately calving.If there is a large number of cyclic cows in a herd, this treatment does not give such a precise synchronisation of oestrus.Limited superovulation using PMSG is not restricted to the production of twins, but includes triplets and quadruplets. However, the viability of embryos and calves in multifoetal pregnancy can be improved by choosing a proven bull, a particular breed of cow, and by super-alimentation towards the end of pregnancy. All can be won or lost at the time of parturition.Such methods for the control of certain phases of the reproductive cycle are useful in improved cattle production.     

Locating QTLs controlling constitutive root traits in the rice population IAC 165 × Co39

Drought is an important constraint to productivity in rainfed rice environments. Improvement in the various components of rice drought tolerance is now possible through the identification and manipulation of DNA markers linked with genes controlling these quantitative traits. A recombinant inbred line population was derived from the cross IAC165 × Co39. A molecular map was built that contained 182 RFLP and microsatellite markers. Segregation distortions were limited to a few chromosomal segments. Constitutive root traits, including maximum root length, root thickness and root dry weight in various layers, were measured on 125 lines in a greenhouse replicated experiment. QTL analysis was performed using composite interval mapping. Between 1and 4 main effect QTLs, which explained individually between 5.5 and 24.8% of the variability, were identified for each trait. The most important genomic regions, which carried QTLs for several traits, were found on chromosomes 1, 4, 9, 11 and 12.The QTL locations were in good agreement with previous studies on these traits, confirming the value of the QTLs in a different genetic background. Epistasis represented a non-negligible component of the observed variability for some of the traits but was not detected for others. These results add to the understanding of the genetic control of root morphology in rice, which is necessary to strengthen marker-aided selection programs to improve varieties for water-limited environments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantification of pericaudal adipocyte diameter in dairy cattle during peripartum: a complementary method to study energetic status using conventional histology

The energetic status of high‐yielding Holstein‐Friesian dairy cattle was studied during peripartum under field conditions using body condition score (BCS), glycemia, seric ß–hydroxybutyrate and adipose tissue cellularity. This last method was tested as a complementary tool for energetic status assessment. Biopsies of pericaudal subcutaneous adipose tissue were obtained from 25 multiparous animals at 28 days before and 21 days after parturition. Samples were routinely processed for histological examination and stained with haematoxylin and eosin (H&E). The mean diameter of adipocytes (MDA) was measured with the aid of a digital image processor. During the same period, blood samples were collected weekly for metabolite determinations. The MDA at 28 days pre‐partum and 21 days post‐partum were 72.1 vs. 66.2 μm respectively (p = 0.055), and the corresponding BCS at these moments was 3.32 vs. 3.19 (p = 0.068). At ?28 days pre‐partum, the BCS was positively correlated with MDA (Pearson’s r = 0.521, p = 0.016) and with glycemia (Pearson’s r = 0.404, p = 0.056). Correlations between BCS and MDA, and between BCS and glycemia, with ß–hydroxybutyrate although not significant, suggest that routine histological preparations of biopsies from subcutaneous adipose tissue could be included as an easy and valuable tool for research purposes to evaluate metabolic adaptation of dairy cows to peripartum, as well as the incidence of metabolic disorders and productive performance.     

Genotypic variability and aggressiveness of Bipolaris oryzae in the Philippines

The genotypic diversity of a collection of 352 isolates of Bipolaris oryzae obtained from 11 locations in the Philippines was estimated. The isolates could be divided into 50 haplotypes based on variation in microsatellite DNA with a moderately high genotypic diversity value of 0.88. Thirty nine haplotypes were represented by three or fewer isolates, whereas 80 % of the isolates belonged to only eight haplotypes, each containing 10 to 88 isolates indicating the prevalence of clonality. AMOVA revealed that the greatest variation was associated with the brown spot isolates collected within provinces (50.81 %), among varieties within provinces (48.17 %) and within ecosystems (49.33 %). Intensive sampling from a single field showed that the population was mostly clonal with about 98 % of the isolates belonging to a single VNTR haplotype. However, isolates within this haplotype exhibited a continuous range of aggressiveness when inoculated onto susceptible rice variety IR72. Several types of lesions were observed in the field during sampling, but the isolates obtained from each type of lesion produced a range of different lesion types when inoculated onto leaves of IR72, indicating that the type of lesion observed in the field was not related to the genotype of the pathogen. These results show that rice fields across the Philippines may contain B. oryzae isolates with considerable genotypic diversity, but an individual field may have both clonal and unique genotypes.     

A Recessive Mutation in Rice Conferring Non-Race-Specific Resistance to Bacterial Blight and Blast

To understand the basis of broad-spectrum disease resistance in rice, we isolated a gamma-ray-induced IR64 mutant G978 that showed enhanced resistance to blast and bacterial blight. The resistance is quantitative and non-race specific against the bacterial and fungal pathogens. The mutation is inherited as a single recessive gene, designated as Bsdr1 and causes shorter stature relative to the wild type; however, it does not show lesion mimics phenotype under the conditions tested. The mutation was mapped as a quantitative trait locus to a 3.8-Mb region on chromosome 12. By comparing the gene expression profiles of the mutant and wild type, we identified a candidate gene encoding a U-box domain-containing protein. The disrupted gene showed a loss of expression in the mutant and co-segregated with mutant phenotype. The mutant provides a useful tool for investigating the important genes responsible for non-race specific resistance to two distinct diseases.