Four QTL in Rice Associated with Broad Spectrum Resistance to Blast Isolates from Rice and Barley

Pyricularia grisea is the most destructive and cosmopolitan fungal pathogen of rice and it can also cause disease on other agriculturally important cereals. We determined the number, location and interaction of quantitative trait loci (QTL) associated with resistance to P. grisea isolates obtained from rice (THL142 and THL222) and barley (TH16 and THL80) grown in Thailand. The isolates showed a spectrum of virulence when used to inoculate a series of differentials. We used a reference blast resistance mapping population of rice (IR64 × Azucena). IR64 was highly resistant, and Azucena was highly susceptible, to all four isolates. The numbers of resistant vs. susceptible progeny suggest that the resistance of IR64 is determined by two or three genes with additive effects. The correlation coefficients for all pairwise comparisons of disease severity were high and highest between barley isolates and between rice isolates. Four QTL were detected, one on each of the following chromosomes 2, 8, 9 and 10. IR64 contributed resistance alleles at three of the QTL (chromosomes 2, 8 and 9). Azucena contributed the resistance allele at the QTL on chromosome 10 in response to inoculation with isolate THL142. The results of the QTL analysis support interpretation of the phenotypic frequency distributions regarding the number of genes determining resistance to the four isolates in this population. Our results are novel in adding blast isolates from barley to the catalogue of pathogen specificities to which a gene, or genes, from IR64 confer resistance.     

Sequence-based genetic markers for genes and gene families: single-strand conformational polymorphisms for the fatty acid synthesis genes of Cuphea

 Gene sequences are rapidly accumulating for many commercially and scientifically important plants. These resources create the basis for developing sequence-based markers for mapping and tracking known (candidate) genes, thereby increasing the utility of genetic maps. Members of most of the gene families underlying the synthesis of seed oil fatty acids have been cloned from the medium-chain oilseed Cuphea. Allele-specific-PCR (AS-PCR) and single-strand conformational polymorphism (SSCP) markers were developed for 22 fatty acid synthesis genes belonging to seven gene families of Cuphea using homologous and heterologous DNA sequences. Markers were developed for 4 fatty-acyl-acyl carrier protein thioesterase, 2 β-ketoacyl-acyl carrier protein synthase I, 4 β-ketoacyl-acyl carrier protein synthase II, 3 β-ketoacyl-acyl carrier protein synthase III, 3 acyl carrier protein, 2 β-ketoacyl-acyl carrier protein reductase, and 4 enoyl-acyl carrier protein reductase loci. Eighty-eight percent (14 of 16) of the SSCP loci were polymorphic, whereas only 9% (2 of 22) of the AS-PCR loci were polymorphic. These markers were mapped using a Cuphea viscosissima×C. lanceolata F₂ population and produced linkage groups of 10, 3, and 2 loci (3 loci segregated independently). The 10-locus linkage group had every gene but one necessary for the synthesis of 2- to 16-carbon fatty acids from acetyl-CoA and malonyl-ACP (the missing gene family was not mapped). SSCP analysis has broad utility for DNA fingerprinting and mapping genes and gene families. Received: 3 May 1996 / Accepted: 30 August 1996     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.     

Amplified fragment length polymorphisms as a tool for DNA fingerprinting sunflower germplasm: genetic diversity among oilseed inbred lines

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient method for producing DNA fingerprints. The AFLP diversity of sunflower has not been described, and much of the public germ plasm of sunflower has not yet been fingerprinted. Our objectives were to: (1) estimate genetic similarities, polymorphism rates, and polymorphic information contents (PICs) for AFLP markers among elite public oilseed inbred lines, and (2) assess the genetic diversity of inbred lines using genetic similarities estimated from AFLP fingerprints. We produced fingerprints for 24 public inbred lines of sunflower (Helianthus annuus L.) using six AFLP primer combinations. These primers produced a total of 359 AFLP markers or about 60 markers per primer combination. Genetic similarities ranged from 0.70 to 0.91, polymorphism rates ranged from 7 to 24%, and PICs ranged from 0.0 to 0.5. Genetic similarities were lower overall for maintainer (B)×restorer (R) crosses than for B×B or R×R crosses. Principal-coordinate and cluster analyses separated lines into two groups, one for B-lines and another for R-lines. These groupings illustrate the breeding history and basic heterotic pattern (B×R) of sunflower and the widespread practice of using B×B and R×R crosses to develop new lines. There were, nevertheless, distinct subgroups within these groups. These subgroups may represent unique heterotic groups and create a basis for formally describing heterotic patterns in sunflower. Received: 10 June 1996 / Accepted: 4 April 1997     

Molecular identification of the economically important freshwater mussels (Mollusca–Bivalvia–Unionoida) of Thailand: developing species‐specific markers from AFLPs

Shells of certain freshwater mussel (Unionoida) species are highly demanded and serve as raw material for a range of decorative and pharmaceutical products. In Thailand, most animals for this purpose are currently harvested from wild populations, with unionoid culture still being in its infancy. Whilst reliable species identification is a prerequisite for developing a large‐scale industry, identification by morphological means is hampered by extensive phenotypic plasticity and poor knowledge of species delimitations. To facilitate alternative molecular identification, we developed species‐specific markers for the three Thai unionoids with considerable economic potential (CEP): that is, Chamberlainia hainesiana, Hyriopsis desowitzi and Hyriopsis myersiana. For this purpose, amplified fragment length polymorphism (AFLP) fingerprints using 24 specific primer pairs were generated for eight samples of each CEP species and four samples of the closely related, non‐CEP species Contradens contradens. Cloning and sequencing of 13 CEP species‐specific AFLP bands revealed fragment collision at three occasions. In total, 16 species‐specific primer pairs were designed and tested on 92 Thai specimens spanning seven species and four genera. Thereby, specificity of (1) three primers to C. hainesiana, (2) one primer to H. desowitzi + Hyriopsis bialata, (3) one primer to H. myersiana + H. bialata and (4) four primers to all three Hyriopsis species tested was confirmed. Respective multiplex PCR protocols are provided. The developed primers enable cheap, quick and reliable identification of the Thai CEP species by one to three PCRs and offer a tool for a range of additional applications within mussel culture and ecological and evolutionary research on these important organisms.     

DFLP,SSCP, and SSR markers for Δ9-stearoyl-acyl carrier protein desaturases strongly expressed in developing seeds of sunflower: intron lengths are polymorphic among elite inbred lines

Stearoyl-acyl carrier protein desaturase (SAD, EC 1.14.99.6) produces oleic acid (18:1 9) by desaturating 18:0. SAD genes have been targets for breeding and engineering oilseed crops with increased stearic acid (18:0). Our aim was to clone, describe, and develop genetic markers for the SAD genes of sunflower (Helianthus annuus L.). Nineteen SAD cDNA clones were partially sequenced and found to belong to two groups. Full-length cDNAs from each group (SAD6 and SAD17) were completely sequenced. The amino acid identity of SAD6 and SAD17 was 89%. Both genes were strongly expressed in developing seeds, moderately expressed in leaves and flowers, and weakly expressed in cotyledons, roots, and stems. One intron was found in SAD6 and two introns were found in SAD17. The SAD introns from two inbred lines (HA370 and HA372) were sequenced and found to vary in length and nucleotide sequence. The length variants were caused by monomeric repeat length differences, insertions, and deletions. Three long poly-T repeats (T₉ to T₃₉) were found in one of the SAD17 introns. Three short adjacent CA repeats were found in the 5-untranslated region of SAD6. DNA fragment length polymorphism (DFLP), single-strand conformational polymorphism (SSCP), and simple sequence repeat (SSR) markers were developed for SAD6 and SAD17 by developing primers to flank introns or the CA repeats. Two of six DFLP, four of six SSCP, and one of two SSR markers were polymorphic among eight elite inbred lines. The polymorphic information contents for DFLP, SSCP, and SSR markers were 0.18, 0.37, and 0.30, respectively. Most of the polymorphisms were caused by intron fragment length polymorphisms. Introns may be an excellent source of hypervariable markers in sunflower and other crop plants.