William D. Park

Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.).

Abstract In order to enhance the resolution of an existing genetic map of rice, and to obtain a comprehensive picture of marker utility and genomic distribution of microsatellites in this important grain species, rice DNA sequences containing simple sequence repeats (SSRs) were extracted from several small-insert genomic libraries and from the database. One hundred and eighty eight new microsatellite markers were developed and evaluated for allelic diversity. The new simple sequence length polymorphisms (SSLPs) were incorporated into the existing map previously containing 124 SSR loci. The 312 microsatellite markers reported here provide whole-genome coverage with an average density of one SSLP per 6 cM. In this study, 26 SSLP markers were identified in published sequences of known genes, 65 were developed based on partial cDNA sequences available in GenBank, and 97 were isolated from genomic libraries. Microsatellite markers with different SSR motifs are relatively uniformly distributed along rice chromosomes regardless of whether they were derived from genomic clones or cDNA sequences. However, the distribution of polymorphism detected by these markers varies between different regions of the genome.

Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.)

Abstract The growing number of rice microsatellite markers warrants a comprehensive comparison of allelic variability between the markers developed using different methods, with various sequence repeat motifs, and from coding and non-coding portions of the genome. We have performed such a comparison over a set of 323 microsatellite markers; 194 were derived from genomic library screening and 129 were derived from the analysis of rice-expressed sequence tags (ESTs) available in public DNA databases. We have evaluated the frequency of polymorphism between parental pairs of six inter- subspecific crosses and one inter-specific cross widely used for mapping in rice. Microsatellites derived from genomic libraries detected a higher level of polymorphism than those derived from ESTs contained in the GenBank database (83.8% versus 54.0%). Similarly, the other measures of genetic variability [the number of alleles per locus, polymorphism information content (PIC), and allele size ranges] were all higher in genomic library-derived microsatellites than in their EST-database counterparts. The highest overall degree of genetic diversity was seen in GA-containing microsatellites of genomic library origin, while the most conserved markers contained CCG- or CAG-trinucleotide motifs and were developed from GenBank sequences. Preferential location of specific motifs in coding versus non-coding regions of known genes was related to observed levels of microsatellite diversity. A strong positive correlation was observed between the maximum length of a microsatellite motif and the standard deviation of the molecular-weight of amplified fragments. The reliability of molecular weight standard deviation (SDmw) as an indicator of genetic variability of microsatellite loci is discussed.

Microsatellites and a single-nucleotide polymorphism differentiate apparentamylose classes in an extended pedigree of US rice germ plasm

Abstract The Waxy gene (Wx) encodes the granule-bound starch synthase responsible for the synthesis of amylose in rice (Oryza sativa). Recently, a polymorphic microsatellite sequence closely linked to the Wx gene was reported. To determine whether polymorphism in this sequence correlates with variation in apparent amylose content, we tested an extended pedigree of 92 current and historically important long-, medium- and short-grain US rice cultivars representing the efforts of many breeders over more than 80 years. Seven Wx microsatellite alleles were identified which together explained 82.9% of the variation in apparent amylose content of the 89 non-glutinous rice cultivars tested. Similar results were also obtained with 101 progenyof a cross between low- and intermediate-amylose breeding lines. An additional, unique microsatelliteallele, (CT)16, was detected in one glutinous cultivar,CI 5309. However, the other glutinous cultivars,Calmochi 101 and Tatsumi mochi, were in the (CT)17 class along with three other cultivars that contained15–16.5% amylose.We sequenced a 200-bp PCR-amplified fragment containing the CT microsatellite and the putative 5′ splice site of the Wx leader intron from a subset of 42 cultivars representing all eight microsatellite alleles. All of the cultivars with 18% or less amylose had the sequence AGTTATA at the putative leader intron 5′ splice site, while all cultivars with a higher proportionof amylose had AGTTATA. This single nucleotidesubstitution could also be assayed by AccI digestion of the amplified fragment. Overall, this single nucleotide polymorphism could explain 79.7% of the variation in the apparent amylose content of the 89 non-glutinous cultivars tested. Interestingly, cultivars in the (CT)19 microsatellite classes that differed substantially in amylose content still showed the correlation between this G-T polymorphism and apparent amylose content. The G-T polymorphism at this site was not, however, able to explain the very low amylose contents of the three glutinous cultivars tested, all of which had the sequence AGTTATA.

Identification of quantitative trait loci (QTLs) for heading date and plant height in cultivated rice (Oryza sativa L.)

Abstract‘Lemont’ and ‘Teqing’ are both semidwarf rice varieties that differ in heading date by only 6 days. However, when ‘Lemont’ and ‘Teqing’ are crossed there is transgressive segregation for both heading date (HD) and plant height (PH). By testing 2418 F4 lines with 113 well-distributed RFLP markers, we identified and mapped chromosomal regions that were largely responsible for this transgressive segregation. QHd3a, a QTL from ‘Lemont’ that gives 8 days earlier heading, was identified on chromosome 3 approximately 3 cM from the marker RG348. Another QTL with a large effect, QHd8a, which gives 7 days earlier heading, was identified on chromosome 8 of ‘Teqing’ between RG20 and RG1034. Along with a QTL, QHd9a with a phenotypic effect of 3.5 days, these genomic regions collectively explain 76.5% of the observed phenotypic variance in heading date. Four QTLs which altered plant height from 4 to 7 cm were also mapped; these collectively explain 48.8% of the observed phenotypic variation in plant height. None of the QTLs for plant height mapped to chromosome 1, the location of the semidwarf gene sd-1. All three of the HD loci mapped to approximately the same genomic locations as PH QTLs, and in all cases, there was a reduction in height of approximately 1 cm for every day of earlier heading. The correspondence between the HD and some of the PH loci suggests that genes at these chromosome locations may have pleiotropic effects on both HD and PH. The observed heterosis in the F1 plants for HD can be largely explained by the dominance for earliness of the identified HD loci and distribution of earlier heading alleles in the parents. However, overdominance observed at one of the PH QTL may, at least in part, be responsible for the observed heterosis in PH.

Sink- and vascular-associated sucrose synthase functions are encoded by different gene classes in potato.

Two differentially regulated classes of sucrose synthase genes, Sus3 and Sus4, were identified in potato. They cannot be classified as Sus1 and Sus2 types based on sequence homology and appear to have evolved after the divergence of the major families of dicotyledonous plants but before the divergence of tomato and potato. The potato sucrose synthase clones Sus3-65 and Sus4-16 share an 87% nucleotide identity in the coding regions, and both are interrupted by 13 introns, including a long leader intron. Potato Sus3 genes are expressed at the highest levels in stems and roots and appear to provide the vascular function of sucrose synthase. In contrast, Sus4 genes are expressed primarily in the storage and vascular tissue of tubers and appear to facilitate sink function. The genes are differentially regulated in root tips, with Sus3 expressed at high levels in the cell division zone and Sus4 expressed at high levels in the meristem and cap.

Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani).

Sheath blight, caused by Rhizoctonia solani, is one of the most important diseases of rice. Despite extensive searches of the rice germ plasm, the major gene(s) which give complete resistance to the fungus have not been identified. However, there is much variation in quantitatively inherited resistance to R. solani, and this type of resistance can offer adequate protection against the pathogen under field conditions. Using 255 F4 bulked populations from a cross between the susceptible variety ‘Lemont’ and the resistant variety ‘Teqing’, 2 years of field disease evaluation and 113 well-distributed RFLP markers, we identified six quantitative trait loci (QTLs) contributing to resistance to R. solani. These QTLs are located on 6 of the 12 rice chromosomes and collectively explain approximately 60% of the genotypic variation or 47% of the phenotypic variation in the ‘Lemont’x‘Teqing’ cross. One of these resistance QTLs (QSbr4a), which accounted for 6% of the genotypic variation in resistance to R. solani, appeared to be independent of associated morphological traits. The remaining five putative resistance loci (QSbr2a, QSbr3a, QSbr8a, QSbr9a and QSbr12a) all mapped to chromosomal regions also associated with increased plant height, three of which were also associated with QTLs causing later heading. This was consistent with the observation that heading date and plant height accounted for 47% of the genotypic variation in resistance to R. solani in this population. There were also weak associations between resistance to R. solani and leaf width, which were likely due to linkage with a QTL for this trait rather than to a physiological relationship.

Analysis of a chimeric class-I patatin-GUS gene in transgenic potato plants: High-level expression in tubers and sucrose-inducible expression in cultured leaf and stem explants.

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. To examine the regulation of the patatin genes, we constructed a chimeric gene containing 2.5 kb of 5′ flanking sequence from the class I patatin genomic clone PS20 transcriptionally fused to β-glucuronidase (GUS) and introduced it into potato plants using an Agrobacterium tumefaciens Tiplasmid vector. While the chimeric gene was expressed at high levels in tubers and in stolons attached to developing tubers, it was not normally expressed in leaves, stems, roots, or in stolons before tuberizatization. However, the expression of the class I patatin-GUS construct was not “tuber-specific” since leaf and stem explants cultured on medium containing 300 to 400 mM sucrose showed GUS activity equal or greater than that of tubers. The sucrose induction of GUS activity in leaf and stem explants was accompanied by the accumulation of patatin protein and large amounts of starch, but not by the morphological changes that normally are associated with tuberization. In contrast, the GUS reporter gene under the control of the 35S promoter of cauliflower mosaic virus showed an essentially uniform pattern of expression in transgenic potato plants and was not induced by sucrose.

Anchor probes for comparative mapping of grass genera

Abstract Comparative mapping of cDNA clones provides an important foundation for examining structural conservation among the chromosomes of diverse genera and for establishing hypotheses about the relationship between gene structure and function in a wide range of organisms. In this study, “anchor probes” were selected from cDNA libraries developed from rice, oat, and barley that were informative for comparative mapping in the grass family. One thousand eight hundred probes were screened on garden blots containing DNA of rice, maize, sorghum, sugarcane, wheat, barley, and oat, and 152 of them were selected as “anchors” because (1) they hybridized to the majority of target grass species based on Southern analysis, (2) they appeared to be low or single copy in rice, and (3) they helped provide reasonably good genome coverage in all species. Probes were screened for polymorphism on mapping parents, and polymorphic markers were mapped onto existing species-specific linkage maps of rice, oat, maize, and wheat. In wheat, both polymorphic and monomorphic markers could be assigned to chromosomes or chromosome arms based on hybridization to nullitetrasomic and ditelosomic stocks. Linkage among anchored loci allowed the identification of homoeologous regions of these distantly related genomes. Anchor probes were sequenced from both ends, providing an average of 260 bp in each direction, and sequences were deposited in GenBank. BLAST was used to compare the sequences with each other and with a non-redundant protein sequence database maintained at the European Molecular Biology Laboratory (EMBL). Of the anchor probes identified in this study 78% showed significant similarity to protein sequences for known genes with BLASTX scores exceeding 100.

Phosphate modulates transcription of soybean VspB and other sugar-inducible genes

The soybean vegetative storage protein genes VspA and VspB encode vacuolar glycoprotein acid phosphatases. Transcription of the Vsp is synergistically activated by jasmonic acid or methyl jasmonate (MeJA) and soluble sugars. The action of these modulators is mediated by two different DNA domains in the VspB promoter. In this study, we present new data regarding VspB regulation by sucrose and inorganic phosphate, which suggest a common mechanism of transcriptional control for Vsp and other sugar-inducible genes. We found that the sugar-mediated activation of VspB expression was inhibited by phosphate. Deletion analysis and transient assays in tobacco protoplasts identified a 130-bp DNA domain in the VspB promoter that mediates both sucrose induction and phosphate inhibition. Transcription mediated by this DNA domain was induced by phosphate elimination from the protoplast incubation medium, even in the absence of sucrose. The effect of sucrose and phosphate on VspB expression was studied in vivo in several ways. Depletion of phosphate from soybean cell cultures by the addition of mannose stimulated VspB expression, even in the absence of sucrose or MeJA. In illuminated soybean leaves treated with MeJA, inhibition of photosynthetic electron transport by DCMU decreased VspB expression. In contrast, VspB expression in soybean leaves stimulated by phosphate depletion was not influenced by DCMU. Moreover, sucrose-stimulated expression of the sugar-responsive genes lipoxygenase A and chalcone synthase of soybean and proteinase inhibitor II and class I patatin of potato was inhibited by phosphate. Like VspB, these genes were stimulated by phosphate depletion in the absence of exogenous sucrose. We propose that sugar-responsive genes are activated, in part, by accumulation of sugar-phosphates and concomitant reduction of cellular phosphate levels. These data may help explain recruitment of the Vsp, which encode acid phosphatases, as vegetative storage proteins.

Is all red rice found in commercial rice really Oryza sativa

Abstract All red rice found in commercial rice in the United States has traditionally been classified as Oryza sativa ssp. indica. This assumption was tested by analyzing red rice samples collected from across the southern United States rice belt with 18 simple sequence length polymorphism (SSLP) markers distributed across all 12 chromosomes. The results clearly demonstrate that the traditional classification of red rice is inadequate. Some red rice is closely related to O. sativa ssp. indica cultivated rice. However, other red rice is more closely related to O. sativa ssp. japonica. Most importantly, some red rice samples collected from Arkansas, Louisiana, Mississippi, and Texas form a distinct group that includes a number of Oryza nivara and Oryza rufipogon accessions from the National Small Grains Center. In particular, red rice samples from three states were identified that for all 18 markers are identical to the O. rufipogon accession IRGC 105491. These different classes of red rice are intermingled across the southern U.S. rice belt and within individual production fields. Oryza sativa ssp. indica-like red rice and O. rufipogon-like red rice have been found within a single 9-m2 collection site. While the classification of red rice as O. sativa ssp. indica, O. sativa ssp. japonica, or O. rufipogon using DNA markers is generally in agreement with classification based on simple morphological traits, readily observed morphological traits alone are not sufficient to reliably classify red rice. Because red rice is much more diverse than previously assumed, this diversity must be considered when developing red rice management strategies. Nomenclature: Glufosinate; Oryza nivara L.; Oryza rufipogon L.; Oryza sativa L. ORYSA, red rice; Oryza sativa L., rice.