Rice DNA Research Articles

Identification of Protein Coding Regions of Rice Genes Using Alternative Spectral Rotation Measure and Linear Discriminant Analysis

An improved method, called Alternative Spectral Rotation (ASR) measure, for predicting protein coding regions in rice DNA has been developed. The method is based on the Spectral Rotation (SR) measure proposed by Kotlar and Lavner, and its accuracy is higher than that of the SR measure and the Spectral Content (SC) measure proposed by Tiwari et al. In order to increase the identifying accuracy, we chose three different coding characters, namely the asymmetric, purine, and stop-codon variables as parameters, and an approving result was presented by the method of Linear Discriminant Analysis (LDA).

Development of genome-wide DNA polymorphism database for map-based cloning of rice genes.

DNA polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. A genome-wide rice (Oryza sativa) DNA polymorphism database has been constructed in this work using the genomes of Nipponbare, a cultivar of japonica, and 93-11, a cultivar of indica. This database contains 1,703,176 single nucleotide polymorphisms (SNPs) and 479,406 Insertion/Deletions (InDels), approximately one SNP every 268 bp and one InDel every 953 bp in rice genome. Both SNPs and InDels in the database were experimentally validated. Of 109 randomly selected SNPs, 107 SNPs (98.2%) are accurate. PCR analysis indicated that 90% (97 of 108) of InDels in the database could be used as molecular markers, and 68% to 89% of the 97 InDel markers have polymorphisms between other indica cultivars (Guang-lu-ai 4 and Long-te-pu B) and japonica cultivars (Zhong-hua 11 and 9522). This suggests that this database can be used not only for Nipponbare and 93-11, but also for other japonica and indica cultivars. While validating InDel polymorphisms in the database, a set of InDel markers with each chromosome 3 to 5 marker was developed. These markers are inexpensive and easy to use, and can be used for any combination of japonica and indica cultivars used in this work. This rice DNA polymorphism database will be a valuable resource and important tool for map-based cloning of rice gene, as well as in other various research on rice (http://shenghuan.shnu.edu.cn/ricemarker).

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol β

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS–PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase β (pol β) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol β inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol β type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase β of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol β in plant and animal.

Genetic variation in microsatellite DNA, physiology and morphology of coastal saline rice (Oryza sativa L.) landraces of Bangladesh

Genetic variation of Oryza sativa L. landraces (LRs) collected from the saline coastal belt of Bangladesh, modern varieties (MVs), as well as Pokkali, Nona Bokra and salt tolerant modern varieties (SMVs) derived from the last two were analyzed with 60 evenly distributed rice microsatellite DNA markers. A total of 196 reproducible polymorphic alleles were identified from the band loci. Heterozygosity among the 31 LRs was found to be 0.57, 0.46 in the 5 MVs and 0.40 in the 8 SMVs. Computation of genetic similarity with this data, using Jaccard’s coefficient followed by UPGMA clustering, divided the landraces into 6 distinct groups. Three groups were composed of LRs only from the highly saline southwest. Two groups consisted of LRs from the mild to moderately saline mid-east and northeast coasts. The sixth group was heterogeneous, with LRs from the northeast, LRs from the southwest and Nona Bokra. Pokkali and Gunshi, a LR of the southwest, branched out individually. When all the 46 O. sativa L. cultivars were clustered together, most of the MVs and SMVs were found to be linked within the heterogeneous group. The measure of seedling Na and K concentration, Na/K ratios, affected leaf area as well as survival under salinity stress in hydroponics identified 6 LRs from the highly saline southwest as the most tolerant. These group with Pokkali when UPGMA clustering using the Pearson product-moment correlation coefficient suitable for the quantitative physiological data on seedling saline stress was computed. Morphological observations of plant type and height, days to maturity and yield components in non-saline soil indicated low variability among the different LRs. When yield performance as well as tolerance scores were considered, 7 LRs from the southwest and 1 LR from the mid-northeast show potential as donors for breeding salt tolerant rice. The microsatellite fingerprinting analysis thus revealed that some of the salt tolerant landraces of the coastal region have unique polymorphic loci, quite distinct from the popular salt tolerance donor Pokkali. The similarity matrices between the O. sativa L. cultivars chosen for the study can be used as a valuable tool for the proper choice of parents for mapping or breeding purposes.

Polymorphism and genetic relatedness among wild and cultivated rice species determined by AP-PCR analysis.

We have applied the arbitrarily primed polymerase chain reaction (AP-PCR) technique to the analysis of the relationships among six japonica and indica cultivars, and four wild species of rice. Chosen were four primers of arbitrary sequence that gave multiple amplification products when rice DNA was used as template. Among a total of 50 bands scored, 44 were polymorphic, which was sufficient to distinguish the species used in this study. It is apparent from the comparisons of genetic distances that cultivated rice (Oryza sativa) exhibits closest molecular affinity to wild O. rufipogon, suggesting that the origin of cultivated rice is from O. rufipogon. In a dendrogram of cultivated rice and O. rufipogon from different regions, japonica and indica rice were most closely clustered with O. rufipogon from China and India, respectively. Japonica and indica subspecies showed closer affinity with O. rufipogon from different origins than with each other, supporting a hypothesis of multi-centers of the origin of rice cultivation.

An overview of gibberellin metabolism enzyme genes and their related mutants in rice.

To enhance our understanding of GA metabolism in rice (Oryza sativa), we intensively screened and identified 29 candidate genes encoding the following GA metabolic enzymes using all available rice DNA databases: ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox). In contrast to the Arabidopsis genome, multiple CPS-like, KS-like, and KO-like genes were identified in the rice genome, most of which are contiguously arranged. We also identified 18 GA-deficient rice mutants at six different loci from rice mutant collections. Based on the mutant and expression analyses, we demonstrated that the enzymes catalyzing the early steps in the GA biosynthetic pathway (i.e. CPS, KS, KO, and KAO) are mainly encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by gene families. The remaining CPS-like, KS-like, and KO-like genes were likely to be involved in the biosynthesis of diterpene phytoalexins rather than GAs because the expression of two CPS-like and three KS-like genes (OsCPS2, OsCPS4, OsKS4, OsKS7, and OsKS8) were increased by UV irradiation, and four of these genes (OsCPS2, OsCPS4, OsKS4, and OsKS7) were also induced by an elicitor treatment.

Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus.

Two genomic clones ( OsMET1-1, AF 462029 and OsMET1-2, TPA BK001405), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice ( Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with 12 exons and 11 introns while OsMET1-2 has an open reading frame of 4,491 nucleotides with 11 exons and 10 introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1, and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56-75% identity) of rice MET1 proteins to other plant MET1 sequences but limited similarity (approx. 24% identity) to animal Dnmt1 proteins. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady-state level of OsMET1-2 was 7- to 12-fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the N- or C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.

Splicing-site recognition of rice (Oryza sativa L.) DNA sequences by support vector machines.

It was found that high accuracy splicing-site recognition of rice (Oryza sativa L.) DNA sequence is especially difficult. We described a new method for the splicing-site recognition of rice DNA sequences. Based on the intron in eukaryotic organisms conforming to the principle of GT-AG, we used support vector machines (SVM) to predict the splicing sites. By machine learning, we built a model and used it to test the effect of the test data set of true and pseudo splicing sites. The prediction accuracy we obtained was 87.53% at the true 5' end splicing site and 87.37% at the true 3' end splicing sites. The results suggested that the SVM approach could achieve higher accuracy than the previous approaches.

The sense and antisense expression of gibberellin 20-oxidase gene (rga5) in rice and its effects on GA1 level and agronomic traits

A gibberellin 20-oxidase generga5 was isolated by PCR from genomic DNA of rice (Oryza sativa ssp indica) cultivars ‘Aizizhan’ and ‘Nante’. Compared with the reported OsGA20ox, therga5 was partial-frame-shifted with 11 different amino acids. Then therga5 with CaMV 35S promotor and NOS terminator was inserted into the polylinker site of pCambia1301 to construct sense and antisense gene expressing vectors pSrga5 and pArga5. The transgenic plants were obtained by biolistic bombardment with pSrga5 or pArga5. The transgenic rice plants showed that the overexpression and antisense-expression ofrga5 have remarkable effects on the biological characters of rice. The sense transgenic plants showed heightening with longer spike, more seed-bearing and unaffected flowering, whereas antisense transgenic plants showed dwarfing, early-flowering with slender stem, dark leaf color, shorter leaf and shorter spike. The PCR amplification and Southern blot hybridization showed that therga5 has been integrated into the transgenic rice genome and the transcription ofrga5 was identified by Northern blot hybridization. In the sense transgenic plants the GA1 content increased of about 50%, however, the antisense transgenic rice decreased of 90% approximately compared with control plant ‘Zhonghua 8’. These results demonstrated that therga5 is a functional gene encoding gibberellin 20-oxidase in rice. Over-expressingrga5 significantly increases the endogenous GA1 level and plant height of rice, whereas the expression of antisenserga5 decreases the GA1 level and plant height of rice dramatically.

Molecular Lymphatic Mapping of the Sentinel Lymph Node

Intraoperative lymphatic mapping to identify the sentinel lymph node (SLN) has significantly changed the management of regional lymph node basin of patients with various types of solid tumors such as melanoma and breast cancer. The procedure has improved the diagnosis of micrometastasis in the regional tumor-draining lymph nodes by providing a focused histopathological assessment of select lymph nodes most likely to harbor occult disease. Blue dye and/or radioisotopes are efficient mapping agents but the lack of accurate methods to quantify their presence and the potential for dissolution and decay, respectively, throughout time limit their role as reliable markers for identifying a sentinel node from additional secondary lymph nodes that may be either blue and/or radioactive to some degree. A consistently durable marker is needed that can be introduced during surgery and successfully quantitated among tumor-draining lymph nodes to permit a more accurate assessment of hierarchical organization. This may be of particular importance in retrospective analysis of archival tissues as there are no inherent markers to denote the SLN from successive echelon nodes. A procedure of molecular lymphatic mapping (MLM) was developed in a rat model to label the SLN preoperatively with rice gene DNA containing plasmid or linear rice DNA fragment (rDNA). The MLM efficiency was demonstrated by polymerase chain reaction (PCR) detection of the molecular marker in both frozen and paraffin-embedded SLN; 1.25 micro g of rDNA injected with blue dye could be reproducibly detected by PCR. The MLM procedure was validated in a rat breast tumor model with lymph node metastasis. The procedure was successful in permanently labeling and identifying by PCR both frozen and paraffin-embedded SLN. MLM in conjunction with a conventional mapping agent can be used as a valuable asset for molecular assessment of the SLN and retrospective analysis of paraffin-embedded specimens.

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum.

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsisthaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34560 and 1.2×105 clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30–50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2–9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.

Resistance gene analogs in barley and their relationship to rust resistance genes

Regions of amino acid conservation in the NBS domain of NBS-LRR resistance proteins facilitated the PCR isolation of eight resistance gene analog (RGA) sequences from genomic DNA of rice, barley, and Aegilops tauschii. These clones and other RGAs previously isolated from maize, rice, and wheat were assigned to 13 classes by DNA-sequence comparison and by their patterns of hybridisation to restricted barley DNA. Using a doubled-haploid mapping population, probes from 12 RGA classes were used to map 17 loci in the barley genome. Many of these probes have been used for mapping in wheat, and the collective data indicate that the positions of orthologous RGAs are conserved between barley and wheat. RGA loci were identified in the vicinity of barley leaf rust resistance loci Rph4, Rph7, and Rph10. Recombinants were identified between RGA loci and Rph7 and Rph10, while a cluster of RGA sequences detected by probe 5.2 cosegregated with Rph4 in 55 F2 lines.

A Widely Distributed Lineage of Xanthomonas oryzae pv. oryzae in India May Have Come from Native Wild Rice.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have collected leaf blight-affected samples from wild rice (Oryza nivara) plants growing naturally at 22 locations in five revenue districts (Nalgonda, Ranga Reddy, Medak, Nizamabad, and Adilabad) in the Telangana Region of Andhra Pradesh, India. Pathotype analysis on a set of differential rice cultivars and DNA fingerprinting with two multilocus restriction fragment length polymorphism probes indicated that the X. oryzae pv. oryzae strains from the O. nivara plants belonged to a pathotype and lineage previously widely distributed among cultivated rice in India. This suggests that the lineage may be native to wild rice and may have been transferred subsequently to cultivated rice plants.

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

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.

Tn10 transpososome assembly involves a folded intermediate that must be unfolded for target capture and strand transfer.

Tn10 transposition, like all transposition reactions examined thus far, involves assembly of a stable protein-DNA transpososome, containing a pair of transposon ends, within which all chemical events occur. We report here that stable Tn10 pre-cleavage transpososomes occur in two conformations: a folded form which contains the DNA-bending factor IHF and an unfolded form which lacks IHF. Functional analysis shows that both forms undergo double strand cleavage at the transposon ends but that only the unfolded form is competent for target capture (and thus for strand transfer to target DNA). Additional studies reveal that formation of any type of stable transpososome, folded or unfolded, requires not only IHF but also non-specific transposase-DNA contacts immediately internal to the IHF-binding site, implying the occurrence of a topo- logically closed loop at the transposon end. Overall, transpososome assembly must proceed via a folded intermediate which, however, must be unfolded in order for intermolecular transposition to occur. These and other results support key features of a recently proposed model for transpososome assembly and morphogenesis.

Molecular cloning, chromosomal mapping and expression analysis of disease resistance gene homologues in rice (Oryza sativa L.)

Resistance-like sequences have been amplified from first strand cDNA and genomic DNA of rice by PCR using oligonucleotide primers designed from sequence motifs conserved between resistance genes of tobacco andArabidopsis thaliana. 3 PCR clones, designatedOsr1, Osr2 andOsr3 which were 98% identical in nucleotide sequence level, have been found to be significantly homologous to known plant resistance genes and all contained the conserved motifs of NBS-LRR type resistance genes, such as P-loop, kinase2a, kinase3a and transmembrane domain.Southern hybridization revealed that rice resistance gene hornologueswere organized as a cluster in the genome. RFLP mapping using a DH population derived from anindica/japonka cross (Zhaiyeqing 8/Jingxi 17) and an RFLP linkage map assigned two copies ofOsrl and one copy ofOsr3 to the distal position of chromosome 12 where a blast resistance QTL has been mapped previously. Northern blot analysis showed thatOsrl gene was constitutively transcribed in rice leaves, shoots and roots. Further study concerning isolation of full-length cDNAs would be conducive to elucidating the functions of these genes.

Comparative genome mapping of Sorghum bicolor (L.) Moench using an RFLP map constructed in a population of recombinant inbred lines

AbstractA restriction fragment length polymorphism (RFLP) linkage map of Sorghum bicolor (L.) Moench was constructed in a population of 137 F6‐8 recombinant inbred lines using sorghum, maize, oat, barley and rice DNA clones. The map consists of 10 linkage groups (LGs) and 323 markers, 247 of which (76.5%) were ordered at a LOD score ≥ 3.0. The LGs comprise from 61 (LG A) to 13 markers (J), which range in length from 205 (A) to 55 cM (J) and have a combined total length of 1347 cM. Highly significant distorted segregation was detected at all of the 38 loci in a 103‐cM segment of LG A, the allelic ratios in the segment ranging from approximately 3:1 (one end) to 19:1 (middle) to 2:1 (other end). Duplicated loci located in different LGs have been mapped with 55 of the 295 DNA probes used in the study (18.6%). The distribution of these loci does not provide support for the hypothesis that Sorghum bicolor (L.) Moench is of tetraploid origin. Comparison of the map with RFLP maps of maize, rice, and oat produced evidence for sorghum‐maize LG rearrangements and homoeologies not reported previously, including evidence that: (1) a segment of maize 5L and a segment of 5S may be homoeologous to sorghum LGA; (2) maize LGs 4 and 6 are partly homoeologous to sorghum LGE; (3) the short arm of maize LG 2 is partly homoeologous to sorghum LGF; (4) maize LG 4 may be partly homoeologous to sorghum LG G; (5) maize LG 5 and sorghum LG G contain a larger amount of homoeologous genetic material than previously indicated; and (6) a short segment of maize LG 1 may be homoeologous to a short segment of sorghum LG I.

The Fate of 14C-Labelled Hurnic Substances in Rice Cells in Culture

Summary Peat and aquatic humic acids covering a molecular mass range from approximately 40,000 to 8,000 were found to stimulate the growth of rice calli and rice cells in suspension culture. Growth stimulation was investigated as a function of humic acid concentration up to 100 ppm and growth time up to 60 days. The humic acids increase calli weight by approximately 80% and cell numbers in culture approximately 160 × after 42 days. The humic acids had no effect on the protein and DNA contents of rice cells; however, by using the radioactive 14 C N-methylamide derivatives of the humic acids it has been found that both rice cell protein and DNA become radiolabelled during culture. The incorporation of 14 C into DNA is approximately 10× that into protein. Extraction of cell DNA followed by hydrolysis with DNAase I and HPLC analysis of the resulting deoxynucleotides has demonstrated that approximately 70% of the 14 C activity is incorporated into the purine deoxyribonucleotides dAMP and dGMP. Analysis of the humic acids in the culture medium during growth showed that while a major proportion of the humic molecules remained, there was some breakdown to lower molecular mass species and possibly some release of the radiolabelled methylamine. However, the asymmetric incorporation of the major proportion of the 14 C into the purine-containing deoxynucleotides did not arise from the putative release of the label from the humic acids, since 14 C-methylamine itself incorporated 14 C evenly into the four deoxynucleotides. It is suggested that aromatic moeties derived from the humic acids could result in phenylalanine and tyrosine entering the metabolic pathways to glycine and aspartate, which are used in the de novo synthesis of the deoxynucleotide bases. The incorporation of 14 C from 14 C-glycine and 14 C-aspartate into rice DNA deoxynucleotides was confirmed in control experiments. The results suggest that rice cells can use humic breakdown products in the synthesis of proteins and DNA.

Anchor probes for comparative mapping of grass genera

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.