Application of denaturing high-performance liquid chromatography for rice variety identification and seed purity assessment

Recent DNA sequencing projects and the establishment of high-throughput assays have provided an abundance of sequence information and data on nucleotide polymorphisms in rice. Based on previously identified single-nucleotide polymorphisms (SNPs) and insertions/deletions, we employed denaturing high-performance liquid chromatography (DHPLC) to genotype rice varieties using a chromatographic pattern-based strategy. In this study, 12 amplicons harboring multiple and informative SNPs were screened. PCR products of the 12 amplicons from 47 rice varieties were analyzed by DHPLC and DNA sequencing. Each homozygous sample with a single peak pattern in the initial DHPLC analysis was mixed with zhenshan97 for a second DHPLC analysis. The 12 amplicons were found to be polymorphic across the hybrids, and mixed homozygous samples with 43 distinct DHPLC elution profiles detected. Sequence analysis confirmed that the distinct DHPLC patterns corresponded to different DNA sequences. A set of distinct characteristic profiles in six amplicons differentiated between all of the hybrids, inbred lines, and restorer lines and produced unique a fingerprint for these lines. In addition, we found that the DHPLC pattern of the hybrid was in accordance with the results obtained by DHPLC analysis of a mixed sample of the two parents. These results demonstrate that DHPLC can be efficiently applied for the rapid and automated identification of diverse rice varieties and could possibly be utilized for seed genetic purity testing on a high-throughput scale.     

Dormancy genes from weedy rice respond divergently to seed development environments

Genes interacting with seed developmental environments control primary dormancy. To understand how a multigenic system evolved to adapt to the changing environments in weedy rice, we evaluated genetic components of three dormancy QTL in a synchronized nondormant genetic background. Two genetically identical populations segregating for qSD1, qSD7-1, and qSD12 were grown under greenhouse and natural conditions differing in temperature, relative humidity, and light intensity during seed development. Low temperatures tended to enhance dormancy in both conditions. However, genotypes responded to the environments divergently so that two populations displayed similar distributions for germination. Additive and/or dominance effects of the three loci explained approximately 90% of genetic variances and their epistases accounted for the remainder in each environment. The qSD1 and qSD7-1 main effects were increased, while the qSD12 additive effect was decreased by relatively low temperatures. Both gene main and epistatic effects were involved in G x E interactions, which in magnitude were greater than environmental main effect. The divergent responses of dormancy genes observed in this simple multigenic system presumably have selective advantages in natural populations adapted to changing environments and hence represent a genetic mechanism stabilizing the dormancy level of weedy rice ripened in different seasons or temperature regimes.     

Studies on rice seed protease V. Protease inhibitor in rice seed

Protease inhibitor in rice seed was investigated physiologicallyin connection with rice seed protease. In rice seed, the inhibitorwas localized in the embryo and its activity decreased on germination.The inhibitor was a protein-like substance which, was non-diffusiblethrough cellophane membrane, salted out on addition of ammoniumsulfate, was denatured on heating it at 100°C and adsorbedonto ion-exchange cellulose. During incubation at 70°C for30 min, inhibitor activity remained unchanged, while the proteaselost its activity within 10 min. The protease and the inhibitorof rice seed were separated from each other by chromatographyon a TEAE-cellulose column. The inhibitor reduced the activityof rice seed protease. ¹ This report was presented in part at the annual meeting ofthe Society of the Science of Soil and Manure of Japan at Tokyoon April 1, 1968.² Present address: Faculty of Agriculture, Kagoshima University,Kagoshima, Japan. (Received June 8, 1971; )     

Inheritance of seed color in Capsicum

The mode of seed color inheritance in Capsicum was studied via an interspecific hybridization between C. pubescens Ruiz and Pav. (black seed color) and C. eximium Hunz. (yellow seed color). Black seed color was dominant over yellow seed color. The F(2) segregation pattern showed continuous variation. The generation means analysis indicated the presence of a significant effect of additive [d], dominance [h], and additive x additive [i] interaction for seed color inheritance. The estimate for a minimum number of effective factors (genes) involved in seed color inheritance was approximately 3.     

Genome-wide identification and characterization of novel microRNAs in seed development of soybean

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in eukaryotes. However, the information about miRNAs population and their regulatory functions involving in soybean seed development remains incomplete. Base on the Dicer-like1-mediated cleavage signals during miRNA processing could be employed for novel miRNA discovery, a genome-wide search for miRNA candidates involved in seed development was carried out. As a result, 17 novel miRNAs, 14 isoforms of miRNA (isomiRs) and 31 previously validated miRNAs were discovered. These novel miRNAs and isomiRs represented tissue-specific expression and the isomiRs showed significantly higher abundance than that of their miRNA counterparts in different tissues. After target prediction and degradome sequencing data-based validation, 13 novel miRNA–target pairs were further identified. Besides, five targets of 22-nt iso-gma-miR393h were found to be triggered to produce secondary trans-acting siRNA (ta-siRNAs). Summarily, our results could expand the repertoire of miRNAs with potentially important functions in soybean.     

Dynamic quantitative trait locus analysis of seed dormancy at three development stages in rice

In this study, a rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of seed dormancy (SD) at 4 (early), 5 (middle) and 6 (late) weeks after heading stages. Dynamic analysis showed that the indica IR28 variety tended to have deeper dormancy than the japonica Daguandao at the middle and late development stages. The level of SD decreased with the process of seed development. The significant interaction between heading date (HD) and SD occurred only in those seeds collected at the early development stage. A total of nine additive quantitative trait loci (QTLs) and eight epistatic QTLs for SD were identified at three seed development stages. Of them, one additive and four epistatic QTLs were identified for the early stage, six additive and one epistatic QTL for the middle stage and two additive and three epistatic QTLs for the late stage. The phenotypic variation explained by each additive and epistatic QTL ranged from 5.8 to 30.6 % and from 3.8 to 13.1 %, respectively. Compared with the additive QTLs, epistatic interactions were much more important for SD at the early and late development stages. Two major additive QTLs, qSD3.1 and qSD4.1, were identified; each QTL could explain more than 20 % of the total phenotypic variance and each dormancy-enhancing allele could decrease the germination percentage by about 10 %. By comparing the chromosomal positions of these additive QTLs with those previously identified, five additive QTLs, qSD1.2, qSD2.1, qSD3.2, qSD4.1 and qSD9.1, might represent novel genes. One QTL identified here, qHD1, and nine QTLs identified in previous studies for HD were co-located with our QTLs for SD, which indicated that the significant correlation between SD and HD might be due to the linkage of QTLs for SD and HD. Four RILs with deep dormancy at development stages but non-dormancy after post-ripening under different germination conditions were selected. Using the selected RILs, three cross combinations of SD for the development of RIL populations were predicted. The selected RILs and the identified QTLs might be applicable for the improvement of pre-harvest sprouting tolerance by marker-assisted selection in rice.     

SoyProDB: A database for the identification of soybean seed proteins

Soybean continues to serve as a rich and inexpensive source of protein for humans and animals. A substantial amount of information has been reported on the genotypic variation and beneficial genetic manipulation of soybeans. For better understanding of the consequences of genetic manipulation, elucidation of soybean protein composition is necessary, because of its direct relationship to phenotype. We have conducted studies to determine the composition of storage, allergen and anti-nutritional proteins in cultivated soybean using a combined proteomics approach. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) was implemented for the separation of proteins along with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS) for the identification of proteins. Our analysis resulted in the identification of several proteins, and a web based database named soybean protein database (SoyProDB) was subsequently built to house and allow scientists to search the data. This database will be useful to scientists who wish to genetically alter soybean with higher quality storage proteins, and also helpful for consumers to get a greater understanding about proteins that compose soy products available in the market. The database is freely accessible.

Availability

http://bioinformatics.towson.edu/Soybean_Seed_Proteins_2D_Gel_DB/Home.aspx     

Genomic regions affecting seed shattering and seed dormancy in rice

Non-shattering of the seeds and reduced seed dormancy were selected consciously and unconsciously during the domestication of rice, as in other cereals. Both traits are quantitative and their genetic bases are not fully elucidated, though several genes with relatively large effects have been identified. In the present study, we attempted to detect genomic regions associated with shattering and dormancy using 125 recombinant inbred lines obtained from a cross between cultivated and wild rice strains. A total of 147 markers were mapped on 12 rice chromosomes, and QTL analysis was performed by simple interval mapping and composite interval mapping. For seed shattering, two methods revealed the same four QTLs. On the other hand, for seed dormancy a number of QTLs were estimated by the two methods. Based on the results obtained with the intact and de-hulled seeds, QTLs affecting hull-imposed dormancy and kernel dormancy, respectively, were estimated. Some QTLs detected by simple interval mapping were not significant by composite interval mapping, which reduces the effects of residual variation due to the genetic background. Several chromosomal regions where shattering QTLs and dormancy QTLs are linked with each other were found. This redundancy of QTL associations was explained by ”multifactorial linkages” followed by natural selection favoring these two co-adapted traits. Received: 23 November 1998 / Accepted: 27 August 1999     

Improving the performance of transplanted rice by seed priming

Transplanting is the major method of rice cultivation in the world, in which seedlings are raised in nursery and then transplanted into well puddle and prepared fields. The traditional nursery sowing method is tedious and produces week seedlings that reduce the final yield due to high mortality. The potential of seed priming to improve the nursery seedlings and thus the transplanted rice was evaluated in the present study. The experiment was conducted in the rice growing area (31.45° N, 73.26° E, and 193 m) of Pakistan, during 2004–2005. Seed priming tools employed during the investigation included traditional soaking, hydropriming for 48 h, osmohardening with KCl or CaCl₂ (Ψ_s −1.25 MPa) for 24 h (one cycle), 10 ppm ascorbate for 48 h or seed hardening for 24 h. Priming improved the initial seedling vigor and resulted in improved growth, yield and quality of transplanted fine rice while traditional soaking behaved similar to that of untreated control. Osmohardening with CaCl₂ resulted in the best performance, followed by hardening, ascorbate priming and osmohardening with KCl. Osmohardening with CaCl₂ produced 3.75 t ha⁻¹ (control: 2.87 t ha⁻¹) kernel yield, 11.40 t ha⁻¹ (control: 10.03 t ha⁻¹) straw yield and 24.57% (control: 22.27%) harvest index. The improved yield was attributed due to increase in the number of fertile tillers. Significant positive correlation was found between mean emergence time of nursery seedlings and kernel yield, nursery seedling dry weight and kernel yield, fertile tillers and kernel yield, and leaf area duration and kernel yield.     

Rice LIM protein OsPLIM2a is involved in rice seed and tiller development

Yield of major monocotyledonous crops including wheat, rice, barley, and sorghum is greatly influenced by tillering. However, deciphering the underlying mechanisms of the tillering has long been hindered because many changeable factors are involved in the process. Plant two LIM-domain-containing proteins bind to and stabilize actin filaments that are major constituents in the formation of higher-order actin cytoskeleton. Here, we report that rice LIM-domain protein, OsPLIM2a, is involved in rice tillering likely through actin cytoskeleton organization. OsPLIM2 genes (OsPLIM2a, OsPLIM2b, and OsPLIM2c) expressed in reproductive organs including anthers, but not in other tissues. Analysis of both OsPLIM2a and OsPLIM2c promoter fused to GUS reporter revealed that both promoters directed strong and specific GUS expression in pollens. Transient expression of OsPLIM2a-GFP and OsPLIM2c-GFP in tobacco leaves showed that OsPLIM2a and OsPLIM2c could bind to actin filaments, which is consistent with other plant LIM proteins with actin-binding property. To examine further physiological roles of rice OsPLIM2a and OsPLIM2c, transgenic rice plants with 35S:OsPLIM2a or 35S:OsPLIM2c were examined for any phenotypic changes. Transgenic plants overexpressing OsPLIM2a produced bigger seeds than wild type, whereas they exhibited reduction in tiller numbers. These results suggest that OsPLIM2a may participate positively in seed development but negatively in tiller differentiation. Protein interaction assays using OsPLIM2c proteins revealed that OsPLIM2c interacted with at least three proteins including rice Fimbrin, of which homologs in Arabidopsis play crucial roles in pollen tube growth, implying that rice OsPLIM2c and Fimbrin may exert roles together in pollen tube growth.     

Temporal and spatial expression pattern of the OSVP1 and OSEM genes during seed development in rice

The spatial and temporal expression patterns of the rice VP1 (OSVP1) gene, as well as the OSEM gene which it controls, were studied during seed development by in situ hybridization and immuno-localization techniques. The expression of OSVP1 could be detected in embryos as early as 2-3 d after pollination (DAP) and thereafter became preferentially localized to shoot, radicle and vascular tissues during the embryo development at both the mRNA and protein levels. In the aleurone layers, OSVP1 mRNA and protein were detected after 6 DAP. OSEM mRNA was detectable after 6 DAP in the embryo and aleurone tissue. The spatial distribution within the embryo of OSEM mRNA and OSVP1 mRNA/protein was very similar after 6 DAP. Transgenic rice carrying a beta-glucuronidase (GUS) gene transcribed from a chimeric promoter consisting of the CaMV 35S minimal promoter (-46) and the 55-bp promoter fragment of OSEM, minimally required for ABA and VP1 regulation, also exhibited a spatial pattern of GUS expression similar to that of OSEM and OSVP1. These results suggest that (OS)VP1 is a major determinant not only of the seed specificity but also of the spatial pattern of OSEM expression in the developing seed.     

Ethylene control of seed coat development in low and high sterile semidwarf indica rice cultivars

Although basally positioned inferior spikelets of rice panicles emerge late from the flag leaf enclosure (boot), they mature early which precludes adequate grain filling. It is assumed that extended exposure to ethylene inside the boot restricts assimilate partitioning to the endosperm in basal spikelets by affecting the functions of seed coat. In the present study, ethylene concentration inside the boot was measured in two high yielding rice cultivars differing in percentage of spikelet sterility. To manipulate the concentration/action of ethylene, silver nitrate, aminoethoxyvinyl glycine and 2-chloroethylphosphonic acid were injected into the boot. The effect of these chemicals on the concentration of photosynthetic pigments, lipid peroxidation and peroxidase activity of the seed coat and lemma and palea were measured to monitor development. Ethylene reduced development during the juvenile phase but accelerated degradation of the photosynthetic tissues of the spikelets in the senescent phase. Boot ethylene correlated positively with number of barren spikelets in the panicle and negatively with concentration of photosynthetic pigments of the seed coat of inferior spikelets. The concentration of ethylene was higher in the high sterile cultivar Mahalaxmi than that of the low sterile Mahanadi. Inhibition of ethylene action or synthesis improved grain filling. The chemicals were most effective on the inferior spikelets. It was concluded that ethylene retarded seed coat development during the prestorage phase and reduced grain filling of basal spikelets.     

Calcium peroxide as a seed coating material for padi rice

Summary Measurement of oxygen uptake by germinating rice seed (Oryza sativa L) suggests that oxygen requirement is independent of temperature of incubation. However, the rate of oxygen consumption is dependent upon incubation temperature and, after an initial lag phase, is exponential with time. Although rice seed can germinate and grow at low oxygen concentrations, germination is poor and seedlings exhibit low vigour. An oxidized zone may be observed around the seed when sown in an anoxic environment but coated with a layer of calcium peroxide. The seed germinates readily and develops normally when a sufficient level of calcium peroxide is used.