Marker-assisted selection for identification of plant regeneration ability of seed-derived calli in rice (Oryza sativa L.).

Quantitative trait loci (QTL), associated with the ability of plant regeneration from seed-derived callus of rice, were mapped using a recombinant inbred (RI) population from Milyang 23/Gihobyeo. Each flanking marker, RZ474 and RZ575, tightly linked to two QTLs (qSGR-3-1 and qSGR-3-2) that are located on chromosome 3 was used in marker-assisted selection (MAS). These markers were tested on IR 36/MG RI036 (F3), Milyang 23/MG RI036 (F3), and forty-one rice cultivars. A restriction fragment length polymorphism (RFLP) marker, RZ575, that is located on chromosome 3 could effectively differentiate lines with high and poor regeneration ability, based on marker genotypes. This marker might be applicable for screening rice germplasms with high regeneration ability. Its introgression into elite lines might also be valuable in breeding programs to develop highly responsive genotypes to tissue culture.

Quantitative trait loci mapping associated with plant regeneration ability from seed derived calli in rice (Oryza sativa L.).

Quantitative trait loci (QTLs), which are associated with the ability of plant regeneration from seed derived calli, were detected using a recombinant inbred (RI) population from a cross between 'Milyang 23 (toingil)' and 'Gihobyeo (japonica)' in rice (Oryza sativa L.). A tongil type cultivar, 'Milyang 23', has a lower frequency of callus induction and plant regeneration than those of japonica 'Gihobyeo'. Transgressive segregations were observed for the callus induction rate and plant regeneration ability from seed derived calli of the RI population. An interval mapping analysis was used to identify the QTL controlling the plant regeneration ability. Two QTLs for the callus induction rate were detected on chromosomes 1 and 2, explaining the 10.9% total phenotypic variation. Four QTLs that are associated with the plant regeneration ability were located on chromosomes 2, 3, and 11, accounting for 25.7% of the total phenotypic variation.

Analysis of expressed sequence tags from Brassica rapa L. ssp. pekinensis.

Non-redundant expressed sequence tags (ESTs) were generated from six different organs at various developmental stages of Chinese cabbage, Brassica rapa L. ssp. pekinensis. Of the 1,295 ESTs, 915 (71%) showed significantly high homology in nucleotide or deduced amino acid sequences with other sequences deposited in databases, while 380 did not show similarity to any sequences. Briefly, 598 ESTs matched with proteins of identified biological function, 177 with hypothetical proteins or non-annotated Arabidopsis genome sequences, and 140 with other ESTs. About 82% of the top-scored matching sequences were from Arabidopsis or Brassica, but overall 558 (43%) ESTs matched with Arabidopsis ESTs at the nucleotide sequence level. This observation strongly supports the idea that gene-expression profiles of Chinese cabbage differ from that of Arabidopsis, despite their genome structures being similar to each other. Moreover, sequence analyses of 21 Brassica ESTs revealed that their primary structure is different from those of corresponding annotated sequences of Arabidopsis genes. Our data suggest that direct prediction of Brassica gene expression pattern based on the information from Arabidopsis genome research has some limitations. Thus, information obtained from the Brassica EST study is useful not only for understanding of unique developmental processes of the plant, but also for the study of Arabidopsis genome structure.

Hormonal cross-talk between auxin and ethylene differentially regulates the expression of two members of the 1-aminocyclopropane-1-carboxylate oxidase gene family in rice (Oryza sativa L.).

Two cDNA clones, pOS-ACO2 and pOS-ACO3, encoding 1-aminocyclopropane-1-carboxylate (ACC) oxidase were isolated from rice seedling cDNA library. pOS-ACO3 is a 1,299 bp full-length clone encoding 321 amino acids (Mr=35.9 kDa), while pOS-ACO2 is 1,072 bp long and is a partial cDNA clone encoding 314 amino acids. These two deduced amino acid sequences share 70% identity, and display a high degree of sequence identity (72-92%) with previously isolated pOS-ACO1 of deepwater rice. The chromosomal location studies show that OS-ACO2 is positioned on the long arm of chromosome 9, while OS-ACO3 on the long arm of chromosome 2 of rice genome. A marked increase in the level of OS-ACO2 transcript was observed in IAA-treated etiolated rice seedlings, whereas the OS-ACO3 mRNA was greatly accumulated by ethylene treatment. Results of ethylene inhibitor studies indicated that auxin promotion of the OS-ACO2 transcription was not mediated through the action of auxin-induced ethylene. Thus, it appears that there are two groups of ACC oxidase transcripts in rice plants, either auxin-induced or ethylene-induced. The auxin-induced OS-ACO2 expression was partially inhibited by ethylene, while ethylene induction of OS-ACO3 transcription was completely blocked by auxin. These results indicate that the expression of ACC oxidase genes is regulated by complex hormonal networks in a gene specific manner in rice seedlings. Okadaic acid, a potent inhibitor of protein phosphatase, effectively suppressed the IAA induction of OS-ACO2 expression, suggesting that protein dephosphorylation plays a role in the induction of ACC oxidase by auxin. A scheme of the multiple regulatory pathways for the expression of ACC oxidase gene family by auxin, ethylene and protein phosphatase is presented.

Structure and expression of the AWI 31 gene specifically induced by wounding in Arabidopsis thaliana.

Differential screening of an Arabidopsis cDNA library constructed from the plant tissues harvested 1 h after wounding resulted in the isolation of wound-inducible cDNA clones (Kim et al., 1994). The cDNA clones could be broadly classified into two groups according to the expression time of their transcripts. Nine clones from the 10 different wound-inducible cDNAs were rapidly induced, reaching a maximum level in approximately 1-1.5 h and then were progressively reduced after wounding. The cDNA clone AWI 31 showed steady accumulation of the transcripts and reached the maximum value at a later time point of 2.5 h and then started to decline. The corresponding gene of the AWI 31 in which the coding region was interrupted by an intron, had an open reading frame that predicted a protein of 386 amino acids. However, the gene product did not show any significant homology to other known proteins in the database. Northern hybridization study using the cDNA probe revealed that the gene was not regulated by other environmental stresses such as drought, high salt, low temperature, or a DPE herbicide treatment, indicating that the cDNA clone AWI 31 was specifically induced by wounding.

The semidwarf gene, sd-1, of rice (Oryza sativa L.). I. Linkage with the esterase locus, Estl-2.

The linkage relationship between the semidwarf gene (sd-1) and the isozyme locus EstI-2 was elucidated using segregating populations derived from crosses between several semidwarf testers and tall rice varieties. Bimodal distributions for culm length were observed in F2 populations of three cross combinations, including Shiokari/Shiokari (sd-1), Taichung 65 (A,Pn,Pau)/Taichung 65 (sd-1), and Milyang 23/Kasalath. Taking the valley of the distribution curves as the dividing point, two height classes were apparent with a segregation ratio of 3 tall∶1 short, demonstrating this character to be under the control of a single recessive gene. An inheritance study of esterase isozymes, based on isoelectric focusing (IEF), showed that the EstI-2 locus had two active allozymes of monomeric structure and one null form, which were designated "a", "b", and "n", respectively (Eun et al. 1990). Semidwarf testers such as Shiokari (sd-1), Taichung 65 (sd-1) and Milyang 23 have an active allozyme designated as EstI-2(aa), while the tall parents, Shiokari and Taichung 65 (A,Pn,Pau), have the active allozyme, EstI-2(bb), and Kasalath has a null form of the allozyme, EstI-2(nn). By dividing F2 populations based on EstI-2 allozyme patterns, culmlength distributions exhibited trimodal curves. Most of the short plants had the homozygous EstI-2(aa) pattern of the short parents, most of the tall plants had the homozygous pattern, EstI-2(bb) or EstI-2(nn), and most of the intermediate plants had the heterozygous EstI-2(ab) or EstI-2(an) banding pattern. Einkage analysis indicated that sd-1 and EstI-2 were tightly linked. These findings were also confirmed by segregation analyses in F3 progenies. No recombinants among 171 F3 families from the Shiokari/Shiokari (sd-1) combination, five recombinants among 267 F3 families from Taichung 65 (A,Pn,Pau)/Taichung 65(sd-1), and only two recombinants out of 237 F3 families from Milyang 23/Kasalath, were found. The recombination values were 0, 1.87 and 0.8%, respectively.

The semidwarf gene, sd-1, of rice (Oryza sativa L.). II. Molecular mapping and marker-assisted selection.

To establish the location of the semidwarf gene, sd-1, the anthocyanin activator (A), purple node (Pn), purple auricle (Pau), and the isozyme locus, EstI-2, in relation to DNA markers on the molecular linkage map of rice, 20 RFLP markers, previously mapped to the central region of chromosome 1 (McCouch et al. 1988), were mapped onto an F2 population derived from the cross Taichung 65 (A,Pn,Pau)/Taichung 65 (sd-1). sd-1 and EstI-2 were determined to be linked most tightly to RFLP markers RG 109 and RG 220, which cosegregated with each other. The distance between these RFLP markers and sd-1 was estimated to be 0.8 cM, based on an observed recombination value of 0.8%. The order of genes and markers in this region of chromosome 1 was determined to be sd-1 - (EstI-2 - RG220 - RG109) - RG381 - A - Pn - Pau. To test the efficacy of selection for sd-1 based on these linked markers, 50-day-old F2 seedlings derived from another cross, Milyang 23/Gihobyeo, were analyzed for marker genotype. At this age, the semidwarf character could not be clearly detected based on phenotype. In addition, plant height was normally distributed in this population, making it difficult to unambiguously identify plants carrying sd-1. Thirteen seedlings homozygous for the sd-1-associated allele at EstI-2, RG220 and RG109, and 13 seedlings homozygous for the Sd-1-associated allele at all three marker loci were selected for further genetic analysis. At 20 days after heading, the culm lengths of these 26 plants were measured and the expected phenotype was confirmed in every case. These 26 plants were then selfed for four generations and F6 lines were again evaluated to determine whether any recombination among the three molecular markers, or between these markers and the sd-1 gene, could be detected. No recombinants were identified, confirming the tight linkage of these loci and the usefulness of genotypic selection for this recessive semidwarf character prior to the time when it can be evaluated based on phenotype.