ipt Gene transformation in petunia by an Agrobacterium mediated method.

To prevent leaf senescence of petunia, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of 35S promoter and introduced into petunia. PCR analysis showed an expected 0.5 Kb fragment of ipt gene in transgenic petunia. RT-PCR analysis indicated the expression of ipt gene in the transgenic lines. Leaves from transgenic plants remained green and healthy in normal culture condition, while the non-transformed plants turned to yellow. Transgenic plants showed a reduction in height and smaller leaf sizes. In transgenic lines, the internodes were shorter, and the roots grew slower than the non-transformed plants.

Characterization and mapping of Rpi1, a gene that confers dominant resistance to stalk rot in maize.

The maize inbred lines 1145 (resistant) and Y331 (susceptible), and the F(1), F(2) and BC(1)F(1) populations derived from them were inoculated with the pathogen Pythium inflatum Matthews, which causes stalk rot in Zea mays. Field data revealed that the ratio of resistant to susceptible plants was 3:1 in the F(2) population, and 1:1 in the BC(1)F(1)population, indicating that the resistance to P. inflatum Matthews was controlled by a single dominant gene in the 1145xY331 cross. The gene that confers resistance to P. inflatum Matthews was designated Rpi1 for resistance to P. inflatum) according to the standard nomenclature for plant disease resistance genes. Fifty SSR markers from 10 chromosomes were first screened in the F(2) population to find markers linked to the Rpi1 gene. The results indicated that umc1702 and mmc0371 were both linked to Rpi1, placing the resistance gene on chromosome 4. RAPD (randomly amplified polymorphic DNA) markers were then tested in the F(2)population using bulked segregant analysis (BSA). Four RAPD products were found to show linkage to the Rpi1 gene. Then 27 SSR markers and 8 RFLP markers in the region encompassing Rpi1 were used for fine-scale mapping of the resistance gene. Two SSR markers and four RFLP markers were linked to the Rpi1 gene. Finally, the Rpi1 gene was mapped between the SSR markers bnlg1937 and agrr286 on chromosome 4, 1.6 cM away from the former and 4.1 cM distant from the latter. This is the first time that a dominant gene for resistance to maize stalk rot caused by P. inflatum Matthews has been mapped with molecular marker techniques.

Genetic analysis and molecular mapping of maize (Zea mays L.) stalk rot resistant gene Rfg1.

One single pathogen Fusarium graminearum Schw. was inoculated to maize inbred lines 1,145 (Resistant) and Y331 (Susceptive), and their progenies of F(1), F(2) and BC(1)F(1) populations. Field statistical data revealed that all of the F(1) individuals were resistant to the disease and that the ratio of resistant plants to susceptive plants was 3:1 in the F(2) population, and 1:1 in the BC(1)F(1 )population. The results revealed that a single dominant gene controls the resistance to F. graminearum Schw. The resistant gene to F. graminearum Schw. was denominated as Rfg1 according to the standard principle of the nomenclature of the plant disease resistant genes. RAPD (randomly amplified polymorphic DNA) combined with BSA (bulked segregant analysis) analysis was carried out in the developed F(2) and BC(1)F(1 )populations, respectively. Three RAPD products screened from the RAPD analysis with 820 Operon 10-mer primers showed the linkage relation with the resistant gene Rfg1. The three RAPD amplification products (OPD-20(1000), OPA-04(1100) and OPY-04(900)) were cloned and their copy numbers were determined. The results indicated that only OPY-04(900) was a single-copy sequence. Then, OPY-04(900) was used as a probe to map the Rfg1 gene with a RIL F(7) mapping population provided by Henry Nguyen, which was developed from the cross "S3xMo17". Rfg1 was primarily mapped on chromosome 6 between the two linked markers OPY-04(900) and umc21 (Bin 6.04-6.05). In order to confirm the primary mapping result, 25 SSR (simple sequence repeat) markers and six RFLP (restriction fragment length polymorphism) markers in the Rfg1 gene-encompassing region were selected, and their linkage relation with Rfg1 was analyzed in our F(2) population. Results indicated that SSR marker mmc0241 and RFLP marker bnl3.03 are flanking the Rfg1 gene with a genetic distance of 3.0 cM and 2.0 cM, respectively. This is the first time to name and to map a single resistant gene of maize stalk rot through a single pathogen inoculation and molecular marker analysis.

Molecular tagging and genetic mapping of the disease resistance gene RppQ to southern corn rust.

Southern corn rust (SCR), Puccinia polysora Underw, is a destructive disease in maize ( Zea mays L.). Inbred line Qi319 is highly resistant to SCR. Results from the inoculation test and genetic analysis of SCR in five F(2) populations and five BC(1)F(1 )populations derived from resistant parent Qi319 clearly indicate that the resistance to SCR in Qi319 is controlled by a single dominant resistant gene, which was named RppQ. Simple sequence repeat (SSR) analysis was carried out in an F(2) population derived from the cross "Qi319x340". Twenty SSR primer pairs evenly distributed on chromosome10 were screened at first. Out of them, two primer pairs, phi118 and phi 041, showed linkage with SCR resistance. Based on this result, eight new SSR primer pairs surrounding the region of primers phi118 and phi 041 were selected and further tested regarding their linkage relation with RppQ. Results indicated that SSR markers umc1,318 and umc 2,018 were linked to RppQ with a genetic distance of 4.76 and 14.59 cM, respectively. On the other side of RppQ, beyond SSR markers phi 041 and phi118, another SSR marker umc1,293 was linked to RppQ with a genetic distance of 3.78 cM. Because the five linkage SSR markers (phi118, phi 041, umc1,318, umc 2,018 and umc1,293) are all located on chromosome 10, the RppQ gene should also be located on chromosome 10. In order to fine map the RppQ gene, AFLP (amplified fragment length polymorphism) analysis was carried out. A total 54 AFLP primer combinations were analyzed; one AFLP marker, AF1, from the amplification products of primer combination E-AGC/M-CAA, showed linkage with the RppQ gene in a genetic distance of 3.34 cM. Finally the RppQ gene was mapped on the short arm of chromosome 10 between SSR markers phi 041 and AFLP marker AF1 with a genetic distance of 2.45 and 3.34 cM respectively.

Genetic analysis, molecular tagging and mapping of the thermo-sensitive genic male-sterile gene (wtms1) in wheat.

A thermo-sensitive genic male-sterile (TGMS) wheat line ( Triticum aestivum L.) BNY-S was obtained from the spontaneous mutant of BNY-F. Its fertility was decided by the temperature during the differentiation stage of the spikelets. BNY-S was completely sterile when the temperature was lower than 10 degrees C during the differentiation stage of the spikelets, but fertile when the temperature was higher than 10 degrees C. Genetic analysis indicated that the sterility of BNY-S was controlled by a single recessive gene, which was named as wtms1. An F(2) population, consisting of 3,000 individuals from the cross between BNY-S and Lankao 52-24, was used for genetic analysis and statistical analysis of the TGMS and, out of them, 158 sterile and 93 fertile extremes were present for molecular tagging and mapping of the wtms1 gene. SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) techniques combined with BSA (bulked segregant analysis) were used to screen markers linked to the target gene. As a result, wtms1 was preliminarily mapped on chromosome 2B according to SSR analysis. In AFLP analysis, 14 polymorphic AFLP loci were identified with a linkage relation to the wtms1 gene. Then linkage analysis using the F(2) population showed that three of them, E: AAG/M: CTA(163), E: AGG/M: CTC(220) and E: ACA/M: CTA(160), were linked to the wtms1 gene relatively close to a genetic distance of 6.9 cM, 6.9 cM and 13.9 cM, respectively. Finally, the wtms1 gene was mapped between the SSR marker Xgwm 374 and the AFLP marker E: AAG/M: CTA(163) with the distance of 4.8 cM and 6.9 cM, respectively. A partial linkage map was constructed according the SSR and AFLP data.

Expression of insulin-like growth factor-II and insulin-like growth factor binding protein-1 in the placental basal plate from pre-eclamptic pregnancies.

OBJECTIVES: To determine whether expressions of insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) are altered in pre-eclamptic placenta and to elucidate the possible relationship between their expressions and a mechanism for inadequate trophoblast invasion in pre-eclampsia. METHODS: Placental tissues were obtained at cesarean delivery from five normotensive, nine mild pre-eclamptic and five severe pre-eclamptic women at 33-39 completed weeks of gestation. After total ribonucleic acid was extracted, reverse transcriptase-polymerase chain reaction was performed to determine IGF-II and IGFBP-1 mRNA expression. Product bands were quantitated by scanning densitometry and results were expressed as ratio of cytokines/beta-actin. Western blot analysis was also done to determine IGF-II and IGFBP-1 protein expression. Statistical analysis was determined by Kruskal-Wallis analysis of variance with the Scheffe multiple post-hoc test. RESULTS: The IGF-II mRNA levels of mild and severe pre-eclamptic placenta were significantly lower than that of uncomplicated placenta (P<0.005, P<0.001, respectively), with the level of severe pre-eclamptic placenta being significantly lower than that of mild pre-eclamptic placenta (P<0.05). As for the IGF-II protein expression, a significant decrease was found among the three groups (P<0.001), correlating with the IGF-II mRNA results. However, the mean IGFBP-1 mRNA levels of mild and severe pre-eclamptic placenta were significantly higher than that of uncomplicated placenta (P<0.05, P<0.005, respectively), with the level of severe pre-eclamptic placenta being significantly raised compared with that of mild pre-eclamptic placenta (P<0.05). Finally, a significant increase of IGFBP-1 protein expression was noted among the three groups (P<0.001), correlating with the IGFBP-1 mRNA results. CONCLUSIONS: This study suggests that IGF-II and IGFBP-1 might be associated with the impaired trophoblastic invasion that may lead to pathogenesis of pre-eclampsia.

[Application of isonucleus and isocytoplasmic lines in the study of maize CMS].

Between wild fertile type (F) and its sterile mutant (cms), if their nucleus and cytoplasm are the same, this wild fertile type (F) and its sterile mutant (cms) are called isonucleus and isocytoplasmic lines. The maize mtDNAs of isonucleus and isocytoplasmic lines (I), wild fertile type 478(F) and its sterile mutant 478-cms, were analyzed by RAPD. 94 primers were screened, 3 polymorphic products, OPZ-19(420), OPAA-15(600) and OPS-01(400), were amplified between 478(F) and 478-cms. The results showed that mtDNAs in isonucleus and isocytoplasmic lines were more homologous than that in others. There is minor mtDNA difference between the fertile type (F) and its sterile type (cms) in a pair of isonucleus and isocytoplasmic lines. The polymorphism detected in isonucleus and isocytoplasmic lines may be more closely linked with the gene of fertility. Therefore, isonucleus and isocytoplasmic line is an excellent system in the study of CMS. Sister isonucleus and isocytoplasmic lines are consisted of 2 groups of isonucleus and isocytoplasmic lines in which their nucleus are not all the same but closely related, their cytoplasm are the same. Using sister isonucleus and isocytoplasmic lines is equal to determining the change of fertility by transferring one cytoplasm into the isonucleus. Isonucleus and isocytoplasmic lines (II) are consisted of Su478(F) and Su478-cms. Isonucleus and isocytoplasmic lines (I) and (II) are sister isonucleus and isocytoplasmic lines. The polymorphic products, OPZ-19(420) and OPAA-15(600), can also be obtained in isonucleus and isocytoplasmic lines (II). The 2 polymorphic products OPZ-19(420) and OPAA-15(600) are existed in both of the isonucleus and isocytoplasmic lines. This showed that isonucleus and isocytoplasmic lines are practicable in the study of CMS, and that common polymorphism in isonucleus and isocytoplasmic lines may be related more directly to fertility.