A total of 26 stably transformed callus lines were obtained. In the condition without DEX treatment, five calli were randomly selected from each callus line and GUS-assayed for detection of transposant cells. Transposant cells were detected in 84.6% of callus lines but mosaic GUS patterns occurred at low frequency as compared with the GUS patterns of untreated pJJ86 calli . GUS assays were also carried out on 14 of pHPT-Ds1 transformed plantlets; 57.1% plantlets contained transposant cells that were rarely distributed in the tissue . The results of pJJ86 transformants and pHPT-Ds1 transformants indicated that there was background transposition activity in the rice calli and plantlets selected from hygromycinmedia, and that the growth of rice cells containing HPT-Ds transposition events were partially suppressed by hygromycin counter selection. To characterize the HPT-Ds excision events, rice genomic DNA of eight GUS-positive pHPT-Ds1 transformants was extracted and examined in nested polymerase chain reaction reactions using Ubi- and GUS-specific primers . Reconstructed Ubi:GUS sequence containing the HPT-Ds empty donor site was confirmed by sequencing the 657-bp PCR product . These results suggested that HPT-Ds elements in the pHPT-Ds1 transformants excised from the T-DNA. To get more information about the background transposition in the GVG-inducible AcPTase system, we constructed pHPT-Ds3 and pHPT-Ds4 by removing the 35S:GVG from pHPT-Ds1 and pHPT-Ds2, respectively. According to the GUS assay results of pHPT-Ds3 transformed callus lines, 57.1% of the callus lines showed somatic transposition. The mosaic GUS patterns of pHPT-Ds3 transformants were similar to those of pHPT-Ds1 transformants and the transposition frequency was a little lower than 84.6% of the pHPT-Ds1 calli. Our explanation for the results of pHPT-Ds1 and pHPT-Ds3 is that the background transposition in the GVG-inducible Ac-Ds system was primarily due to a low-level leaky expression of 4xUAS:AcTPase.
The system of bacterial Cre-lox site-specific recombination was shown to be a useful tool for the generation of chromosomal rearrangements in plants . To stabilize transposed HPT-Ds,indoor vertical farming we used Cre-lox system to delete AcTPase after HPT-Ds transposition. pHPT-Ds5 and pHPT-Ds6 carry AcTPase flanked by two lox sites and the Cre gene that is separated from the upstream Ubi by HPT-Ds. To control AcTPase, the two vectors were designed in such a way that HPT-Ds can transpose in the rice genome and excision of HPT-Ds reconstructs Ubi:Cre and Cre recombinase mediates lox-lox recombination and thereby deletes AcTPase . For examination of rice cells containing deletion events, GUS and Bar were used in pHPTDs5 and pHPT-Ds6, respectively. We transformed pHPT-Ds5 into rice cultivars Taipei 309 and Nipponbare. Three of four Taipei 309 transformants and 23 of 30 Nipponbare transformants showed transposition as shown by mosaic GUS patterns . Genomic DNA of the Taipei 309 transformants was examined in nested PCR reactions using Ubi- and Cre-specific primers. Reconstructed Ubi:Cre sequence containing the HPT-Ds EDS was confirmed by sequencing the 0.6-kb PCR product . A 4.3-kb fragment containing the HPT-Ds full donor site in T-DNA was also amplifified in PCR of the transformants. Additionally, the pHPT-Ds6 vector was transformed into Nipponbare and the Ubi:Cre sequence was detected in genomic DNA of five of the eight pHPT-Ds6 transformants . Using adaptor-ligation PCR , we successfully cloned the rice genomic sequences flanking the HPT-Ds terminus from one pHPT-Ds5-transformant and one pHPT-Ds6- transformant . BLAST analysis showed that the flanking sequences were from the rice chromosomes 6 and 4, respectively, thereby confirming the reinsertion of excised HPT-Ds in the rice genome.
In analysis of T1 populations of pHPT-Ds5 plants, four of six T1 families showed transposition based on spotted GUS staining of the leaf tissues. These results indicated that the HPT-Ds element in pHPT-Ds5 and pHPTDs6 transformants transposed in rice and that the transposition restored Cre expression and induced deletion of AcTPase.During plant transformation and selection, HPT expression relies on the upstream Ubi promoter to confer resistance to hygromycin in selection media. In case of transposition, the HPT gene may be inactive because the 5 flanking sequence of HPT-Ds at a new genomic site may not be able to provide promoter activity. It is conceivable that most of the transposant cells become sensitive to hygromycin. Therefore, the counter-selection nature of the HPT gene in HPT-Ds can be used to diminish transposant cells in newly transformed rice calli on hygromycin media. In testing pHPT-Ds1 and pHPT-Ds3, it was observed that early transposition events in transformed calli and plantlets were suppressed by hygromycin. Few transposant cells in the calli and plantlets were able to grow under the hygromycin selection pressure, which might be due to escaping transposant cells or because of promoter activity of the 5 transposon flanking sequence. Because transposition requires transposase, an important theme in transposon tagging research is how to efficiently control transposase activity. It was reported that AcTPase driven by strong promoters mediated high-frequency Ds excision in several dicot plants . Strong double enhancers of CaMV 35S promoter adjacent to wildtype Ac element induced high-frequency Ac excision in rice transformation . In the present study, we have used the GVG-inducible promoter to control AcTPase expression and transposition was induced to high levels by DEX treatment of pJJ86 transformed callus.
However, we also observed a leaky expression of AcTPase in the GVG-inducible Ac system in the transformants of pJJ86, pHPT-Ds1 and pHPT-Ds7 based on GUS assay results. Our explanation is that the transposition background was primarily from a low level of leaky expression of 4xUAS:AcTPase. Consistently, in the pHPT-Ds3 and pHPT-Ds5 vectors that do not have 35S:GVG, 57.1% of the pHPT-Ds3 transformants and 76.6% of the pHPT-Ds5 transformants still showed transposition in somatic cells. In spite of the wild type Ac element having a weak promoter that supports only 0.2% expression of the CaMV 35S promoter , the wildtype Acitself can transpose in rice with a relatively low activity for three successive generations . This indicates that a weak expression of AcTPase can cause transposition events. In Southern blot analysis of genomic DNA of pHPT-Ds7 and pHPT-Ds8 transformants, the 5.4 kb hybridizing band represented the HPT-Ds at FDS in T-DNA. For the hybridizing bands larger or smaller than 5.4 kb, we explain that some of the bands might be from transposed HPT-Ds. The pHPTDs7 transformants showed transposition in somatic cells as suggested by GUS assay results. Because the rice genomic DNA for Southern hybridization was extracted from few leavesof a transformant, transposition in other leaves might not have been detected in the results. Also, since a rice transformant may have more than one T-DNA copy and may contain rearranged T-DNA,hydroponic vertical farming the hybridizing bands larger or smaller than 5.4 kb might possibly be from transgene rearrangement. Nevertheless, the efficacy of the HPT-Ds element when it was brought together with the GVG-inducible-AcTPase and the Cre-lox recombination system in pHPT-Ds7 and pHPT-Ds8 was confirmed by GUS assay and Southern blot analysis. For inducible Ac-Ds system, it was reported that in Arabidopsis AcTPase controlled by a heat shock promoter transactivated Ds upon heat shock treatment of flowering plants and the transposition was subsequently stabilized by release of the heat shock treatment . The heat shock method used in Arabidopsis seems impractical for rice because of the difficulty of heat shock treatment of a large number of rice plants. But for the GVG-inducible Ac-Ds system, the transgenic rice plants can be treated with DEX by hydroponics or by spray to induce transposition to higher frequency given that the treatment condition is optimized. Because the Cre-lox-based strategy will help delete AcTPase and thereby stabilize transposed HPTDs elements, we will be able to use GVG-inducible AcTPase to induce higher levels of transposition while using the Cre-lox system to stabilize transposition. The pHPT-Ds7 and pHPTDs8 vectors contain both GVG-inducible AcTPase and Crelox systems and therefore provide a good solution to major drawbacks in the Ac-Ds system. Further work needs to be done with the pHPT-Ds8 vector to determine how to enhance transposition by DEX induction and how to use the Bar gene to select Basta-resistant transposant progeny. In summary, we have constructed a series of Ac-Ds transposon tagging vectors and tested individual approaches to control AcTPase expression and transposition in transgenic rice. The pJJ86 and pDs-Ac-GVG vectors were made for testing GVGinducible AcTPase; the pHPT-Ds1 vector was for testing both GVG-inducible AcTPase and HPT-Ds that contains a dualfunctional HPT gene; the pHPT-Ds5 and pHPT-Ds6 vectors were for testing the deletion of AcTPase via Cre-lox recombination.
The pHPT-Ds7 and pHPT-Ds8 vectors contain all the features of GVG-inducible AcTPase, HPT-Ds and Cre-lox recombination and were tested for comprehensive control of AcTPase and HPT-Ds. The Ac-Ds transposon tagging vectors described in the present paper are publicly available, and provide useful resources for the functional genomics of a wide range of plants and especially for that of monocot plants.Introgressions from wild species are important resources for broadening the genetic base of cultivated species, particularly for traits where little variability currently exists. This is certainly the case for cultivated tomato , an economically important vegetable crop species with limited genetic variability . The genetic diversity of tomato has been augmented through introgression of alleles from several closely related wild species . One of these species, Solanum habrochaites, has been an important source of favorable alleles for horticultural traits such as yield, fruit size, and fruit quality . This wild species also contains genes for resistance to major tomato diseases such as late blight, bacterial canker, gray mold, and early blight . In cultivated tomato, genetic diversity is particularly lacking for resistance to late blight disease caused by Phytophthora infestans . Late blight is an economically important and devastating disease of both tomato and potato because it results in approximately $5 billion in annual crop losses and chemical control costs . S. habrochaites has genetic resistance to P. infestans. QTL for quantitative resistance to P. infestans from S. habrochaites have been mapped on each of tomato’s 12 chromosomes . Three of these QTL were then fifine-mapped by Brouwer and St.Clair using near-isogenic lines . QTL affecting horticultural traits including plant height, plant shape, maturity, yield, and fruit size were co-located and/or linked with each of these resistance QTL, suggesting the potential for linkage drag in crosses between S. lycopersicum and S. habrochaites. Subsequently, we mapped the QTL on S. habrochaites chromosome 11 at higher resolution using sub-NILs and detected multiple closely linked QTL controlling both foliar and stem resistance to P. infestans within a 9.4-cM region . To gain a better understanding of the genetic basis of QTL controlling horticultural traits and their linkage relationships with QTL for resistance to P. infestans, we used this same set of sub-NILs in the present study to map loci controlling horticultural traits and determine linkage relationships among them and with P. infestans resistance QTL. We also sought to identify useful breeding material with improved late blight resistance in this set of sub-NILs. In the present study, we further investigated the P. infestans resistance QTL lb11 region identified by Brouwer and St. Clair , conferred by a S. habrochaites introgression on tomato chromosome 11 as a potential source of useful quantitative resistance to late blight disease of tomato. Specifically, our goals in this study were to: assess the effects and extent of linkage drag of QTL controlling horticultural traits with P. infestans resistance QTL on S. habrochaites chromosome 11; identify markers closely linked to P. infestans resistance QTL and to positive alleles at horticultural QTL to facilitate MAS breeding; and identify potentially useful breeding lines for future breeding of tomato cultivars with improved quantitative resistance to late blight disease.We developed a set of sub-near-isogenic lines in S. lycopersicum for a chromosome 11 introgression containing resistance QTL from P. infestans-resistant S. habrochaites accession LA2099 via marker assisted selection during back crossing and selfing generations, as described by Johnson et al. . Methods used for genomic DNA extractions, genotyping with chromosome 11 PCR-based markers , primer sequences, enzymatic reaction conditions, and restriction enzymes used for each marker were described by Johnson et al. . We genotyped 1902 BC6S1 progeny to identify recombinant subNIL progeny for the chromosome 11 introgression from S. habrochaites; of these progeny, a subset of 852 progeny was used to construct a linkage map for the introgressed region .