Transformation-competent Artificial Chromosome Research Articles

The Arabidopsis T-DNA mutant SALK_008491 carries a 14-kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress.

In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light‐dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer‐DNA (T‐DNA) insertion mutant line SALK_008491, previously known as nhd1‐1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non‐photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14‐kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD‐dependent D‐3‐phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow‐up studies employing respective single mutants and complementation with overlapping transformation‐competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion‐carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T‐DNA lines. Although second‐site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T‐DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences.

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

The Arabidopsis TAC Position Viewer: a high-resolution map of transformation-competent artificial chromosome (TAC) clones aligned with the Arabidopsis thaliana Columbia-0 genome.

We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression.

Drought-tolerant rice germplasm developed from an Oryza officinalis transformation-competent artificial chromosome clone

Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.

Delivery of multiple transgenes to plant cells by an improved version of MultiRound Gateway technology

At present, only few methods for the effective assembly of multigene constructs have been described. Here we present an improved version of the MultiRound Gateway technology, which facilitates plant multigene transformation. The system consists of two attL-flanked entry vectors, which contain an attR cassette, and a transformation-competent artificial chromosome based destination vector. By alternate use of the two entry vectors, multiple transgenes can be delivered sequentially into the Gateway-compatible destination vector. Multigene constructs that carried up to seven transgenes corresponding to more than 26kb were assembled by seven rounds of LR recombination. The constructs were successfully transformed into tobacco plants and were stably inherited for at least two generations. Thus, our system represents a powerful, highly efficient tool for multigene plant transformation and may facilitate genetic engineering of agronomic traits or the assembly of genetic pathways for the production of biofuels, industrial or pharmaceutical compounds in plants.

Transgenic Rice Plants Harboring Genomic DNA from Zizania latifolia Confer Bacterial Blight Resistance

Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia (Griseb.), a pair of specific PCR primers FZ14P 1/FZ14P 2 was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome (TAC) library derived from Z. latifolia. A positive TAC clone (ZR1) was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop (kinase 1a), kinase 2, kinase 3a and GLPL (Gly-Leu-Pro-Leu), suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic T 0 plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.

Integration of cytogenetic and genetic linkage maps of Lotus japonicus, a model plant for legumes

To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.

Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants. In order to clone genes highly tolerant to barley yellow dwarf virus (BYDV), Aphids, drought and salt from Leymus multicaulis, the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB, which contain about 165000 and 236000 recombinant clones separately. The genome coverage of the two libraries was totally estimated to be about 3-5 haploid genome equivalents, as size selection of genomic DNA fragments was approximately from 9 to 300 kb. Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so, stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3x3 pattern using a GeneTACtrade mark G3 arraying robot after being transferred manually into three 384-well plates. Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6x6 pattern after a replication of plates. Nineteen positive clones were detected by using the probe glutahione reductase gene of L. multicaulis. TAC libraries constructed here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning. Once the target TAC clones were isolated, they could be immediately transferred into plant genomes with the Agrobacterium system.

Development of transgenic rice (Oryza sativa L.) expressing wheat high-and low-molecular-weight glutenin subunit proteins

We isolated transformation-competent artificial chromosome (TAC) clones harboring a high-molecular-weight glutenin subunit (HMW-GS) gene and a low-molecular-weight GS (LMW-GS) gene from wheat genomic DNA, and developed transgenic rice lines with these genes using a transformation system mediated by Agrobacterium tumefaciens. We developed two lines of transgenic rice, one expressing the gene coding for HMW-GS and the other expressing the gene coding for LMW-GS. By crossing these transgenic lines, a novel line harboring both genes was developed and the expression of GS proteins was analyzed. This is the first study indicating that the two kinds of wheat GSs, HMW-GS and LMW-GS, accumulated in rice endosperm. In all the transgenic lines, the introduced GS genes were expressed in the rice endosperm, and the expressed proteins were processed at the same site as the mature GS protein in wheat seeds, forming insoluble polymeric proteins similar to those found in wheat. It was suggested that the protein-processing system was conserved between rice and wheat, and that it was possible to produce wheat gluten using the protein-processing system of rice.

Structures of the three homoeologous loci of wheat benzoxazinone biosynthetic genes TaBx3 and TaBx4 and characterization of their promoter sequences

Common wheat (2n=6x=42, genome formula AABBDD) accumulates benzoxazinones (Bxs) as defensive compounds. There are five Bx biosynthetic genes (TaBx1-TaBx5), and their homoeologous alleles are located on all three homoeologous chromosomes of the A, B and D genomes. Here the molecular structures of the TaBx3 and TaBx4 loci, both of which are located on chromosomes 5A, 5B and 5D, were revealed by sequencing transformation-competent artificial chromosome (TAC) clones. In all homoeologous chromosomes, TaBx3 existed downstream of TaBx4 in a tail-to-head manner, and the two genes were separated from each other by 9.0 kb in 5A, 7.3 kb in 5B and 11.3 kb in 5D. Among the three homoeologs of TaBx3 and TaBx4, the promoter sequences were less conserved than the coding sequences. The promoter sequences of TaBx3 and TaBx4 were highly similar to those of their respective orthologs in the diploid progenitors of common wheat, but were not similar to those of the maize orthologs. Sequence similarity was found between the TaBx3 and TaBx4 coding sequences, but not between their promoter sequences despite their similar transcription pattern at the seedling stage. Some putative cis-elements were found to be shared by all TaBx3 and TaBx4 promoter regions. These results imply that stage-specific transcription of TaBx3 and TaBx4 is not controlled by global sequence similarity of their promoters but by some essential cis-elements. The promoter activity measured by transient assays in wheat protoplasts was similar among the three homoeologs of TaBx3 and TaBx4 in spite of their differential transcript levels in wheat seedlings.

Phytochelatin Synthases of the Model Legume Lotus japonicus. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants

The biosynthesis of phytochelatins and homophytochelatins has been studied in nodulated plants of the model legume Lotus (Lotus japonicus). In the first 6 to 24 h of treatment with cadmium (Cd), roots started to synthesize elevated amounts of both polypeptides, with a concomitant increase of glutathione and a decrease of homoglutathione, indicating the presence of active phytochelatin synthase (PCS) genes. Screening of transformation-competent artificial chromosome libraries allowed identification of a cluster of three genes, LjPCS1, LjPCS2, and LjPCS3, which were mapped at 69.0 cM on chromosome 1. The genes differ in exon-intron composition and responsiveness to Cd. Gene structures and phylogenetic analysis of the three protein products, LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N, are consistent with two sequential gene duplication events during evolution of vascular plants. Two sites for alternative splicing in the primary transcripts were identified. One of them, involving intron 2 of the LjPCS2 gene, was confirmed by the finding of the two predicted mRNAs, encoding LjPCS2-7R in roots and LjPCS2-7N in nodules. The amino acid sequences of LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share 90% identity, but have only 43% to 59% identity with respect to the typical PCS1 enzymes of Lotus and other plants. The unusual LjPCS2-7N and LjPCS3-7N proteins conferred Cd tolerance when expressed in yeast (Saccharomyces cerevisiae) cells, whereas the alternatively spliced form, LjPCS2-7R, differing only in a five-amino acid motif (GRKWK) did not. These results unveil complex regulatory mechanisms of PCS expression in legume tissues in response to heavy metals and probably to other developmental and environmental factors.

Construction and characterization of a transformation-competent artificial chromosome (TAC) library ofZizania latifolia (Griseb.)

The tribe Oryzeae consists of 12 genera and 71 species with a world distribution.Zizania latifolia (Griseb.) Turcz. ex Stapf is included in this tribe and possesses numerous traits valuable for rice breeding, such as disease and insect resistance, cold and flood tolerance, and high grain quality. The genetics and breeding ofZ. latifolia are still in their infancy. To facilitate genomic studies ofZizania, a genomic DNA library was constructed using a transformation-competent artificial chromosome (TAC) vector system. The TAC library contains 91, 584 TAC clones with an average insert size of approximately 45 kb, covering six haploidZizania genome equivalents. Very low signals after hybridization with chloroplast and mitochondrial genes indicate that the TAC library is predominantly composed of nuclear DNA. The TAC clones were stable inE. coli for 100 generations. Clones containing thedihydrodipicolinate synthase (DHPS) gene were screened by pooled PCR. The positive clones can be used forZ. latifolia DHPS gene cloning and functional analysis. The library will be useful in studies of genome structure, gene cloning and evolution of rice.

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.

Application of comparative genomics in developing molecular markers tightly linked to the virus resistance geneRsv4 in soybean

The Rsv4 gene confers resistance to all the known strain groups of soybean mosaic virus in soybean (Glycine max (L.) Merr.). To construct a fine genetic map near Rsv4 in soybean, we employed a comparative genomics approach that used genome sequence information of the model legume Lotus japonicus. Sequences of the soybean expressed sequence tags (ESTs) AI856415 and BF070293 mapping to one side of the Rsv4 gene showed high similarity with gene sequences of the transformation-competent artificial chromosome (TAC) clone LjT32P24 of Lotus. LjT32P24 is tightly linked to another sequenced TAC clone, LjT26I01, in Lotus. A new marker, AW307114A, developed from soybean EST AW307114, which is homologous to a Lotus gene within LjT26I01, was mapped to the other side of the Rsv4 gene. The identification of the microsyntenic relationship facilitated the development of additional 2 EST markers between BF070293-S and AW307114A bracketing the Rsv4 gene. Several other markers developed in this study were mapped to putative homoeologous or duplicated chromosomal regions in soybean. Alignment between the soybean maps indicated that Rsv4 is located near a local chromosomal rearrangement. This targeted comparative mapping serves to provide a foundation for marker-assisted selection and cloning of the Rsv4 gene.

Molecular cytogenetic characterization of the Antirrhinum majus genome.

As a model system in classical plant genetics, the genus Antirrhinum has been well studied, especially in gametophytic self-incompatibility, flower development biology, and transposon-induced mutation. In contrast to the advances in genetic and molecular studies, little is known about Antirrhinum cytogenetics. In this study, we isolated two tandem repetitive sequences, CentA1 and CentA2, from the centromeric regions of Antirrhinum chromosomes. A standard karyotype has been established by anchoring these centromeric repeats on meiotic pachytene chromosome using FISH. An ideogram based on the DAPI-staining pattern of pachytene chromosomes was developed to depict the distribution of heterochromatin in the Antirrhinum majus genome. To integrate the genetic and chromosomal maps, we selected one or two molecular markers from each linkage group to screen an Antirrhinum transformation-competent artificial chromosome (TAC) library. These genetically anchored TAC clones were labeled as FISH probes to hybridize to pachytene chromosomes of A. majus. As a result, the relationship between chromosomes and the linkage groups (LGs) in Antirrhinum has been established.

Identification of a 98-kb DNA segment containing the rice Eui gene controlling uppermost internode elongation, and construction of a TAC transgene sublibrary.

The recessive 'tall rice' phenotype associated with the mutation eui (elongated upper-most internode) is an important agronomic trait that has been introduced into hybrid rice to eliminate panicle enclosure in all types of male-sterile lines and produce good-quality seeds in high yield and at low cost. Based on our previous Eui mapping data, we conducted fine-structure mapping and positional cloning of the gene using an F2 population comprising more than 5000 individuals derived from a cross of the near-isogenic lines 307T (eui/eui) with the recurrent parent Zhenshan 97 (Eui/Eui). In total 45 CAPS (cleaved amplified polymorphic sequences) markers located within an interval of 14.5 cM were analyzed in the subpopulation of 1298 homozygous recessive plants. The resulting high-resolution map defined a 98-kb interval containing the Eui locus flanked by the markers M0387 and M01, and three markers were found to co-segregate with Eui. In order to facilitate the identification of the Eui gene, we used a transformation-competent artificial chromosome (TAC) vector to construct a set of contiguous TAC clones from the Nipponbare BACs (obtained from the Clemson University Genome Institute; CUGI) spanning this region. These clones can be used to streamline complementation testing. The markers tightly linked to the Eui locus can also be used in breeding male-sterile lines with the elongated uppermost internode.

The F-box protein AhSLF-S2 controls the pollen function of S-RNase-based self-incompatibility.

Recently, we have provided evidence that the polymorphic self-incompatibility (S) locus-encoded F-box (SLF) protein AhSLF-S(2) plays a role in mediating a selective S-RNase destruction during the self-incompatible response in Antirrhinum hispanicum. To investigate its role further, we first transformed a transformation-competent artificial chromosome clone (TAC26) containing both AhSLF-S(2) and AhS(2)-RNase into a self-incompatible (SI) line of Petunia hybrida. Molecular analyses showed that both genes are correctly expressed in pollen and pistil in four independent transgenic lines of petunia. Pollination tests indicated that all four lines became self-compatible because of the specific loss of the pollen function of SI. This alteration was transmitted stably into the T1 progeny. We then transformed AhSLF-S(2) cDNA under the control of a tomato (Lycopersicon esculentum) pollen-specific promoter LAT52 into the self-incompatible petunia line. Molecular studies revealed that AhSLF-S(2) is specifically expressed in pollen of five independent transgenic plants. Pollination tests showed that they also had lost the pollen function of SI. Importantly, expression of endogenous SLF or SLF-like genes was not altered in these transgenic plants. These results phenocopy a well-known phenomenon called competitive interaction whereby the presence of two different pollen S alleles within pollen leads to the breakdown of the pollen function of SI in several solanaceaous species. Furthermore, we demonstrated that AhSLF-S(2) physically interacts with PhS(3)-RNase from the P. hybrida line used for transformation. Together with the recent demonstration of PiSLF as the pollen determinant in P. inflata, these results provide direct evidence that the polymorphic SLF including AhSLF-S(2) controls the pollen function of S-RNase-based self-incompatibility.

TheVER2 promoter contains repeated sequences and requires vernalization for its activity in winter wheat (Triticum aestivum L.)

Previously, we isolated a vernalization-related gene,VER2, from winter wheat (Triticum aestivum L.) and its expression was restricted in the immature leaves of vernalized wheat seedlings. To further investigate the regulation ofVER2 expression and the function of its promoter, we isolated a 41.7 kb genomic clone containingVER2 gene from a transformation-competent artificial chromosome (TAC) library of wheat (Triticum aestivum-Haynaldia villosa). The sequence analysis showed that there were eleven predicted genes in the TAC. The exons of gene 3 corresponded to the cDNA sequence ofVER2 gene. Analysis ofVER2 promoter structure showed that there were three small repeat sequences divided by two large repeat sequences. The putative response elements, such as abscisic acid response elements (ABRE), MeJA-response elements (Me-JARE), low-temperature response elements (LTR), endosperm expression elements, MYB binding sites and similar elements to GA response elements (GARE), were involved in theVER2 promoter region. Construct containing theVER2 promoter (-5895 to +73) drivingGFP reporter gene was bombarded into vernalized or non-vernalized immature leaves in wheat. The vernalized immature leaves showed bright green fluorescence after incubation for 24 h, however, the green fluorescence was not observed in the non-vernalization leaves under the same condition. These results suggested that vernalization was essential for the function ofVER2 promoter in the immature leaves of winter wheat.

Development of a new transformation-competent artificial chromosome (TAC) vector and construction of tomato and rice TAC libraries

Recent research has shown that BIBAC (binary bacterial artificial chromosome) and TAC (transformation-competent artificial chromosome) vector systems are very useful tools for map-based cloning of agronomically important genes in plant species. We have developed a new TAC vector that is suitable for both dicot and monocot transformation. Using this new TAC vector, we constructed large-insert genomic libraries of tomato and rice. The tomato library contains 96,996 clones (28.3-38.5 kb insert size) and has 3.18 haploid genome equivalents. The rice TAC library has 32.7 kb average insert size and has 9.24 haploid genome equivalents. The quality of these two libraries was tested using PCR to verify genome coverage. Individual clones were characterized to confirm insert integrity by Southern analysis, end sequencing and genetic mapping. To investigate the potential application of these TAC libraries in map-based cloning, TAC constructs containing a 45 kb fragment were introduced into the rice genome via Agrobacterium-mediated transformation. Molecular analysis indicates that the 45 kb fragment was successfully transferred into the rice genome. Although rearrangements of the introduced DNA were detected, 50% of regenerated plants contained at least one intact copy of the 45 kb clone and associated vector sequences. These libraries provide us with a valuable resource to rapidly isolate important genes in tomato and rice.

Structural and transcriptional analysis of S -locus F-box genes in Antirrhinum

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S 2, very similar to S 2 -RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S 2 also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S 1 S 5 and S 2 S 4, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S 2-specific primers, TAC clones containing both AhSLF-S 2 and its homologs were subsequently identified (S 2 TAC, S 5 TACa, S 4 TAC, and S 1 TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S 2, -S 1, -S 4 and -S 5) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S 2 were identified. In total, three F-box genes (AhSLF-S 2, -S 2 A and -S 2 C) in S 2 TAC (51 kb), three (AhSLF-S 4, -S 4 A and -S 4 D) in S 4 TAC (75 kb), two (AhSLF-S 5 and -S 5 A) in S 5 TACa (55 kb), and two (AhSLF-S 1 and -S 1 E) in S 1 TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S 2 C, -S 4 D and -S 1 E) were specifically expressed in pollen, similar to AhSLF-S 2, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.