Genomic prediction and genome-wide association study using combined genotypic data from different genotyping systems: application to apple fruit quality traits.

With advances in next-generation sequencing technologies, various marker genotyping systems have been developed for genomics-based approaches such as genomic selection (GS) and genome-wide association study (GWAS). As new genotyping platforms are developed, data from different genotyping platforms must be combined. However, the potential use of combined data for GS and GWAS has not yet been clarified. In this study, the accuracy of genomic prediction (GP) and the detection power of GWAS increased for most fruit quality traits of apples when using combined data from different genotyping systems, Illumina Infinium single-nucleotide polymorphism array and genotyping by random amplicon sequencing-direct (GRAS-Di) systems. In addition, the GP model, which considered the inbreeding effect, further improved the accuracy of the seven fruit traits. Runs of homozygosity (ROH) islands overlapped with the significantly associated regions detected by the GWAS for several fruit traits. Breeders may have exploited these regions to select promising apples by breeders, increasing homozygosity. These results suggest that combining genotypic data from different genotyping platforms benefits the GS and GWAS of fruit quality traits in apples. Information on inbreeding could be beneficial for improving the accuracy of GS for fruit traits of apples; however, further analysis is required to elucidate the relationship between the fruit traits and inbreeding depression (e.g. decreased vigor).

Corrigendum: Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus.

[This corrects the article DOI: 10.3389/fpls.2023.1163358.].

Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus.

In citrus breeding programs, male sterility is an important trait for developing seedless varieties. Sterility associated with the male sterile cytoplasm of Kishu mandarin (Kishu-cytoplasm) has been proposed to fit the cytoplasmic male sterility (CMS) model. However, it remains undetermined whether CMS in citrus is controlled by interactions between sterile cytoplasm and nuclear restorer-of-fertility (Rf) genes. Accordingly, mechanisms underlying the control of the wide phenotypic variation in pollen number for breeding germplasm should be elucidated. This study aimed to identify complete linkage DNA markers responsible for male sterility at the MS-P1 region based on fine mapping. Two P-class pentatricopeptide repeat (PPR) family genes were identified as candidates for Rf based on predicted mitochondrial localization and higher expression in a male fertile variety/selected strain than in a male sterile variety. Eleven haplotypes (HT1-HT11) at the MS-P1 region were defined based on genotyping of DNA markers. Association analysis of diplotypes at the MS-P1 region and the number of pollen grains per anther (NPG) in breeding germplasms harboring Kishu-cytoplasm revealed that the diplotypes in this region influenced NPG. Among these haplotypes, HT1 is a non-functional restorer-of-fertility (rf) haplotype; HT2, a less-functional Rf; HT3-HT5 are semi-functional Rfs; and HT6 and HT7 are functional Rfs. However, the rare haplotypes HT8-HT11 could not be characterized. Therefore, P-class PPR family genes in the MS-P1 region may constitute the nuclear Rf genes within the CMS model, and a combination of the seven haplotypes could contribute to phenotypic variation in the NPG of breeding germplasms. These findings reveal the genomic mechanisms of CMS in citrus and will contribute to seedless citrus breeding programs by selecting candidate seedless seedlings using the DNA markers at the MS-P1 region.

Dissecting Breeders' Sense via Explainable Machine Learning Approach: Application to Fruit Peelability and Hardness in Citrus.

"Genomics-assisted breeding", which utilizes genomics-based methods, e.g., genome-wide association study (GWAS) and genomic selection (GS), has been attracting attention, especially in the field of fruit breeding. Low-cost genotyping technologies that support genome-assisted breeding have already been established. However, efficient collection of large amounts of high-quality phenotypic data is essential for the success of such breeding. Most of the fruit quality traits have been sensorily and visually evaluated by professional breeders. However, the fruit morphological features that serve as the basis for such sensory and visual judgments are unclear. This makes it difficult to collect efficient phenotypic data on fruit quality traits using image analysis. In this study, we developed a method to automatically measure the morphological features of citrus fruits by the image analysis of cross-sectional images of citrus fruits. We applied explainable machine learning methods and Bayesian networks to determine the relationship between fruit morphological features and two sensorily evaluated fruit quality traits: easiness of peeling (Peeling) and fruit hardness (FruH). In each of all the methods applied in this study, the degradation area of the central core of the fruit was significantly and directly associated with both Peeling and FruH, while the seed area was significantly and directly related to FruH alone. The degradation area of albedo and the area of flavedo were also significantly and directly related to Peeling and FruH, respectively, except in one or two methods. These results suggest that an approach that combines explainable machine learning methods, Bayesian networks, and image analysis can be effective in dissecting the experienced sense of a breeder. In breeding programs, collecting fruit images and efficiently measuring and documenting fruit morphological features that are related to fruit quality traits may increase the size of data for the analysis and improvement of the accuracy of GWAS and GS on the quality traits of the citrus fruits.

QTL mapping of male sterility and transmission pattern in progeny of Satsuma mandarin.

Seedlessness is one of the important traits in citrus breeding. Male sterility derived from Satsuma mandarin (Citrus unshiu) has been used in Japanese citrus breeding programs to obtain seedless cultivars. The efficiency of seedless cultivar breeding would be improved by developing a selection marker linked to seedlessness. In this study, we performed QTL mapping in 'Okitsu No. 46' × 'Okitsu No. 56' (O46-O56) crosses for the number of pollen grains per anther (NPG) and apparent pollen fertility (APF), two traits used as an index of male sterility, and detected a candidate QTL for NPG (MS-P1) on linkage group 8 with a significant LOD score (7.31) and 47% of variance explained. The QTL for APF (MS-F1) was detected on linkage group 6 with a significant LOD score (5.71) and 63.6% of variance explained. The role of both MS-P1 in reducing NPG and MS-F1 in decreasing APF were confirmed with the 'Okitsu No.46' × 'Kara' (O46-K) cross. Pedigree analysis inferred that both MS-P1 and MS-F1 in 'Okitsu No. 46' were derived from kunenbo (Citrus nobilis) through hassaku (C. hassaku) and 'Sweet Spring'. Cytoplasm analysis revealed that both male-sterile 'Sweet Spring' and 'Okitsu No. 46' have cytoplasm derived from Kishu (C. kinokuni hort. ex Tanaka), but the cytoplasm of male-sterile kunenbo and hassaku were derived from other varieties rather than Kishu. These results suggest that MS-P1 and MS-F1 primarily reduce the NPG and decrease APF, but their expression requires a cytoplasm derived from Kishu. These findings will improve our understanding of the molecular mechanism of male sterility in citrus and help to develop a DNA marker for seedless breeding in citrus.

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach.

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma ("Miyagawa Wase") was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome.

Genome-wide association study and genomic prediction in citrus: Potential of genomics-assisted breeding for fruit quality traits.

Novel genomics-based approaches such as genome-wide association studies (GWAS) and genomic selection (GS) are expected to be useful in fruit tree breeding, which requires much time from the cross to the release of a cultivar because of the long generation time. In this study, a citrus parental population (111 varieties) and a breeding population (676 individuals from 35 full-sib families) were genotyped for 1,841 single nucleotide polymorphisms (SNPs) and phenotyped for 17 fruit quality traits. GWAS power and prediction accuracy were increased by combining the parental and breeding populations. A multi-kernel model considering both additive and dominance effects improved prediction accuracy for acidity and juiciness, implying that the effects of both types are important for these traits. Genomic best linear unbiased prediction (GBLUP) with linear ridge kernel regression (RR) was more robust and accurate than GBLUP with non-linear Gaussian kernel regression (GAUSS) in the tails of the phenotypic distribution. The results of this study suggest that both GWAS and GS are effective for genetic improvement of citrus fruit traits. Furthermore, the data collected from breeding populations are beneficial for increasing the detection power of GWAS and the prediction accuracy of GS.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives.

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes.

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

Segregation and Heritability of Male Sterility in Populations Derived from Progeny of Satsuma Mandarin.

Male sterility derived from Satsuma mandarin (Citrus unshiu) has been used in Japanese citrus breeding programs to obtain seedless cultivars, which is a desirable trait for consumers. Male sterility has often been evaluated by anther development or pollen fertility; however, the inheritance and heritability of male sterility derived from Satsuma is poorly understood. In this study, we investigated the mode of inheritance and broad-sense heritability of male sterility derived from Satsuma. Initially, we evaluated the total number of pollen grains per anther and apparent pollen fertility, as indicated by lactophenol blue staining, in 15 citrus cultivars and selections to understand the male sterility of Satsuma. The results indicated that male sterility was primarily caused by decreased number of pollen grains per anther in progeny of Satsuma. We also evaluated these traits in three F1 populations (hyuganatsu × 'Okitsu No. 56', 'Okitsu No. 46' × 'Okitsu No. 56' and 'Okitsu No. 46' × 'Kara'), of which the parents are derived from Satsuma. Individuals in these populations showed strong segregation for number of pollen grains per anther. The apparent fertility of pollen also showed segregation but was almost constant at 70%-90%. The estimated broad-sense heritability for the number of pollen grains per anther was as high as 0.898 in the 'Okitsu No. 46' × 'Okitsu No. 56' and 'Okitsu No. 46' × 'Kara' populations. These results indicated that the number of pollen grains per anther primarily determined male sterility among progeny of Satsuma, and this trait was inherited by the progeny. Development of DNA markers closely linked to male sterility using the F1 populations of 'Okitsu No. 46' × 'Okitsu No. 56' and 'Okitsu No. 46' × 'Kara' is expected to contribute to the breeding of novel seedless citrus cultivars.

Minimal marker: an algorithm and computer program for the identification of minimal sets of discriminating DNA markers for efficient variety identification.

DNA markers are frequently used to analyze crop varieties, with the coded marker data summarized in a computer-generated table. Such summary tables often provide extraneous data about individual crop genotypes, needlessly complicating and prolonging DNA-based differentiation between crop varieties. At present, it is difficult to identify minimal marker sets--the smallest sets that can distinguish between all crop varieties listed in a marker-summary table--due to the absence of algorithms capable of such characterization. Here, we describe the development of just such an algorithm and MinimalMarker, its accompanying Perl-based computer program. MinimalMarker has been validated in variety identification of fruit trees using published datasets and is available for use with both dominant and co-dominant markers, regardless of the number of alleles, including SSR markers with numeric notation. We expect that this program will prove useful not only to genomics researchers but also to government agencies that use DNA markers to support a variety of food-inspection and -labeling regulations.

H2DB: a heritability database across multiple species by annotating trait-associated genomic loci.

H2DB (http://tga.nig.ac.jp/h2db/), an annotation database of genetic heritability estimates for humans and other species, has been developed as a knowledge database to connect trait-associated genomic loci. Heritability estimates have been investigated for individual species, particularly in human twin studies and plant/animal breeding studies. However, there appears to be no comprehensive heritability database for both humans and other species. Here, we introduce an annotation database for genetic heritabilities of various species that was annotated by manually curating online public resources in PUBMED abstracts and journal contents. The proposed heritability database contains attribute information for trait descriptions, experimental conditions, trait-associated genomic loci and broad- and narrow-sense heritability specifications. Annotated trait-associated genomic loci, for which most are single-nucleotide polymorphisms derived from genome-wide association studies, may be valuable resources for experimental scientists. In addition, we assigned phenotype ontologies to the annotated traits for the purposes of discussing heritability distributions based on phenotypic classifications.

Characterization of genomic sequence showing strong association with polyembryony among diverse Citrus species and cultivars, and its synteny with Vitis and Populus.

Polyembryony, in which multiple somatic nucellar cell-derived embryos develop in addition to the zygotic embryo in a seed, is common in the genus Citrus. Previous genetic studies indicated polyembryony is mainly determined by a single locus, but the underlying molecular mechanism is still unclear. As a step towards identification and characterization of the gene or genes responsible for nucellar embryogenesis in Citrus, haplotype-specific physical maps around the polyembryony locus were constructed. By sequencing three BAC clones aligned on the polyembryony haplotype, a single contiguous draft sequence consisting of 380 kb containing 70 predicted open reading frames (ORFs) was reconstructed. Single nucleotide polymorphism genotypes detected in the sequenced genomic region showed strong association with embryo type in Citrus, indicating a common polyembryony locus is shared among widely diverse Citrus cultivars and species. The arrangement of the predicted ORFs in the characterized genomic region showed high collinearity to the genomic sequence of chromosome 4 of Vitis vinifera and linkage group VI of Populus trichocarpa, suggesting that the syntenic relationship among these species is conserved even though V. vinifera and P. trichocarpa are non-apomictic species. This is the first study to characterize in detail the genomic structure of an apomixis locus determining adventitious embryony.

A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag).

BACKGROUND: Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence. RESULTS: A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size. CONCLUSIONS: BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs.

Molecular characterization of FLOWERING LOCUS T-like genes of apple (Malus x domestica Borkh.).

The two FLOWERING LOCUS T (FT)-like genes of apple (Malus x domestica Borkh.), MdFT1 and MdFT2, have been isolated and characterized. MdFT1 and MdFT2 were mapped, respectively, on distinct linkage groups (LGs) with partial homoeology, LG 12 and LG 4. The expression pattern of MdFT1 and MdFT2 differed in that MdFT1 was expressed mainly in apical buds of fruit-bearing shoots in the adult phase, with little expression in the juvenile tissues, whereas MdFT2 was expressed mainly in reproductive organs, including flower buds and young fruit. On the other hand, both genes had the potential to induce early flowering since transgenic Arabidopsis, which ectopically expressed MdFT1 or MdFT2, flowered earlier than wild-type plants. Furthermore, overexpression of MdFT1 conferred precocious flowering in apple, with altered expression of other endogenous genes, such as MdMADS12. These results suggest that MdFT1 could function to promote flowering by altering the expression of those genes and that, at least, other genes may play an important role as well in the regulation of flowering in apple. The long juvenile period of fruit trees prevents early cropping and efficient breeding. Our findings will be useful information to unveil the molecular mechanism of flowering and to develop methods to shorten the juvenile period in various fruit trees, including apple.

Transcriptional changes in CiFT-introduced transgenic trifoliate orange (Poncirus trifoliata L. Raf.).

Ectopic expression of a Flowering Locus T (CiFT) from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). To profile transcriptional effects of CiFT, mRNA extracted from the shoots of the transgenic trifoliate orange was subjected to genetic analysis using a 22-K oligo DNA microarray. Microarray results and reverse transcription polymerase chain reaction analysis indicated that genes relating to transcription, cell wall modification and defense responses were expressed at higher levels in the transgenic shoots than in the control wild-type shoots. Of the genes showing ectopic mRNA accumulation, two SEPALLATA (SEP) and one FRUITFULL (FUL) homologues (CuSEP1, CuSEP3 or CuFUL) were introduced to Arabidopsis thaliana. Constitutive expression of each gene caused early flowering in transgenic Arabidopsis, suggesting that these genes could function as regulators of flowering time.

Differences in seasonal expression of flowering genes between deciduous trifoliate orange and evergreen Satsuma mandarin.

To determine differences in seasonal flowering between evergreen and deciduous woody perennials, endogenous expression of flowering-related genes was investigated in Satsuma mandarin (Citrus unshiu Marc.) and its close relative, trifoliate orange (Poncirus trifoliata (L.) Raf.), which are evergreen and deciduous, respectively, and show different seasonal flowering characteristics. In Satsuma mandarin, in which floral induction is triggered by low temperatures during fall and winter, mRNA levels of the citrus FLOWERING LOCUS T homologue CiFT increased during fall and winter, corresponding to the floral induction period, and mRNA levels of citrus LEAFY and SEPALLATA homologues (CsLFY and CuSEP) increased during early spring just before blooming. Citrus APETALA1 and FRUITFULL homologues (CsAP1 and CuFUL) did not show a significant association with seasonal flowering. In trifoliate orange, in which floral induction and flower bud development occur during early summer as in many deciduous trees, expression of CiFT, CsLFY, CsAP1, CuSEPs and CuFUL increased during early summer, corresponding to the period of floral induction and flower bud development. The CuSEPs expression peaked again during early spring just before blooming. In both species, the citrus TERMINAL FLOWER1 homologue (CsTFL), which acts as a floral repressor, showed low transcript levels during the period of floral induction and flower bud development. Thus, despite the difference in flowering season, in both species transcriptional changes in CiFT, CsLFY, CsTFL and CuSEPs were correlated with seasonal flowering. In contrast, the correspondence between CsAP1 and CuFUL expression and seasonal flowering differed between the species.

Increased CiFT abundance in the stem correlates with floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.).

After several years in the juvenile phase, adult citrus trees show seasonal periodicity of flowering. A prolonged exposure to low temperature is one of the most important environmental cues for floral induction in citrus. In the present study, the expression of flowering-related genes during the annual cycle of flowering and inductive low-temperature treatment in Satsuma mandarin (Citrus unshiu Marc.) trees is investigated. Simultaneously, floral induction, which occurs before the period of morphological flower development, was estimated as the number of flowers after the forcing of sprouting by defoliation at 25 degrees C. The expression of citrus FLOWERING LOCUS T homologues, CiFT, showed a seasonal increase during the floral induction period and was also induced by an artificial low-temperature treatment (15 degrees C) at which floral induction occurred. By contrast, the mRNA level of CiFT did not show any distinct changes following a warm-temperature treatment (25 degrees C) for 2.5 months, during which time floral induction was completely suppressed. Changes in the expression of the citrus homologues of TERMINAL FLOWER 1, LEAFY, and APETALA1 did not show any correlation with floral induction in the field or under artificial low-temperature conditions. In juvenile seedlings of Satsuma mandarin, which does not flower even under inductive low-temperature conditions, the mRNA levels of CiFT were not affected by the low-temperature treatment, unlike adult tissues. These results suggest that low temperature promotes floral induction via the activation of CiFT transcription in adult Satsuma mandarin trees and that, in the juvenile plant, CiFT transcription does not respond to low temperature.

Tolerance to Citrus mosaic virus in Transgenic Trifoliate Orange Lines Harboring Capsid Polyprotein Gene.

Trifoliate orange plants (Poncirus trifoliata) were transformed with a binary vector containing the capsid polyprotein (pCP) gene of Citrus mosaic virus (CiMV) via Agrobacterium tumefaciens LBA4404. Transformation was performed on the epicotyl segments obtained from a young seedling that was grown in the dark. Southern blot hybridization analysis showed that the transgene was stable in the transgenic lines after regeneration and propagation by grafting. Transgenic lines were screened for tolerance to CiMV by mechanical inoculation. Infection was monitored 30, 60, 90, and 120 days after inoculation by reverse transcription-polymerase chain reaction. The transgenic line 24 had the lowest infection rate (7.1%) at 60 days after inoculation, in contrast to that of nontransgenic plants (65.1%).The response of other lines to inoculation ranged from susceptibility to moderate tolerance.