Development and Assessment of SNP Genotyping Arrays for Citrus and Its Close Relatives.

Rapid advancements in technologies provide various tools to analyze fruit crop genomes to better understand genetic diversity and relationships and aid in breeding. Genome-wide single nucleotide polymorphism (SNP) genotyping arrays offer highly multiplexed assays at a relatively low cost per data point. We report the development and validation of 1.4M SNP Axiom® Citrus HD Genotyping Array (Citrus 15AX 1 and Citrus 15AX 2) and 58K SNP Axiom® Citrus Genotyping Arrays for Citrus and close relatives. SNPs represented were chosen from a citrus variant discovery panel consisting of 41 diverse whole-genome re-sequenced accessions of Citrus and close relatives, including eight progenitor citrus species. SNPs chosen mainly target putative genic regions of the genome and are accurately called in both Citrus and its closely related genera while providing good coverage of the nuclear and chloroplast genomes. Reproducibility of the arrays was nearly 100%, with a large majority of the SNPs classified as the most stringent class of markers, "PolyHighResolution" (PHR) polymorphisms. Concordance between SNP calls in sequence data and array data average 98%. Phylogenies generated with array data were similar to those with comparable sequence data and little affected by 3 to 5% genotyping error. Both arrays are publicly available.

Real-Time PCR to Phenotype Resistance to the Citrus Nematode Tylenchulus semipenetrans Cobb.

As pest management options, such as nematicides, become more restrictive, developing rootstocks resistant to the citrus nematode is fundamental for citrus production. This study provides an updated methodology to screen for citrus nematode resistance in rootstock-breeding programs. We developed a novel method to extract female citrus nematodes from roots that is suitable for molecular work and a real-time-PCR-based nematode quantification method for Tylenchulus semipenetrans. These procedures allow scaling up screening to high-throughput workflows, increasing the chances of finding rootstock candidates that combine all the desired traits. Our results contribute to the growing literature supporting quantification of nematodes with molecular methods.

An Improved Reference Gene for Detection of "Candidatus Liberibacter asiaticus" Associated with Citrus Huanglongbing by qPCR and Digital Droplet PCR Assays.

Citrus huanglongbing (HLB) disease associated with the 'Candidatus Liberibacter asiaticus' (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.

Inference of Chromosome-Length Haplotypes Using Genomic Data of Three or a Few More Single Gametes.

Compared with genomic data of individual markers, haplotype data provide higher resolution for DNA variants, advancing our knowledge in genetics and evolution. Although many computational and experimental phasing methods have been developed for analyzing diploid genomes, it remains challenging to reconstruct chromosome-scale haplotypes at low cost, which constrains the utility of this valuable genetic resource. Gamete cells, the natural packaging of haploid complements, are ideal materials for phasing entire chromosomes because the majority of the haplotypic allele combinations has been preserved. Therefore, compared with the current diploid-based phasing methods, using haploid genomic data of single gametes may substantially reduce the complexity in inferring the donor's chromosomal haplotypes. In this study, we developed the first easy-to-use R package, Hapi, for inferring chromosome-length haplotypes of individual diploid genomes with only a few gametes. Hapi outperformed other phasing methods when analyzing both simulated and real single gamete cell sequencing data sets. The results also suggested that chromosome-scale haplotypes may be inferred by using as few as three gametes, which has pushed the boundary to its possible limit. The single gamete cell sequencing technology allied with the cost-effective Hapi method will make large-scale haplotype-based genetic studies feasible and affordable, promoting the use of haplotype data in a wide range of research.

Hyperacidification of Citrus fruits by a vacuolar proton-pumping P-ATPase complex.

The sour taste of Citrus fruits is due to the extreme acidification of vacuoles in juice vesicle cells via a mechanism that remained elusive. Genetic analysis in petunia identified two vacuolar P-ATPases, PH1 and PH5, which determine flower color by hyperacidifying petal cell vacuoles. Here we show that Citrus homologs, CitPH1 and CitPH5, are expressed in sour lemon, orange, pummelo and rangpur lime fruits, while their expression is strongly reduced in sweet-tasting "acidless" varieties. Down-regulation of CitPH1 and CitPH5 is associated with mutations that disrupt expression of MYB, HLH and/or WRKY transcription factors homologous to those activating PH1 and PH5 in petunia. These findings address a long-standing enigma in cell biology and provide targets to engineer or select for taste in Citrus and other fruits.

Construction of High-Density Genetic Maps and Detection of QTLs Associated With Huanglongbing Tolerance in Citrus.

Huanglongbing (HLB), or citrus greening, is the most devastating disease in citrus worldwide. Commercial citrus varieties including sweet orange (Citrus sinensis) are highly susceptible to HLB, and trifoliate orange (Poncirus trifoliata, a close Citrus relative) is widely considered resistant or highly tolerant to HLB. In this study, an intergeneric F1 population of sweet orange and trifoliate orange was genotyped by Genotyping-by-Sequencing, and high-density SNP-based genetic maps were constructed separately for trifoliate orange and sweet orange. The two genetic maps exhibited high synteny and high coverage of the citrus genome. Progenies of the F1 population and their parents were planted in a replicated field trial, exposed to intense HLB pressure for 3 years, and then evaluated for susceptibility to HLB over 2 years. The F1 population exhibited a wide range in severity of HLB foliar symptom and canopy damage. Genome-wide QTL analysis based on the phenotypic data of foliar symptom and canopy damage in 2 years identified three clusters of repeatable QTLs in trifoliate orange linkage groups LG-t6, LG-t8 and LG-t9. Co-localization of QTLs for two traits was observed within all three regions. Additionally, one cluster of QTLs in sweet orange (linkage group LG-s7) was also detected. The majority of the identified QTLs each explained 18-30% of the phenotypic variation, indicating their major role in determining HLB responses. These results show, for the first time, a quantitative genetic nature yet the presence of major loci for the HLB tolerance in trifoliate orange. The results suggest that sweet orange also contains useful genetic factor(s) for improving HLB tolerance in commercial citrus varieties. Findings from this study should be very valuable and timely to researchers worldwide as they are hastily searching for genetic solutions to the devastating HLB crisis through breeding, genetic engineering, or genome editing.

Genomics of the origin and evolution of Citrus.

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus.

Long-Term Field Evaluation Reveals Huanglongbing Resistance in Citrus Relatives.

Citrus huanglongbing (HLB) is a destructive disease with no known cure. To identify sources of HLB resistance in the subfamily Aurantioideae to which citrus belongs, we conducted a six-year field trial under natural disease challenge conditions in an HLB endemic region. The study included 65 Citrus accessions and 33 accessions belonging to 20 other closely related genera. For each accession, eight seedling trees were evaluated. Based on quantitative polymerase chain reaction analysis of the pathogen titers and disease symptoms, eight disease-response categories were identified. We report two immune, six resistant, and 14 tolerant accessions. Resistance and tolerance observed in different accessions may be attributed to a multitude of factors, including psyllid colonization ability, absence of pathogen multiplication, transient replication of the bacterium, lack of pathogen establishment in the plant, delayed infection, or recovery from infection. Most citrus cultivars were considered susceptible: 15 citrons, lemons, and limes retained leaves in spite of the disease status. Resistance and high levels of field tolerance were observed in many noncitrus genera. Disease resistance/tolerance was observed in Australian citrus relative genera Eremocitrus and Microcitrus, which are sexually compatible with citrus and may be useful in future breeding trials to impart HLB resistance to cultivated citrus.

Cytological and molecular characterization of three gametoclones of Citrus clementina.

BACKGROUND: Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH). RESULTS: Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. CONCLUSIONS: The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.

A six nuclear gene phylogeny of Citrus (Rutaceae) taking into account hybridization and lineage sorting.

BACKGROUND: Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4-12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species. METHODOLOGY AND PRINCIPAL FINDINGS: (1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test. CONCLUSIONS: We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches-gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer the phylogeny among species of Citrus.

The draft genome of sweet orange (Citrus sinensis).

Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping.

BACKGROUND: Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a 'Mediterranean' mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine. RESULTS: Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. CONCLUSIONS: A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the 'Mediterranean' mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.

Expression of the H+-ATPase AHA10 proton pump is associated with citric acid accumulation in lemon juice sac cells.

The sour taste of lemons (Citrus limon (L.) Burm.) is determined by the amount of citric acid in vacuoles of juice sac cells. Faris is a "sweet" lemon variety since it accumulates low levels of citric acid. The University of California Riverside Citrus Variety Collection includes a Faris tree that produces sweet (Faris non-acid; FNA) and sour fruit (Faris acid; FA) on different branches; it is apparently a graft chimera with layer L1 derived from Millsweet limetta and layer L2 from a standard lemon. The transcription profiles of Faris sweet lemon were compared with Faris acid lemon and Frost Lisbon (L), which is a standard sour lemon genetically indistinguishable from Faris in prior work with SSR markers. Analysis of microarray data revealed that the transcriptomes of the two sour lemon genotypes were nearly identical. In contrast, the transcriptome of Faris sweet lemon was very different from those of both sour lemons. Among about 1,000 FNA-specific, presumably pH-related genes, the homolog of Arabidopsis H(+)-ATPase proton pump AHA10 was not expressed in FNA, but highly expressed in FA and L. Since Arabidopsis AHA10 is involved in biosynthesis and acidification of vacuoles, the lack of expression of the AHA10 citrus homolog represents a very conspicuous molecular feature of the FNA sweet phenotype. In addition, high expression of several 2-oxoglutarate degradation-related genes in FNA suggests activation of the GABA shunt and degradation of valine and tyrosine as components of the mechanism that reduces the level of citric acid in sweet lemon.

Development and implementation of high-throughput SNP genotyping in barley.

BACKGROUND: High density genetic maps of plants have, nearly without exception, made use of marker datasets containing missing or questionable genotype calls derived from a variety of genic and non-genic or anonymous markers, and been presented as a single linear order of genetic loci for each linkage group. The consequences of missing or erroneous data include falsely separated markers, expansion of cM distances and incorrect marker order. These imperfections are amplified in consensus maps and problematic when fine resolution is critical including comparative genome analyses and map-based cloning. Here we provide a new paradigm, a high-density consensus genetic map of barley based only on complete and error-free datasets and genic markers, represented accurately by graphs and approximately by a best-fit linear order, and supported by a readily available SNP genotyping resource. RESULTS: Approximately 22,000 SNPs were identified from barley ESTs and sequenced amplicons; 4,596 of them were tested for performance in three pilot phase Illumina GoldenGate assays. Data from three barley doubled haploid mapping populations supported the production of an initial consensus map. Over 200 germplasm selections, principally European and US breeding material, were used to estimate minor allele frequency (MAF) for each SNP. We selected 3,072 of these tested SNPs based on technical performance, map location, MAF and biological interest to fill two 1536-SNP "production" assays (BOPA1 and BOPA2), which were made available to the barley genetics community. Data were added using BOPA1 from a fourth mapping population to yield a consensus map containing 2,943 SNP loci in 975 marker bins covering a genetic distance of 1099 cM. CONCLUSION: The unprecedented density of genic markers and marker bins enabled a high resolution comparison of the genomes of barley and rice. Low recombination in pericentric regions is evident from bins containing many more than the average number of markers, meaning that a large number of genes are recombinationally locked into the genetic centromeric regions of several barley chromosomes. Examination of US breeding germplasm illustrated the usefulness of BOPA1 and BOPA2 in that they provide excellent marker density and sensitivity for detection of minor alleles in this genetically narrow material.

Genetic diversity and structure in semiwild and domesticated chiles (Capsicum annuum; Solanaceae) from Mexico.

The chile of Mesoamerica, Capsicum annuum, is one of five domesticated chiles in the Americas. Among the chiles, it varies the most in size, form, and color of its fruits. Together with maize, C. annuum is one of the principal elements of the neotropical diets of Mesoamerican civilizations. Despite the great economic and cultural importance of C. annuum both worldwide and in Mexico, however, very little is known about its geographic origin and number of domestications. Here we sampled a total of 80 accessions from Mexico (58 semiwild and 22 domesticated) and examined nucleotide sequence diversity at three single- or low-copy nuclear loci, Dhn, G3pdh, and Waxy. Across the three loci, we found an average reduction of ca. 10% in the diversity of domesticates relative to semiwild chiles and geographic structure within Mexican populations. The Yucatan Peninsula contained a large number of haplotypes, many of which were unique, suggesting an important region of chile domestication and center of diversity. The present sampling of loci did not conclusively resolve the number and location of domestications, but several lines of evidence suggest multiple independent domestications from widely distributed progenitor populations.

Recent history of artificial outcrossing facilitates whole-genome association mapping in elite inbred crop varieties.

Genomewide association studies depend on the extent of linkage disequilibrium (LD), the number and distribution of markers, and the underlying structure in populations under study. Outbreeding species generally exhibit limited LD, and consequently, a very large number of markers are required for effective whole-genome association genetic scans. In contrast, several of the world's major food crops are self-fertilizing inbreeding species with narrow genetic bases and theoretically extensive LD. Together these are predicted to result in a combination of low resolution and a high frequency of spurious associations in LD-based studies. However, inbred elite plant varieties represent a unique human-induced pseudo-outbreeding population that has been subjected to strong selection for advantageous alleles. By assaying 1,524 genomewide SNPs we demonstrate that, after accounting for population substructure, the level of LD exhibited in elite northwest European barley, a typical inbred cereal crop, can be effectively exploited to map traits by using whole-genome association scans with several hundred to thousands of biallelic SNPs.

Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs).

Twenty-four simple sequence repeat (SSR) markers were used to detect molecular polymorphisms among 370 mostly sexually derived Citrus accessions from the collection of citrus germplasm maintained at the University of California, Riverside. A total of 275 alleles were detected with an average of 11.5 alleles per locus and an average polymorphism information content of 0.625. Genetic diversity statistics were calculated for each individual SSR marker, the entire population, and for specified Citrus groups. Phylogenetic relationships among all citrus accessions and putative non-hybrid Citrus accessions were determined by constructing neighbor-joining trees. There was strong support for monophyly at the species level when hybrid taxa were removed from the data set. Both of these trees indicate that Fortunella clusters within the genus Citrus but Poncirus is a sister genus to Citrus. Additionally, Citrus accessions were probabilistically assigned to populations or multiple populations if their genotype indicated an admixture by a model-based clustering approach. This approach identified five populations in this data set. These separate analyses (distance and model based) both support the hypothesis that there are only a few naturally occurring species of Citrus and most other types of Citrus arose through various hybridization events between these naturally occurring forms.

Sequence analysis of a 282-kilobase region surrounding the citrus Tristeza virus resistance gene (Ctv) locus in Poncirus trifoliata L. Raf.

Citrus tristeza virus (CTV) is the major virus pathogen causing significant economic damage to citrus worldwide, and a single dominant gene, Ctv, provides broad spectrum resistance to CTV in Poncirus trifoliata L. Raf. Ctv was physically mapped to a 282-kb region using a P. trifoliata bacterial artificial chromosome library. This region was completely sequenced to about 8x coverage using a shotgun sequencing strategy and primer walking for gap closure. Sequence analysis predicts 22 putative genes, two mutator-like transposons and eight retrotransposons. This sequence analysis also revealed some interesting features of this region of the P. trifoliata genome: a disease resistance gene cluster with seven members and eight retrotransposons clustered in a 125-kb gene-poor region. Comparative sequence analysis suggests that six genes in the Ctv region have significant sequence similarity with their orthologs in bacterial artificial chromosome clones F7H2 and F21T11 from Arabidopsis chromosome I. However, the analysis of gene colinearity between P. trifoliata and Arabidopsis indicates that Arabidopsis genome sequence information may be of limited use for positional gene cloning in P. trifoliata and citrus. Analysis of candidate genes for Ctv is also discussed.