Robert R. Krueger

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.

Genetic Analysis of Egyptian Date (Phoenix dactylifera L.) Accessions Using AFLP Markers

Forty-seven samples of date palm (Phoenix dactylifera L.) collected from eight locations in Egypt were studied using four sets of amplified fragment length polymorphism (AFLP) markers with near infrared fluorescence labeled primers. These samples belonged to 21 named accessions and 9 of unknown pedigrees. A total of 350 bands were scored and 233 (66.6%) were polymorphic. Twenty-seven Egyptian accessions and ‘Medjool’and ‘Deglet Noor’accessions from California could beclassified into the major cluster. This major cluster may represent a major group of date palm germplasm in North Africa. There were four other clusters, each containing one or two accessions. The variety ‘Halawy’and one accession of unknown provenance were most likely from hybridization between two clusters. Six groups of accessions of which had the same names, revealed similar but not identical AFLP profiles suggesting these accessions might derive from seedlings rather thanthrough clonal offshoot propagation.

Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop

Date palms (Phoenix dactylifera) are the most significant perennial crop in arid regions of the Middle East and North Africa. Here, we present a comprehensive catalogue of approximately seven million single nucleotide polymorphisms in date palms based on whole genome re-sequencing of a collection of 62 cultivars. Population structure analysis indicates a major genetic divide between North Africa and the Middle East/South Asian date palms, with evidence of admixture in cultivars from Egypt and Sudan. Genome-wide scans for selection suggest at least 56 genomic regions associated with selective sweeps that may underlie geographic adaptation. We report candidate mutations for trait variation, including nonsense polymorphisms and presence/absence variation in gene content in pathways for key agronomic traits. We also identify a copia-like retrotransposon insertion polymorphism in the R2R3 myb-like orthologue of the oil palm virescens gene associated with fruit colour variation. This analysis documents patterns of post-domestication diversification and provides a genomic resource for this economically important perennial tree crop.

What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles

Sixty-five microsatellite alleles amplified from ancestral citrus accessions classified in three separate genera were evaluated for sequence polymorphism to establish the basis of inter- and intra-allelic genetic variation, evaluate the extent of size homoplasy, and determine an appropriate model (stepwise or infinite allele) for analysis of citrus microsatellite alleles. Sequences for each locus were aligned and subsequently used to determine relationships between alleles of different taxa via parsimony. Interallelic size variation at each SSR locus examined was due to changes in repeat copy number with one exception. Sequencing these alleles uncovered new distinct point mutations in the microsatellite region and the region flanking the microsatellite. Several of the point mutations were found to be genus, species, or allele specific, and some mutations were informative about the inferred evolutionary relationships among alleles. Overall, homoplasy was observed in alleles from all three loci, where the core microsatellite repeat was changed causing alleles of the same size class to be identical in state but not identical by descent. Because nearly all changes in allele size (with one exception) were due to expansion or contraction of the repeat motif, this suggests that a stepwise mutation model, which assumes homoplasy may occur, would be the most appropriate for analyzing Citrus SSR data. The collected data indicate that microsatellites can be a useful tool for evaluating Citrus species and two related genera since repeat motifs were reasonably well retained. However, this work also demonstrated that the number of microsatellite alleles is clearly an underestimate of the number of sequence variants present.

A Genome-Wide Survey of Date Palm Cultivars Supports Two Major Subpopulations in Phoenix dactylifera

The date palm (Phoenix dactylifera L.) is one of the oldest cultivated trees and is intimately tied to the history of human civilization. There are hundreds of commercial cultivars with distinct fruit shapes, colors, and sizes growing mainly in arid lands from the west of North Africa to India. The origin of date palm domestication is still uncertain, and few studies have attempted to document genetic diversity across multiple regions. We conducted genotyping-by-sequencing on 70 female cultivar samples from across the date palm–growing regions, including four Phoenix species as the outgroup. Here, for the first time, we generate genome-wide genotyping data for 13,000–65,000 SNPs in a diverse set of date palm fruit and leaf samples. Our analysis provides the first genome-wide evidence confirming recent findings that the date palm cultivars segregate into two main regions of shared genetic background from North Africa and the Arabian Gulf. We identify genomic regions with high densities of geographically segregating SNPs and also observe higher levels of allele fixation on the recently described X-chromosome than on the autosomes. Our results fit a model with two centers of earliest cultivation including date palms autochthonous to North Africa. These results adjust our understanding of human agriculture history and will provide the foundation for more directed functional studies and a better understanding of genetic diversity in date palm.

Past and future of a century old Citrus tristeza virus collection: a California citrus germplasm tale

Citrus tristeza virus (CTV) isolates collected from citrus germplasm, dooryard and field trees in California from 1914 have been maintained in planta under quarantine in the Citrus Clonal Protection Program (CCPP), Riverside, California. This collection, therefore, represents populations of CTV isolates obtained over time and space in California. To determine CTV genetic diversity in this context, genotypes of CTV isolates from the CCPP collection were characterized using multiple molecular markers (MMM). Genotypes T30, VT, and T36 were found at high frequencies with T30 and T30+VT genotypes being the most abundant. The MMM analysis did not identify T3 and B165/T68 genotypes; however, biological and phylogenetic analysis suggested some relationships of CCPP CTV isolates with these two genotypes. Phylogenetic analysis of the CTV coat protein (CP) gene sequences classified the tested isolates into seven distinct clades. Five clades were in association with the standard CTV genotypes T30, T36, T3, VT, and B165/T68. The remaining two identified clades were not related to any standard CTV genotypes. Spatiotemporal analysis indicated a trend of reduced genotype and phylogenetic diversity as well as virulence from southern California (SC) at early (1907–1957) in comparison to that of central California (CC) isolates collected from later (1957–2009) time periods. CTV biological characterization also indicated a reduced number and less virulent stem pitting (SP) CTV isolates compared to seedling yellows isolates introduced to California. This data provides a historical insight of the introduction, movement, and genetic diversity of CTV in California and provides genetic and biological information useful for CTV quarantine, eradication, and disease management strategies such as CTV-SP cross protection.

Changes in Anthocyanin Production during Domestication of Citrus

Ruby, a regulatory gene encoding a MYB transcription factor, is essential for anthocyanin production, and differences in its activity determine most of the natural variation in pigmentation in Citrus and related genera. Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus. Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

Coconut, Date, and Oil Palm Genomics

The palm family, consisting of over 2,500 species arrayed among ca. 200 genera, is the third most economically important family of plants after the grasses and legumes. Three palm species account for the large majority of the family’s economic importance: coconut (Cocos nucifera), African oil palm (Elaeis guineensis), and date palm (Phoenix dactylifera). Of the three, genomics has been least developed in the coconut, where molecular tools have largely been used to characterize germplasm, and, to a lesser extent, develop quantitative trait loci (QTL). Both date palm and oil palm have recently had their genomes sequenced. The application of genomic tools to these palm species will result in enormous advances in the genetic improvement of all three crops.

Phytoplasmas and Their Insect Vectors: Implications for Date Palm

Phytoplasmas are transmitted chiefly by insects, most commonly planthoppers and leafhoppers. Molecular genetic analyses have improved the understanding of phytoplasma taxonomy, and also enhanced the ability to identify phytoplasmas detected in hosts and insect vectors. Date palm is affected by Al-Wijam disease in Saudi Arabia and molecularly indistinguishable phytoplasmas (16SrI group) were recovered from affected palms and from Cicadulina bipunctata, an insect commonly found on the palms. The phytoplasma that is associated with the lethal yellowing disease in coconut palm (16SrIV-A) can also infect date palm. In the Americas lethal yellowing is likely to be transmitted by Haplaxius crudus (formerly Myndus crudus). Texas phoenix decline is reported from warm regions of South-East USA and may be transmitted by two species of Derbidae. Phytoplasmas belonging to the 16SrIV-F and 16SrXIV groups have also been identified from date palm growing in the USA and Africa respectively, though vectors have not been identified. Preventing spread in infected vegetative planting material and of vectors is key to limiting the impact of phytoplasma diseases. Management in affected areas can use antibiotics on high value trees, but this is not economical for extensive crops. In these situations, vector control by insecticide use or habitat management can be useful, but the long lifespan of individual palms means that even low vector pressure can lead to infection over successive years. The development of resistant varieties and replanting is the most effective long-term approach developed so far for phytoplasma disease management in this plant species.

Mission and Modern Citrus Species Diversity of Baja California Peninsula Oases

The spring-fed mission oases of the Baja California peninsula, Mexico, hold several species, varieties and unique hybrids of heritage citrus, which may represent valuable genetic resources. Citrus species first arrived to the peninsula with the Jesuit missionaries (1697-1768), and new varieties were introduced during the colonial and rancho periods following the mission era. These heritage trees are grown in field and house gardens as ornamental, fruit and shade trees. Commercial citrus varieties introduced from the United States have become a strong source of economic revenue for peninsula agriculturalists. A few of the peninsula oases support commercial citrus groves, though these are primarily found in the broader valleys of the peninsula where groundwater is tapped for irrigation. The isolated environments of the mission oases and surrounding ranches have facilitated propagation of unique citrus types, and these have stabilized through integration into the cultural practices of the region. We identify and document citrus of the peninsula oases to serve as a baseline for those interested in the cultural ecology of citrus and citrus genetic resources.ResumenLos oasis-misiones formados por manantiales en la península de Baja California, México, tienen especies, variedades e híbridos únicos de cítricos antiguos que pueden representar valiosos recursos genéticos. Las primeras especies de cítricos llegaron a la península con los misioneros jesuitas (1697-1768) y, posteriormente, las nuevas variedades se introdujeron durante los períodos colonial y ranchero después de la época misional. Estos árboles de genética antigua se cultivan en las huertas y los solares, como setos y como árboles ornamentales, frutales y de sombra. Las variedades de cítricos comerciales introducidas desde Estados Unidos se han convertido en una fuente importante de ingresos económicos para los agricultores de la península. Algunos de los oasis de la península tienen plantaciones comerciales de cítricos, aunque la mayoría de estas plantaciones comerciales se encuentran en los valles más extensos de la península dónde el agua subterránea es aprovechada para el riego. Los ambientes aislados de los oasis-misiones y de los ranchos circundantes han facilitado la propagación de tipos de cítricos únicos, y éstos se han estabilizado gracias a su integración en las prácticas culturales de la región. Identificamos y documentamos los cítricos de los oasis de la península lo que ha de servir como punto de referencia para los interesados en la ecología cultural y los recursos genéticos de los cítricos.