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United States Department of Agriculture

Agricultural Research Service

Research Project: Use of Molecular Markers to Determine Genetic Relationships of Citrus and Citrus Relatives

Location: National Clonal Germplasm Repository for Citrus & Dates

2010 Annual Report


1a.Objectives (from AD-416)
Provide expertise in the area of genetic analysis of germplasm of citrus, citrus relatives, and date palm. This supports the mission to collect, evaluate, and distribute germplasm of citrus, citrus relatives, and date palm.


1b.Approach (from AD-416)
Use molecular markers and gene sequence information to characterize citrus, citrus relatives, and date palm accessions to help identify redundancies and gaps in the collection, screen potential new accessions, and identify important regulatory genes of metabolic pathways. Documents SCA with UC Riverside. Replaces 5310-21000-008-03S (4/08). FORMERLY 5310-21000-008-12S (8/08).


3.Progress Report

Nuclear gene sequence-based phylogeny of Citrus: Citrus belongs to the family Rutaceae and sub-family Aurantioideae. Members of Rutaceae are of interest as possible sources of superior rootstocks and for their role in spread of diseases like Huanglongbing (HLB). To aid in molecular identification of the citrus relatives and to understand the phylogenetic relationships among members of Aurantioideae, a 1.6 Kb fragment of a nuclear gene, malate dehydrogenase, was PCR amplified, cloned, sequenced and analyzed. The study included taxa belonging to thirty-one genera and seventy-six species. Taxa with heterozygous bases were resolved into two haplotypes. The sequences were aligned and used to construct phylogenetic trees. Interestingly, the general pattern of clustering of the accessions was in agreement with the traditional classification of the sub-family Aurantioideae proposed by Swingle and Reece in 1967 based on morphological characters. However, the position of some of the genera and the relationships among the different subgroups are different. The overall pattern is sometimes consistent with that found using chloroplast DNA sequence analysis, but many differences were noted and it is likely that a clear picture of relationships will require analysis of several nuclear genes. Huanglongbing (HLB) in citrus relatives: HLB is a devastating disease reported predominantly from Citrus species. Effective mitigation of HLB requires information on all possible means of distribution of the disease including spread by alternate hosts. Citrus relatives collected from HLB-infected regions of South Florida were analyzed for the presence of ‘Candidatus Liberibacter asiaticus’ (Las) using quantitative PCR. Molecular confirmation of the presence of Las was carried out by PCR amplification, cloning and sequencing of several other genomic regions of the bacterium. The taxonomic identity of the host plant materials was confirmed by comparing the sequence of a nuclear gene, malate dehydrogenase, with the sequence of known accessions from the Citrus Variety Collection, Riverside, CA. The study helps in generating information about citrus relatives that can serve as alternate hosts for Las. Management of HLB will likely require consideration of bacteria in related species. Marker Analysis of New Citrus Seedling Introductions: In order to identify priority accessions for inclusion in the protected collection, a total of 72 citrus accessions (mostly seedling trees) were compared to each other and to standard accessions. Nine genetic markers were analyzed on a total of 100 samples from the eleven “population” groups. Overall, the marker analysis was very effective for determining relationships among the accessions. For example the group of 15 sweet lime seedlings were identical to each other and to an existing Palestine sweet lime accession, so they must be nucellar seedlings that do not add diversity to the collection, and new accessions thought to be sour orange were different from sour orange and from each other and likely represent novel genotypes.

ADODR monitored activities by meetings, site visits, e-mail correspondence, and phone calls.


Last Modified: 8/22/2014
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