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United States Department of Agriculture

Agricultural Research Service

Research Project: Identifying Genetic Relationships among Citrus and Citrus Relatives Using Molecular Markers

Location: National Clonal Germplasm Repository for Citrus & Dates

2013 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.

Specific objectives: 1. Provide expertise to determine genetic relationships of cultivars of citrus and citrus relatives which appear to have tolerance to huanglongbing. 2. Provide expertise and aid to identify interactions among genes expressed by the bacterium associated with huanglongbing and genes expressed by the citrus host. 3. Provide expertise to determine effect of cryopreservation and/or cryotherapy on genetic trueness of type in cultivars and varieties which have undergone treatment. 4. Aid in identification of possible sources of redundancy in the Repository accessions by use of molecular markers.

1b.Approach (from AD-416):
All research involving huanglongbing (HLB) will be conducted in Florida or other location where HLB is present and widespread. From a four year field screening of 87 genotypes of citrus and citrus relatives in Florida, some accessions appear to have some tolerance to HLB via differing mechanisms: one group became infected but survived and partially recovered; the second group expressed delayed susceptibility where after four years not all replications tested positive; and the third group would become HLB infected but was able to ward off the infection and tested negative. This research will determine the variability of the Candidatus Liberibacter asiaticus (Las) genome in plants that are in each group, and compared to the group which was extremely susceptible to HLB. In greenhouse tests, plants will be challenged using psyllids, and samples taken at time intervals for analyses of genes being expressed by the Las and by the plant host. Genome analysis of the host DNA of selected individual plants will be done by single nucleotide polymorphism (SNP) analysis of several nuclear genes and also by microsatellite or simple sequence repeat (SSR) analysis. Recently we have successfully adapted the methodology to preserve vegetative shoots of citrus in liquid nitrogen, and further adapted this methodology to eliminate graft transmissible pathogens by treatment in liquid nitrogen. A concern is that this treatment may induce genetic mutations in citrus. Citrus accessions which have been treated with liquid nitrogen will be recovered, allowed to grow, and planted in the field for horticultural evaluation. Samples will be collected and analyzed by SNP analyses of selected nuclear genes and/or by SSR markers to evaluate the genetic trueness of type of the source and recovered plants.

3.Progress Report:

This project is related to objective 1 and 2 (identification of priority genetic resources of citrus and characterization of the genotypes of citrus, respectively) of the parent project. Most of the cultivated varieties of Citrus are derived by hybridization of three basic species: citron, mandarin, and pummelo. Citrus can reproduce by both sexual (through seed) and asexual means (polyembryony and budding). Because of its peculiar biology, the species demarcation is not clear in Citrus and sexually nonviable cultivars can be propagated clonally. This makes it difficult to predict the relationships between the different citrus cultivars. In order to understand the phylogenetic relationships of the different Citrus cultivars and their close relatives, we did an analysis with sequences generated from six nuclear genes obtained from 33 cultivars. The phylogenetic trees obtained from the six genes show inconsistencies or, incongruence, since the individual genes are predicted to undergo additional changes due to hybridization, incomplete lineage sorting, gene duplication and loss. Lineage sorting is the process of fixation of alleles along species lineage; for example, alleles specific to mandarins will be found only in the line that evolves into mandarins and not in the line that evolves into pummelos. Most of the cultivated citrus varieties are derived by hybridization – both natural and man-made. During the course of evolution, different lineages of citrus acquire distinct characters. At times, these characters may not be as distinct because of incomplete lineage sorting. If the species being studied are derived from an ancestor that existed a long time ago, it is possible that given sufficient time, the distinct characters of a lineage revert back to the ancestral characters. All these factors can result in incomplete lineage sorting. As we go back in time, two divergent lineages “coalesce” if they are derived from a common ancestor. Using coalescence simulation testing methods we tried to understand the cause of incongruence in the gene trees. Previous study done using simple sequence repeat (SSR) markers has indicated the possible hybrid nature of several citrus accessions. We compared our results with the previous published data using SSR markers and used the patterns observed among the six nuclear loci to identify incomplete lineage sorting, hybridization, or other processes in the different accessions studied. We identified hybrids by coalescence simulation testing that could not be detected by other methods, such as SSR markers. The study leads to a better understanding of the phylogenetic relationships in this biologically complex genus.

Last Modified: 4/16/2014
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