Selection of SSR Markers for the Identification of Zygotic Seedlings in Citrus Rootstock

Most citrus rootstocks are clonally propagated via seed in Florida.  Citrus rootstocks are typically selected to have high levels of nucellar embryony, a type ofP apomixis, to ensure that propagation is true to type.  The spread of the Huang-Long-Bing disease in Florida has significantly increased the demand of citrus rootstocks for the propagation of replants to replace dying trees.  Unfortunately many of the rootstock varieties are also susceptible to citrus greening and this has reduced the availability of rootstock seed.  The increased demand for rootstocks has enhanced interest in using tissue culture for the clonal propagation of citrus rootstocks.  The use of seed to initiate in vitro cultures of citrus rootstocks requires the fingerprinting of the resulting seedlings to ensure that the cultures are initiated from clonal ‘true to type’ seedlings of the seed parent.  Microsatellite (SSR) markers are an excellent tool for the fingerprinting of citrus because the loci typically have multiple alleles, and are likely to be polymorphic in rootstock cultivars. A set of 20 SSR primers was screened on a panel of 619 citrus cultivars and selections to identify highly informative markers that were heterozygous in the rootstock cultivars being tested.  A diagnostic set of 5 makers that were heterozygous in each of the citrus rootstock cultivars was selected.   DNA was extracted from seedlings from 14 different citrus rootstock cultivars.  The SSR genotypes of the seedlings were compared with the seed parent genotype to determine if they were of zygotic or nucellar origin .  The Sour Orange, US 802, US 942, and Cleopatra rootstocks had 100% nucellar progeny, all being genetically identical to the parent, and only two cultivars, Sun Chu Sha and Rough Lemon, had greater than 10% zygotic progeny. The remaining 8 cultivars had between 1-10% zygotic seedlings, which is a relatively low percentage. These results indicate that SSR genotyping of clones derived from embryonic tissue is very effective and efficient for the identification of zygotic progeny.