Verification of Mandarin and Pummelo Somatic Hybrids by Expressed Sequence Tag–Simple Sequence Repeat Marker Analysis

Abstract

<jats:p>Somatic hybridization is a powerful tool for the genetic improvement of citrus rootstocks, and it is part of an efficient in vitro-based breeding system described here. An essential component of the system is the requirement of confirming tetraploidy and the combination of the two donor genomes. Expressed sequence tag–simple sequence repeat (EST-SSR) markers provide a means to accomplish both of these objectives, and their application to a population of pummelo [<jats:italic>Citrus grandis</jats:italic> (L.) Osbeck] + mandarin (<jats:italic>C. reticulata</jats:italic> Blanco) somatic hybrids developed for the specific purpose of providing alternative rootstocks for sour orange (<jats:italic>Citrus aurantium</jats:italic> L.) is detailed. Nineteen new somatic hybrids were produced from various mandarin and pummelo parents, and their ploidy level and the complementation of their nuclear genomes were confirmed using four EST-SSR markers. These markers were selected from markers previously mapped in sweet orange [<jats:italic>C. sinensis</jats:italic> (L.) Osbeck] and trifoliate orange [<jats:italic>Poncirus trifoliata</jats:italic> (L.) Raf.] and prescreened for suitable allelic polymorphism within the mandarin and pummelo lines used. After polymerase chain reaction amplification of sequences from the parents and putative hybrids, the products were separated on a genetic sequencer and visualized electronically. Additionally, EST-SSR markers identified the unexpected zygotic origin of a presumed nucellar embryogenic callus line. Integration of EST-SSR techniques for high-throughput genotyping with previously developed approaches to somatic hybrid creation increases substantially the effectiveness and efficiency of this in vitro-based breeding system for citrus rootstock improvement.</jats:p>

Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus

“…After a significant quantity of callus is obtained, callus lines can be habituated by subculturing on growth regulator free EME or H?H medium (Table 1). At this point, each callus line should be checked by analysis of robust genetic markers, such as SSRs, to assure nucellar rather than zygotic origin; we have occasionally found that embryogenic callus can originate from zygotic embryos in undeveloped aborted ovules (Chen et al 2008). It generally takes a year to have adequate quantities of embryogenic callus for suspension initiation.…”

“…Recovered tetraploids should undergo molecular analysis to determine if they are autotetraploids or allotetraploid somatic hybrids, with both having potential value to subsequent conventional breeding schemes. Previously, somatic hybrids have been validated by isozymes, RAPD or CAPS (Grosser and Gmitter 2005); more recently unambiguous and reliable EST-SSR markers with fragments separated on a sequencer, has been the method of choice (Chen et al 2008). EST-SSRs have a further advantage of ploidy level validation in many instances, as up to four different alleles may be identified at a single locus in a somatic hybrid plant.…”

“…More recently, Chen et al (2008) studied meiosis and microsporogensis in two additional citrus somatic hybrids, Hamlin sweet orange ? Rough Lemon (Citrus jambhiri Lush) (HR) and Key lime (Citrus aurantifolia (Christm.)…”

Investigating the Parentage of 'Orri' and 'Fortune' Mandarin Hybrids

“…Compared to other molecular markers, SSR (simple sequence repeat) markers are now more widely used in genetic, systematic, and comparative genomic studies largely due to their co-dominance, multi-alleles among closely related genomes, and relatively inexpensive development and detection procedures (Chen et al, 2006). These polymorphic, co-dominant, and segregating alleles greatly facilitate authentication and tracking of genetically-related cultivars and hybrids (Chen et al, 2008). 'Orah' mandarin was reportedly bred by Pinhas Spiegel-Roy and Aliza Vardi at The Volcani Center, Israel, with 'Temple' tangor (♀) (Citrus reticulata × C. sinensis) and 'Dancy' mandarin (♂) (C. reticulata) reported as the parents (Jaffa, undated;Saunt, 2000;Greenberg et al, 2004;Tietel et al, 2009;Citrogold, 2012).…”

Novel Peach Flower Types in a Segregating Population from ‘Helen Borchers’

“…Identification of the pollen parent of an outcross can be difficult if there are multiple possible pollen sources nearby with similar traits. Under this circumstance, codominant simple sequence repeat (SSR) markers, along with other types of DNA markers, are useful for distinguishing parentage (Chen et al, 2008a(Chen et al, , 2008bDirlewanger et al, 2004). This approach is more effective and informative if the SSR markers contain polymorphic heterozygous alleles that will help distinguish among the possible pollen parents (Chen et al, 2008a).…”