The rootstock is an important component of a healthy and productive citrus tree, influencing the fruit yield, fruit quality, tree size, and tolerance of diseases (Bowman and Joubert, 2020; Castle et al., 2011; McCollum and Bowman, 2017; Wutscher and Bowman, 1999). HLB disease (also known as citrus greening) is arguably the most important and most destructive disease in much of the world's citrus industry. Some hybrids of trifoliate orange (Poncirus trifoliata) with Citrus spp. have been identified as tolerant to HLB when used as a scion (Albrecht and Bowman, 2019; Folimonova et al., 2009) or as a rootstock (Albrecht and Bowman, 2011, 2012; Bowman et al., 2016a, 2016b). Hybrids of this parentage are known to possess many other outstanding rootstock characteristics. As a consequence, use of HLB-tolerant rootstocks is considered one of the most effective tools currently available to combat the disease. After new rootstocks are tested and released, one of the first challenges is to obtain enough plants of the new rootstock clones in nursery propagation.
Although propagation of citrus rootstocks can be accomplished effectively by stem cuttings or micropropagation (Albrecht et al., 2017; Bowman and Albrecht, 2017), commercial propagation of citrus rootstocks usually depends on the production of genetically uniform clonal plants from seed. Within the genus Citrus, many species show the phenomenon of nucellar polyembryony, which means that seeds contain multiple embryos produced by ordinary mitotic division of cells of the nucellus (nucellar embryos) and no male gamete contributes to their formation (Garcia et al., 1999). For these species, some or a large portion of the seedlings will therefore be genetically identical to the seed parent. Historically, clones have been used as citrus rootstocks only when they provide a relatively high proportion of genetically uniform nucellar seedlings (Bowman and Joubert, 2020). Eliminating the zygotic plants among primarily nucellar rootstock seedling populations in the citrus nursery is important to maintain genetic homogeneity, thereby assuring growers of uniform rootstock performance in the field (Ruiz et al., 2000). In the citrus nursery, zygotic seedlings of rootstocks are eliminated, or rogued, based on visual appearance. However, separating the seedlings only by leaf morphology, size, and growth habit is not always reliable, because some seedlings of zygotic origin for particular rootstocks are difficult to visually identify (Anderson et al., 1991). If these zygotic seedlings are mistakenly used for propagation, the result can be unpredictable or reduced tree performance, including a high level of variability in tree size and health.
Several methods can be used to identify true-to-type and off-type seedlings, including isozyme analysis, random amplified polymorphic DNA (RAPD) analysis, amplified fragment length polymorphisms (AFLP) analysis, or SSR analysis (Rao et al., 2008). Isozyme analysis has limitations because of the small number of available loci in the genome and the scarce variability at those loci. RAPD and AFLP markers have limitations because of their dominant nature (heterozygous and homozygous individuals are not easily distinguished), which reduces in half the ability to detect zygotic plants in some progenies (Ruiz et al., 2000). SSR markers typically have a high number of polymorphic loci with numerous alleles (Karhu et al., 1996; Raybould et al., 1998; White and Powell, 1997), and have proven a useful tool to identify zygotic and nucellar seedlings (Russell et al., 1997).
Six new hybrid rootstocks ‘US-1279’, ‘US-1281’, ‘US-1282’, ‘US-1283’, ‘US-1284’, and ‘US-1516’ were released from the USDA citrus breeding program during 2014 and 2015 to provide improved rootstock tolerance to HLB (Bowman and McCollum, 2015; Bowman et al., 2016b). Sweet orange scions on these rootstocks demonstrated tree health and fruit productivity that was superior to the most widely used rootstocks under conditions severely challenged by HLB. All the new hybrids originated from crosses of mandarin (Citrus reticulata) or pummelo (Citrus maxima), and trifoliate orange (Poncirus trifoliata). At the time of release, fruits and seeds were not available for five of these hybrids, and little was known about their potential for seed propagation. During the 2018 season, fruiting trees were available from these new rootstocks to evaluate fruit, seed, and seedling characteristics, and determine the percentage of true-to-type and off-type seedlings by visual and SSR markers of these new rootstocks, along with four other USDA rootstocks of commercial importance. The information provided in this study is of substantial value to commercial nurseries and others interested in propagation of these rootstocks. In addition, it provides a readily applicable methodology for evaluating nursery characteristics that can be applied to other new rootstocks in the future.
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