Philip L. Forsline

Rupestris stem pitting associated virus-1 is consistently detected in grapevines that are infected with rupestris stem pitting

Rupestris stem pitting (RSP) appears to be the most widespread disease of the rugose wood complex of grapevines. In previous work we showed that rupestris stem pitting associated virus-1 (RSPaV-1), an agent that has similarities in genome structure to apple stem pitting virus is associated with RSP (Meng et al., 1997, 1998). To validate the association of RSPaV-1 with RSP, grapevines that had been indexed on the RSP indicator St. George (Vitis rupestris Scheele) were collected from four major grape-growing countries and tested for RSPaV-1 by reverse transcription-polymerase chain reaction (RT-PCR) assay. Depending on the sets of primers used, we detected RSPaV-1-specific sequences from 85% to nearly 100% of the grapevines that had indexed positive for RSP. These data further demonstrate that RSPaV-1 is closely associated with, and likely to be the causal agent of RSP. We have also shown that RT-PCR is a fast and reliable alternative to biological indexing for the diagnosis of RSP.

Genetic diversity of red-fleshed apples (Malus)

Anthocyanins are flavonoid pigments imparting red, blue, or purple pigmentation to fruits, flowers and foliage. These compounds are powerful antioxidants in vitro, and are widely believed to contribute to human health. The fruit of the domestic apple (Malus x domestica) is a popular and important source of nutrients, and is considered one of the top ‘functional foods’—those foods that have inherent health-promoting benefits beyond basic nutritional value. The pigmentation of typical red apple fruits results from accumulation of anthocyanin in the skin. However, numerous genotypes of Malus are known that synthesize anthocyanin in additional fruit tissues including the core and cortex (flesh). Red-fleshed apple genotypes are an attractive starting point for development of novel varieties for consumption and nutraceutical use through traditional breeding and biotechnology. However, cultivar development is limited by lack of characterization of the diversity of genetic backgrounds showing this trait. We identified and cataloged red-fleshed apple genotypes from four Malus diversity collections representing over 3,000 accessions including domestic cultivars, wild species, and named hybrids. We found a striking range of flesh color intensity and pattern among accessions, including those carrying the MYB10R6 allele conferring ectopic expression of a key transcriptional regulator of anthocyanin biosynthesis. Although MYB10R6 was strongly associated with red-fleshed fruit among genotypes, this allele was neither sufficient nor required for this trait in all genotypes. Nearly all red-fleshed accessions tested could be traced back to ‘Niedzwetzkyana’, a presumed natural form of M. sieversii native to central Asia.

Natural variation in fruit abscission-related traits in apple (Malus)

Abscission or retention of ripening fruit is a major component of seed dispersal strategies and also has important implications for horticultural production. Abscission-related traits have generally not been targeted in breeding efforts, and their genetic bases remain mostly unknown. We evaluated 144 Malus accessions representing wild species, domestic cultivars, and hybrids for abscission-related traits. We found that seasonal timing of fruit abscission in wild species and hybrids showed a broad distribution similar to that seen for domestic cultivars, and that internal ethylene concentration at the time of abscission varied by over three orders of magnitude. Wild species, domestic cultivars, and hybrids all included representatives that showed abscission of fruit prior to substantial production of ethylene, as well as accessions that retained fruit for a significant period of time following ethylene production. For all accessions that retained fruit, fruit removal resulted in abscission of the pedicel, and exogenous ethylene promoted abscission, suggesting that the abscission zone was functional. Our results suggest important roles for mechanisms independent of fruit ethylene production in abscission.

Seeds capture the diversity of genetic resource collections of Malus sieversii maintained in an orchard

Many species in genebank collections are maintained vegetatively either as in vitro cultures or as plants in the field or greenhouse. In these cases, individual genotypes (cultivars) are the focus of conservation. In crop wild relatives, where the value of an accession is in the heterogeneity of segregating genotypes, seeds may be the most representative form of conservation. We used two sets of field-planted Malus sieversii apple trees that originated from seeds collected from two wild populations in Kazakhstan as a model system to determine if seeds from hand-pollinated crosses could be used to represent the allelic diversity of the parent trees in a genebank orchard. A crossing design was developed that used each of the selected trees from the two Kazakhstan collection sites as mothers and also as fathers in the form of bulked pollen pools. The sets of offspring from these crosses were genotyped using microsatellite markers. Paternity analyses revealed the percentage of offspring that was derived from each of the parent trees. We also determined the contribution of each parent to the fertilization events from the pollen pools. Although there were some unintended pollination events (fertilization by trees in unexpected pollen pools) and unequal contribution of pollen parents within pollen pools, we demonstrated that the seedlings effectively captured the allelic frequency and diversity of the parents. A seed collection strategy is the most efficient way to conserve the diversity of crop wild relatives even in collections typically conserved as individual clones.