Fusarium crown and root rot (crown rot) develops on tomato from the fungus Fusarium oxysporum f.sp. radicis-lycopersici (FORL). Genetic resistance to crown rot was previously introduced into the cultivated tomato from the wild species Lycopersicon peruvianum and found to be a single dominant gene, Frl, on the long arm near the centromere of chromosome 9 of the tomato genome. In an effort to identify molecular markers tightly linked to the gene, Ohio 89-1 Fla 7226, Fla 7464, `Mocis', and `Mopèrou', lines homozygous for Frl (resistant), were screened with restriction fragment length polymorphism (RFLP) markers in comparison to Fla 7482B and `Monalbo', lines homozygous for Frl + (susceptible). Frl was determined to be between the RFLP markers CT208 and CD8. These two markers are separated by a genetic map distance of 0.9 cM according to Pillen et al. (1996). In addition, we screened a pool of eight resistant plants against a pool of nine susceptibles from a BC1 population segregating for Frl for amplified fragment length polymorphism (AFLP) markers. Fazio et al. (1998) previously determined that crossover events occurred in these 17 plants between Frl and a rapid amplified polymorphic DNA (RAPD) marker, UBC194. Our research has indicated that UBC194 is also between CT208 and CD8 on the centromeric side of Frl. Of the 62 AFLP primer combinations tested, 34 showed more than 63 strong polymorphisms in linkage to resistant phenotypes.
Matthew D. Robbins, Mikel R. Stevens, Gennaro Fazio and Gennaro Fazio
Michelle M. Leinfelder, Ian A. Merwin, Gennaro Fazio and Terence Robinson*
We are testing control tactics for apple replant disease (ARD) complex, a worldwide problem for fruit growers that is attributed to various biotic and abiotic soil factors. In Nov. 2001, “Empire” apple trees on five rootstocks (M.26, M.7, G.16, CG.6210, and G.30) were planted into four preplant soil treatments—commercial compost at 492 kg/ha soil-incorporated and 492 kg·ha-1 surface-applied), soil fumigation with Telone C-17 (400 L·ha-1 of 1,3-dichloropropene + chloropicrin injected at 30 cm depth five weeks prior to replanting), compost plus fumigant combination, and untreated controls—at an old orchard site in Ithaca, N.Y. Trees were replanted in rows perpendicular to, and either in or out of, previous orchard rows. Irrigation was applied as needed, and N-P-K fertilizer was applied in 2001 to all non-compost treatments to compensate for nutrients in the compost treatment. After two growing seasons, the rootstock factor has contributed most to tree-growth differences. CG.6210 rootstock supported greater growth in trunk diameter, central leader height, and lateral shoot growth (P < 0.05), regardless of preplant soil treatments and replant position. Trees on M.26 grew least over a two year period. Replant growth was greater in old grass lanes than in old tree rows, despite higher root-lesion nematode populations in previous grass lanes. Growth responses to preplant soil fumigation were negligible. Preplant compost did not increase tree growth during year one, but did increase lateral branch growth in year two. Results thus far suggest that replanting apple trees out of the old tree-row locations, and using ARD tolerant rootstocks such as CG.6210, may be more effective than soil fumigation for control of ARD in some old orchard sites.
Gennaro Fazio, Mikel R. Stevens and John W. Scott
Fusarium crown and root rot of tomato is caused by Fusarium oxysporum f.sp. radicis-lycopersici (FORL). A single dominant gene (Frl) derived from L. peruvianum L. (Mill.) was previously identified as a useful source of resistance to FORL. The objective of this research was to identify molecular markers linked to Frl and RAPD markers linked to a new source of resistance to FORL being developed from L. pennellii (Corr.) D'Arcy accession LA1277. The DNAs of resistant (Frl) and susceptible breeding lines were screened for polymorphisms using 1200 RAPD primers. Of these, only 104 yielded polymorphisms between the resistant and susceptible lines. These polymorphisms were then tested on four additional tomato lines homozygous for Frl and an additional pair of near-isogenic lines developed by Dr. Laterrot. Only 13 primers still produced consistent polymorphisms between all resistant and susceptible lines. Four of these polymorphisms (RAPD 116, 194, 405, 655) were determined to be linked to Frl in an F5 segregating population using an inoculation procedure devised to clearly differentiate susceptible and resistant plants. The linkage between ah and Frl reported by Laterrot [Laterrot and Moretti Tomato Genet. Coop. Rep. 45:29 (1995)] places Frl on the long arm of chromosome 9 of the tomato genome. The parent lines were also tested with a sequence tagged site (STS) of TG101, which is tightly linked to Tm2a [Young et al., Genetics 120:579-585 (1988)] and yielded polymorphic codominant bands. This STS was also tested on the F5 segregating population and it cosegregated with the resistance and with the RAPD markers. Breeding of the second source of resistance is still in progress. The DNAs of 30 resistant BC1F5 plants derived from LA1277 were bulked and compared to the recurrent susceptible parent DNA using 800 RAPD primers. Of the 800 RAPD primers, 72 yielded consistent polymorphisms. None of the 72 primers were found to produce polymorphisms similar to those identified from the analysis of Frl, thus suggesting the possibility different genetic control being involved with FORL resistance from LA1277.
Gennaro Fazio, Herb S. Aldwinckle, Terence L. Robinson and James Cummins
The Geneva® Apple Rootstock Breeding program, which was initiated in 1968 by Dr. James Cummins and Dr. Herb Aldwinckle of Cornell University and which has been continued as a joint breeding program with the U.S. Dept. of Agriculture Agricultural Research Service (USDA-ARS) since 1998, has released a new semi-dwarfing apple rootstock which is named Geneva® 935 or G.935. G.935 (a progeny from a 1976 cross of `Ottawa 3' × `Robusta 5') is a selection that has been widely tested at the New York State Agricultural Experiment Station in Geneva, N.Y., in commercial orchards in the United States and at research stations across the United States and Canada. G.935 is a semi-dwarfing rootstock that produces a tree slightly larger than M.26. G.935 is the most precocious and productive semi-dwarf rootstock we have released. It has had similar yield efficiency to M.9 along with excellent fruit size and wide crotch angles. It showed no symptoms of winter damage during the 1994 test winter in N.Y. G.935 is resistant to fire blight and Phytophthora; however. it is susceptible to infestations by woolly apple aphids. G.935 has shown tolerance to replant disease complex in several trials. It has good propagation characteristics in the stool bed and produces a large tree in the nursery. G.935 has better graft union strength than M.9, but will require a trellis or individual tree stake in the orchard to support the large crops when the tree is young. G.935 will be a possible replacement for M.26. Suggested orchards planting densities with this rootstock are 1,500-2,500 trees/ha. It has been released for propagation and sale by licensed nurseries. Liners will be available in the near future.
Renae E. Moran, Bryan J. Peterson, Gennaro Fazio and John Cline
To identify genotypes of apple (Malus ×domestica) rootstock with vulnerability to low temperature, we measured the low temperature tolerance of xylem, phloem and cambium in 2-year-old shoot pieces from cultivars Budagovsky 9 (B.9), M.7 EMLA (M.7), M.9 EMLA (M.9), Geneva® 41 (G.41), Geneva 30 (G.30), Geneva 214 (G.214), Geneva 814 (G.814), and Geneva 935 (G.935), as well as six advanced selections in the Geneva (G.) series and three in the Vineland (V.) series. From Oct. 2013 to Apr. 2014, injury was measured as a 0–10 rating based on percentage of discolored cross-sectional xylem and phloem, and cambial length and circumference with brown discoloration, with 0 indicating no browning and 10 indicating browning in the entire tissue. From Oct. 2014 to Apr. 2015, injury was measured as xylem, phloem and cambium browning using a similar rating scale that accounted for both the percentage of browned tissues and the intensity of browning. Following exposure to −35 to −40 °C, many genotypes, including ‘M.7’, ‘M.9’, ‘G.935’, G.4011, G.4292, G.5087, and V.5, had only partial xylem injury in the fall, whereas others, ‘M.7’, ‘G.41’, ‘G.214’, and G.4011, had more extensive xylem browning at −30 °C and colder. ‘G.30’ had moderate to severe xylem browning at −15 to −19 °C. In late October of both years, G.4013 exhibited severe phloem browning at relatively high temperatures, but accrued additional hardiness by Nov. 2014, whereas genotypes ‘B.9’, ‘M.9’, ‘G.30’, and ‘G.41’ developed considerable phloem hardiness by late October with no additional increase in hardiness in November. Geneva and Vineland genotypes exhibited a low degree of susceptibility to injury at −35 to −40 °C in Jan. 2014 and Mar. 2015. Shoot hardiness in Apr. 2014 and 2015 was highly variable between the 2 years, with severe browning of xylem and cambium at −40 °C in every genotype sampled in Apr. 2014, but not in Apr. 2015. ‘M.9’ and G.3902 appeared to be the least vulnerable to injury in April, whereas ‘G.30’, ‘G.41’, ‘G.814’, G.4292, and G.5257 seem more likely to suffer injury in spring. ‘G.30’ had tender xylem in both fall and spring, G.4013 had the least hardy cambium and phloem in fall, and G.5257 the least hardy cambium in the spring. These genotypes are vulnerable to damaging temperatures during fall acclimation and spring deacclimation.
Gennaro Fazio, Jack E. Staub and Sang Min Chung
Highly polymorphic microsatellites or simple sequence repeat (SSR), along with sequence characterized amplified region (SCAR) and single nucleotide polymorphisms (SNP), markers are reliable, cost-effective, and amenable for large scale analyses. Molecular polymorhisms are relatively rare in cucumber (Cucumis sativus L.) (3% to 8%). Therefore, experiments were designed to develop SSR, SCAR and SNP markers, and optimize reaction conditions for PCR. A set of 110 SSR markers was constructed using a unique, strategically applied methodology that included the GeneTrapper (Life Technologies, Gaithersburg, Md.) kit to select plasmids harboring microsatellites. Of these markers, 58 (52%) contained dinucleotide repeats (CT, CA, TA), 21 (19%) possessed trinucleotide repeats (CTT, ATT, ACC, GCA), 3 (2.7%) contained tetranucleotide repeats (TGCG, TTAA, TAAA), 4 (3.6%) enclosed pentanucleotide repeat (ATTTT, GTTTT, GGGTC, AGCCC), 3 (2.7%) contained hexanucleotide repeats (CCCAAA, TAAAAA, GCTGGC) and 21 possessed composite repeats. Four SCARs (L18-3 SCAR, AT1-2 SCAR, N6-A SCAR, and N6-B SCAR) and two PCR markers based on SNPs (L18-2H19 A and B) that are tightly linked to multiple lateral branching (i.e., a yield component) were also developed. The SNP markers were developed from otherwise monomorphic SCAR markers, producing genetically variable amplicons. The markers L18-3 SCAR and AT1-2 SCAR were codominant. A three-primer strategy was devised to develop a codominant SCAR from a sequence containing a transposable element, and a new codominant SCAR product was detected by annealing temperature gradient (ATG) PCR. The use of a marker among laboratories can be enhanced by methodological optimization of the PCR. The utility of the primers developed was optimized by ATG-PCR to increase reliability and facilitate technology transfer. This array of markers substantially increases the pool of genetic markers available for genetic investigation in Cucumis.
Sang-Min Chung, Jack E. Staub and Gennaro Fazio
Chilling temperatures (≤12°C) can cause substantial economic damage to cucumber (Cucumis sativus L.) plants. Previous studies suggest chilling tolerance trait is controlled by nuclear gene(s). To investigate inheritance of chilling injury in cucumber, cucumber lines [susceptible GY14 (P1), tolerant `Chipper' (P2), and tolerant `Little John' (P3)], and their exact reciprocal F1 and F2 cross-progeny were evaluated to determine the inheritance of chilling injury at the first true-leaf stage when challenged at 4 °C for 5.5 hours. The mean chilling ratings [1(trace) to 9(dead)] of progeny comparisons were F1(P1 × P2) = 6.2 vs. F1(P2 × P1) = 1.6; F2(P1 × P2) = 6.4 vs. F2(P2 × P1) = 2.7; F1(P1 × P3) = 5.4 vs. F1(P3 × P1) = 1.7; and F2(P1 × P3) = 5.8 vs. F2(P3 × P1) = 2.2. These data suggest that chilling tolerance was maternally inherited as is the chloroplast genome in cucumber. Parents, reciprocal F1, and F2 progeny were evaluated for variation using random amplified polymorphism DNA (RAPD). Although no maternally inherited RAPD markers were detected, polymorphic and paternally inherited RAPD bands AD21249, AV8916, and AV8969 amplified by AD2 and AV8 primers were cloned and sequenced. A BLAST search of these sequences suggested that their origin is likely cucumber mitochondrial DNA. These results indicate that the mitochondria genome is not associated with the chilling tolerant trait because this genome is paternally inherited in progeny derived from this reciprocal mating. Therefore, the results of maternally inherited chilling tolerant trait and paternally transmitted mitochondria genome support that the chilling tolerant trait as identified is likely associated with the chloroplast genome which is maternally transmitted in cucumber.
Gennaro Fazio, Herb S. Aldwinckle, Terence L. Robinson and James Cummins
The Geneva® Apple Rootstock Breeding program initiated in 1968 by Cummins and Aldwinckle of Cornell University and continued as a joint breeding program with the USDA-ARS since 1998, has released a new dwarf apple rootstock named Geneva® 41 or G.41. G.41 (a progeny from a 1975 cross of `Malling 27' × `Robusta 5') is a selection that has been tested at the N.Y. State Agricultural Experiment Station, in commercial orchards in the United States, and at research stations across the United States, Canada, and France. G.41 is a fully dwarfing rootstock with vigor similar to M.9 T337, but with less vigor than M.9 Pajam2. It is highly resistant to fire blight and Phytophthora with no tree death from these diseases in field trials or inoculated experiments. G.41 has also shown tolerance to replant disease. Its precocity and productivity have been exceptional, equaling M.9 in all trials and surpassing M.9 in some trials. It also confers excellent fruit size and induces wide crotch angles in the scion. It appears to be very winter hardy and showed no damage following the test winter of 1994 in New York. Propagation by layering in the stool bed G.41 is not consistent and may require higher layering planting densities or tissue culture mother plants to improve its rooting. G.41 also produces some side shoots in the stool bed. The nursery liners of G.41 produce a smaller tree than G.16 liners, but similar to M.9, which is very acceptable. Unlike G.16, G.41 is not sensitive to latent viruses. G.41 has similar graft union strength to M.9 and requires a trellis or individual tree stake when planted in the orchard. Suggested orchards planting densities with this rootstock are 2,000-4,000 trees/ha. This rootstock has been released for propagation and commercial sale by licensed nurseries.
Gennaro Fazio, Yizhen Wan, Dariusz Kviklys, Leticia Romero, Richard Adams, David Strickland and Terence Robinson
The ability of certain apple rootstocks to dwarf their scions has been known for centuries and their use revolutionized apple (Malus ×domestica) production systems. In this investigation, several apple rootstock breeding populations, planted in multiple replicated field and pot experiments, were used to ascertain the degree of dwarfing when grafted with multiple scions. A previous genetic map of a breeding population derived from parents ‘Ottawa 3’ (O.3) and ‘Robusta 5’ (R5) was used for quantitative trait locus (QTL) analysis of traits related to scion vigor suppression, induction of early bearing, and other tree size measurements on own-rooted and grafted trees. The analysis confirmed a previously reported QTL that imparts vigor control [Dw1, log of odds (LOD) = 7.2] on linkage group (LG) 5 and a new QTL named Dw2 (LOD = 6.4) on LG11 that has a similar effect on vigor. The data from this population were used to study the interaction of these two loci. To validate these findings, a new genetic map comprised of 1841 single-nucleotide polymorphisms was constructed from a cross of the dwarfing, precocious rootstocks ‘Geneva 935’ (G.935) and ‘Budagovsky 9’ (B.9), resulting in the confirmation and modeling of the effect of Dw1 and Dw2 on vigor control of apple scions. Flower density and fruit yield data allowed the identification of genetic factors Eb1 (LOD = 7.1) and Eb2 (LOD = 7.6) that cause early bearing of scions, roughly colocated with the dwarfing factors. The major QTL for mean number of fruit produced per tree colocated with Dw2 (LOD = 7.0) and a minor QTL was located on LG16 (LOD = 3.5). These findings will aid the development of a marker-assisted breeding strategy, and the discovery of additional sources for dwarfing and predictive modeling of new apple rootstocks in the Geneva® apple rootstock breeding program.
Renae E. Moran, Youping Sun, Fang Geng, Donglin Zhang and Gennaro Fazio
Winter injury to the root systems of fruit trees can cause significant tree losses and yield reductions in the northern regions of the United States and Canada. To compare the root and trunk cold temperature tolerance, a series of experiments were conducted using ungrafted apple rootstocks. ‘Geneva® 11’ (G.11), ‘Geneva® 30’ (G.30), ‘Geneva® 41’ (G.41), ‘P.2’, and ‘Budagovsky 9’ (B.9) apple (Malus ×domestica Borkh.) rootstocks had root tissue hardiness similar to ‘M.26’, but ‘Geneva® 935’ (G.935) had greater cold-hardiness than M.26 when based on shoot regrowth in ungrafted trees. The LT50 of M.26 and P.2 roots ranged from –12 to –14 °C. The LT50 was –13 °C for B.9, –13.4 to –14.6 °C for G.30, and –12 °C for G.11. The LT50 of G.41 was one of the highest in one experiment, –8 °C, and one of the lowest in another, colder than –15.0 °C. The LT50 of G.935 roots was the lowest and ranged from –16 to –19 °C. Compared with M.26, trunk cold-hardiness in December was greater in B.9 and P.2 and was similar in G.30. Cold-hardiness of G.11 in December was mixed with less injury in the xylem but more injury in the phloem compared with M.26. In October, M.26 and G.935 trunks had little injury after exposure to –24 °C.