W. V. Baird

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Abstract Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)n- and (CA)n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L.

Genetic linkage mapping in peach using morphological, RFLP and RAPD markers.

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F2 individuals derived from the self-fertilization of four F1 individuals of a cross between ‘New Jersey Pillar’ and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color (γ) loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F2 trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 1∶2∶1 or 3∶1) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.

Development and characterization of a codominant marker linked to root-knot nematode resistance, and its application to peach rootstock breeding

Abstract The presence of a codominant AFLP marker, EAA/MCAT10, correlates with the primary source of resistance to root-knot nematodes (Meloidogyne incognita and M. javanica) in rootstock cultivars of peach [Prunus persica (L.) Batsch]. Two allelic DNA fragments of this AFLP marker were cloned, sequenced and converted to sequence tagged sites (STS). Four nucleotide differences (i.e. one addition and three substitutions) were observed between the two clones. Furthermore, there was a diagnostic Sau3 AI cleavage site (GATC) in the large fragment that was absent from the small fragment (GTTC at this site). The applicability of this STS marker system to peach germplasm improvement was evaluated: genomic DNAs of cross parents (i.e. ‘Lovell’ and ‘Nemared’), four F1 hybrids (K62-67, K62-68, P101-40 and P101-41) and two F2 populations (from K62-68 and P101-41), as well as DNA from a test panel of 18 rootstock cultivars or selections, were PCR-amplified with the Mij3F/Mij1R primer pair and then digested with Sau3 AI. The banding patterns showed that the EAA/MCAT10 STS markers can clearly distinguish the three genotypes – homozygous resistant, heterozygous resistant and homozygous susceptible – in the ‘Lovell’בNemared’ cross. In addition, results from the rootstock survey were consistent with each rootstock’s phenotypic response to nematode infection, except for ‘Okinawa’, ‘Flordaguard’ and ‘Yunnan’ where root-knot resistance may have arisen independently. Therefore, the EAA/MCAT10 STS markers will be a useful tool to initiate marker assisted selection studies in peach rootstock breeding for root-knot nematode resistance.

High polymorphism and resolution in targeted fingerprinting with combined β-tubulin introns

Tubulin-Based-Polymorphism (TBP) was originally introduced as a novel method for assaying genetic diversity in plants. TBP is based on polymorphism resulting from the PCR-mediated amplification of the first intron in the coding region of the β-tubulin gene family. Although, the method was successful in genetic assessment of some plant species and varieties, it suffered from low number of molecular markers due to limited variation in the first intron of β-tubulin gene family. We have now rectified this limitation by introducing the second intron of the β-tubulin genes as a valuable source of molecular markers. We show that the combined use of the two introns substantially increases the number of molecular markers and results in a reliable assessment of species/varieties relationships. After a preliminary validation on Brassica, this new combinatorial method was tested on species of Eleusine and Arachis. For both, reliable assessment of species relationships were obtained that were consistent with recently published studies resulting from more elaborated methods including DNA sequencing. Combinatorial TBP is a reliable, reproducible, simple, fast, and easy to score method that is very useful for breeding programs and species and variety assessments.

Surface Characteristics of Root and Haustorial Hairs of Parasitic Scrophulariaceae

The outer wall surfaces of root and haustorial hairs in seven species of parasitic Scrophulariaceae were examined with scanning electron microscopy. The surface of nonhaustorial root hairs is comparatively smooth and free of extracellular coating material; that of haustorial hairs is coated with an extracellular matrix, which has a granular or papillate appearance. Haustorial hairs of attached haustoria show an increased amount of the coating material over that observed for haustorial hairs of nonattached haustoria. Haustorial hairs directly involved in host attachment have the greatest amount of coating material. At the attachment interface between parasite and host, the coating material forms a continuous sheet, thereby enhancing adhesion of the haustorium to the host root by increasing the effective area of contact.

Characterization of Rubisco activase from thermally contrasting genotypes of Acer rubrum (Aceraceae).

The lability of Rubisco activase function is thought to have a major role in the decline of leaf photosynthesis under moderate heat (<35°C). To investigate this further, we characterized Rubisco activase and explored its role in the previously demonstrated thermal acclimation and inhibition of two genotypes of Acer rubrum originally collected from Florida (FL) and Minnesota (MN). When plants were grown at 33/25°C (day/night) for 21 d, the FL genotype compared to the MN genotype maintained about a two-fold increase in leaf photosynthetic rates at 33-42°C and had a 22% increase in the maximal rate of Rubisco carboxylation (V(cmax)) at 33°C under nonphotorespiratory conditions. Both genotypes had two leaf Rca transcripts, likely from equivalent alternative splicing events. The RCA1 and RCA2 proteins increased modestly in FL plants under warmer temperature, while only RCA2 protein increased in MN plants. Rubisco large subunit (RbsL) protein abundance was relatively unaffected in either genotype by temperature. These results support the idea that Rubisco activase, particularly the ratio of Rubisco activase to Rubisco, may play a role in the photosynthetic heat acclimation in A. rubrum and may have adaptive significance. This mechanism alone is not likely to entirely explain the thermotolerance in the FL genotype, and future research on adaptive mechanisms to high temperatures should consider activase function in a multipathway framework.

Development and characterisation of diagnostic AFLP markers in Prunus persica for its response to peach tree short life syndrome

Summary Peach tree short life (PTSL) is a complex disease syndrome caused by multiple factors, the genetics of which is unknown. Amplified fragment length polymorphism (AFLP) technology and bulked segregant analysis (BSA) were used to identify diagnostic markers for PTSL syndrome in peach rootstocks. Forty-four AFLPs were identified as potential PTSL-response associated markers based on the combined results of BSA and screening 11 PTSL tolerant, two intermediately-susceptible and two highly-susceptible genotypes with 60 polymorphic EcoRI/MseI primer combinations. The use of primer combination EcoRI+AC/MseI+CCC in conjunction with BSA, resulted in the identification of a single amplification product in the bulk DNA from ten susceptible trees that died from PTSL. This AFLP fragment was also detected in the intermediately-susceptible rootstock ‘Lovell’, the highly-susceptible rootstock ‘Nemared’, and in the highly-susceptible rootstock ‘Nemaguard’. This 96 bp AFLP fragment was absent in the DNA bulked from ten tolerant trees and in the PTSL-tolerant parents used in four controlled crosses that segregated for their response to PTSL. Testing this AFLP primer pair on an additional 111 trees that died or survived on a severe PTSL site, revealed the presence of the diagnostic fragment in 18 trees, of which 17 were ‘Lovell’. Death due to PTSL syndrome was observed in 61% of those trees with the AFLP fragment, which was significantly higher (P ≤ 0.05) compared to those that did not have the fragment (17%). Furthermore, the PTSL symptom ratings were significantly higher, and cumulative tree-life was significantly shorter, (P ≤ 0.05) for ‘Lovell’ when compared with 93 genotypes that lacked the AFLP fragment. These results suggest an association of this AFLP marker, called EAC/MCCC1-96, with PTSL-susceptibility. An additional 43 marker candidates, diagnostic for the PTSL-response were also selected.