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California Cling Peach Advisory Board 2006 Annual Report Project Titles: Development of New Cling Peach Varieties Project Leaders: Tom Gradziel & Carlos Crisostos Cooperating Personnel: M.A. Thorpe, C. Peace, R. Bostock , J. Adaskaveg C. Peace and E. Ogundiwin Location: Dept. of Pomology, University of California at Davis Breeding Synopsis. Despite the challenging weather conditions during the spring bloom, the breeding program has achieved it’s goal of over 6000 seed from controlled crosses, primarily through the used of protected pollination structures and an emphasis on enforced selfpollinations of previous hybrids. (The hybridization of selected parents possessing distinct but desirable traits allows their consolidation in the hybrids, while the subsequent selfing of the best hybrid progeny allow the concentration of desirable genes from both parents in a genetic background more adapted to California growing conditions). Over 18,000 seedling trees from breeding crosses were evaluated for fruit and tree quality. Almost 500 individuals seedling trees were selected for further evaluation of processing potential using the UCD pilot plant processing facility at Cruess Hall. BioStar initiative to maximize breeding efficiency using recent biotechnologies. To accelerate progress in developing improved fruit firmness disease/pest resistance, the 2006 processing peach Variety Development and Regional Testing programs underwent dramatic expansion in research efforts though without increases in the level of industry support. In previous years we have maximized our variety development and regional testing efforts (also with very limited increases in industry funding) by combining day-today activities of the peach breeding program with a similarly industry-funded almond breeding program. Because the more intensive peach and almond field management operations (planting, pollination at flowering, harvest, etc.) were largely sequential with little overlap between the two crops, we were able to maximize our labor and equipment efficiency. The greater incentive for combining these seemingly disparate breeding programs, however, involved our long-term efforts to enrich the previously very narrow genetic variability within each crop through the exchange of genes between these and related species. This more exotic germplasm also has unique value for basic research on genetic control of plant development, allowing us to secure additional outside funding to further supplement breeding efforts. Previous industry reports have demonstrated the successful transfer to peach of a wide range of useful genes from almond and its wild relatives, including resistance to brown rot, aphids, leaf curl, as well 1 as genes conferring desirable effects on tree structure and fruit quality. Because genes are physically linked together on the DNA strand, the transfer of the small pieces of DNA containing desirable genes inevitably result in some undesirable linked genes being transferred as well. Over the last 6 years we have been involved in a rigorous, recurrent (generation-by-generation) selection for desirable genes and against undesirable traits as a way to purge out unwanted traits. This process, known as gene introgression, is relatively inefficient in tree crops because of the long generation-to-generation time periods involved, and because undesirable traits (such as high chilling requirement) may be difficult to detect in any one season due to their ambiguous expression and/or differences in expression in changing environments. The emerging biotechnologies offer powerful tools to address these problems but are very expensive. In addition, because this technology presently provides the 'cutting-edge' of our understanding of genetic/developmental processes, the majority of current biotech funded research is directed towards more basic questions such as identifying gene function (gene discovery). Rather than a barrier, we saw this emphasis of biotech research on 'gene discovery' as a potential advantage. A major (though frequently poorly appreciated) problem for biotech-based gene discovery research is that to be 'discoverable' the target needs to be a single gene whose expression is pronounced enough to be identifiable in an otherwise chaotic genetic/environment background. Because of the inbred nature of most of today's crops, the genetic variability is low and so presence of these novel genes is less likely. For example, a biotech-based program to identify a commercially useful brown-rot resistance gene in traditional California processing peach germplasm would be unsuccessful simply because no such gene is present in the germplasm. Molecular biologists are realizing that to increase the probability of success, they need to examine genetic populations with the often conflicting attributes of being both genetically diverse yet well characterized enough to demonstrate the presence of genes with significant commercial value. During the last 15 years, the California processing peach and almond breeding programs have successfully introgressed and partially characterized a wealth of new genetic material available for crop improvement. By pooling this resource with the demonstrated expertise in molecular and postharvest fruit analysis of Carlos Crisosto's lab at the Kearney Agricultural Center in Parlier, California, we have recently been successful in securing matching funds to current peach and almond industry support to develop molecular tools to complement our more traditional gene-discovery methods and so more efficiently exploit this rich germplasm. To encourage collaboration with top-notch molecular researchers, (and to facilitate paperwork), matching funds go entirely to the more expensive molecular research with the applied breeding efforts utilizing the industry sponsored funding. A major goal of this report is to document our progress in identifying and characterizing candidate genes for more detailed molecular analysis. A second major objective is to illustrate how this molecular technology can dramatically increase the efficiency and so effectiveness of the applied variety development and regional testing programs. In addition, examples of the frequently significant, though often poorly recognized, limitations of this new biotechnology will be addressed. 2 Breeding objectives. Breeding objectives and their relative priorities are summarized in Fig. 1. A short-term objective is replacement of inferior varieties in the Dixon-Andross and Halford-Corona maturity periods. Longer-term objectives Other include development of commercially New Traits pre-Loadel viable varieties ripening before Loadel (to extend the processing period), the Dixon-Andross Impr. Quality Other development of resistance to fruit brown rot and other commercially important Halford-Starn diseases, and the reduction in labor inputs. In addition to the ongoing Disease Resist. improvement of current breeding lines targeting these objectives, we continue to evaluate new traits for their potential value in solving current and future Fig. 1. Breeding objectives: 2006. production problems. 95 85 75 65 55 45 35 25 15 Number of Trees Replacement of inferior varieties. Progress in breeding efforts to replace inferior varieties is demonstrated by the recent release and positive industry acceptance of the Andross-period variety Goodwin and the Halford-period variety Lilliland, as well as the quantity and quality of advanced selections now in grower tests (see Regional Testing report). Continued breeding progress can also be gauged by the number of breeding progeny in each targeted category demonstrating sufficient quality to merit further canning evaluation. Changes in the numbers and distribution of UCD pilot plant processed selections is shown in Fig. 2. Increased numbers of progeny have occurred in the before-Loadel and after-Corona season-extension periods. (As discussed in the regional Ripe Date of Seedlings testing report, many of the 1993 2007 post-Corona caning evaluations are Halford70 Corona period selections 60 being tested for fruit structural 50 stability, both as post-ripening 40 holding ability on the tree, as 30 well as post-harvest storage). 20 Sizable increases in the 10 number of progenies can also be seen in the targeted early0 harvest season (day 45 to 60) and the extra-late harvest Days from June 1 season (day 80 to 90). In Figure 2. Distribution of processed breeding selections over the 1993, a distinct gap in the 2006 vs. 1993 seasons. number of progeny maturing in 3 the Dixon-Andross period (day 45 to 55) is apparent despite intense efforts to select parents targeting this season. Results from this early work suggested that the DixonAndross gap was the result of genetic deficiencies in the traditional California germplasm and outside germplasm would have to be incorporated to overcome this limitation. Accelerated efforts to incorporate new germplasm, particularly from eastern Europe and Mexico, have been successful and significantly increased both the quantity and quality of breeding progeny within this harvest season (Fig. 2). However, even in advanced breeding lines derived from this new germplasm, a distinct decline in progeny numbers is readily apparent which roughly corresponds with the maturity of the Dixon variety (day 45). Our current working hypothesis is that this period represents a threshold event for clingstone peach development (i.e. if the seedling tree fruit achieves this stage of development by a certain time then development continues; if this developmental threshold is not achieved by the critical time, then continued development is delayed by 3-5 days). While the nature or even existence of this threshold remains uncertain, we speculate that it may involve the transition from initial fruit cell division to later cell specialization (for example, cell lignification in the development of the pit). We have recently selected and placed in regional grower testing a very high quality processing peach, designated Extra-Early#1, which ripens at the targeted Dixon time Figure 3. Extra-Early#1: Dixon-period but inconsistent (Fig. 3). Evidence of a similar maturity time? bimodal distribution of harvest time, even within this genotype or clone, has been collected in the last two years, suggesting an inherent genetic/developmental instability at this ripening time. Other evidence of developmental instabilities may be the preponderance of pit-breakdown (pit fragments and red pit staining) common to this ripening period. Molecular fingerprinting procedures are currently being used to examine the possibility that the two ripening periods represent propagation mistakes. If it proves to be the same clone and it continues to produce two distinctive phenotypes (ripening times) then a careful genetic dissection using molecular techniques (as described in later sections) remains the only option for characterizing and perhaps avoiding this problem. (See regional testing report for more thorough evaluation of the Extra-Early#1, as well as the large number of advanced selections in the Ultra-Early, Extra-Early, Early, Late, and Extra-Late maturity periods). Canning evaluations in 2005 and 2006 at both the UCD pilot plant as well as cooperating commercial processors, have demonstrated an increasing number of breeding selections possessing the fruit size, color and firmness required for commercial acceptability (data and images available upon request). This larger pool of quality selections has allowed the breeding program to put greater emphasis on attributes seen as increasingly important in the maturing industry, such as disease resistance, fruit structural stability with increased mechanical handling and storage, and uniform harvest. 4 Breeding for brown rot resistance. Progress in the breeding program for brown rot resistance is demonstrated in the increasing number of advanced selections showing improved field resistance (Ultra-Early#1, Extra-Late#4, Extra-Late#5, Extra-Late#6, and Extra-Late#7) as well as the numbers and harvest season distribution of breeding progeny screened in 2006 (Fig. 4). As pointed out by Dr. Bostock in his accompanying 2006 report of laboratory screening results for our breeding progeny, uniformly higher levels of brown rot susceptibility were observed during the later harvest season relative to the early-season. This has been attributed to greater disease pressures at these times (latent infections, higher disease inoculum, greater probability of rain events, and greater stress on late-season trees). The skewed distribution also reflects the distribution of our major resistance parents (UltraEarly#1 ripens before Loadel, Bolinha ripens midseason and Extra-Late#5 ripens with or Figure 4. Brown rot severity in 2005 & 2006 genotypes after Corona). The unusually arranged in order of sampling date. cool early-season temperatures followed by very hot midseason temperatures in 2006 also shifted the ripening date distribution towards the late-season. The distribution of disease resistance over the harvest season for 2005 is also shown in Fig. 4 where a more uniform resistance or susceptible distribution is observed except in the third quarter of the season. This is partly a consequence of our targeting resistance for the Extra-Early and Extra-Late maturity seasons (as these are the times when rains and so disease explosions are more likely to occur). The importance of outside germplasm for developing resistance for California adapted processing peaches is demonstrated in Fig. 5 where Dr. Bostock has sorted the genotypes his lab evaluated in their order of resistance. Of the first 150 resistant selections, all but three were derived from outside resistance sources. In addition, examination of the inoculated fruit of these three lines indicates that they were picked prior to the full-ripe stage when their resistance would be expected to be greater. To guard against such false-resistances in immature fruit, we take color spectrum readings of all tested fruit to quantify both their level of maturity, their remnant chlorophyll content, and their potential for final fruit color. Other sources of resistance variability include undetected damages to the fruit epidermis prior to inoculation, latent infections, poor inoculation conditions in lab tests, and inconsistent levels of expression of the resistance 5 genes in different environments. The combination of these different and often independent sources of variation can result in considerable year-to-year variability in resistance scores for individual selections. To buffer against such environmental and genetic variability, we are attempting to combine resistances from several independent sources. Fig. 6 shows the fruit from several advanced resistance lines as well as documented resistance mechanisms identified in Drs. Bostock’s and Adaskaveg’s labs. Figure 5. Composite of all genotypes evaluated during 2006 illustrating the Mechanisms include range in disease severity values. Values are presented from lowest (most (from top to bottomresistant) to highest (most susceptible). small picture sidebar in Fig. 6) thicker layers of epicuticular waxes, denser epidermis cell walls, resistance to epidermis and mesocarp cell wall breakdown with infection, and higher levels of antifungal phytochemicals such as phenolics. Currently, our resistance breeding strategy involves combining resistance from multiple sources and selecting the resultant progeny with the highest level and most stable field resistance. Since total resistance rather than individual resistance mechanism is measured, the integration of multiple resistances is not as efficient as we would like. While the histological and biochemical analysis of individual mechanisms is overly tedious and beyond our level of expertise, the identification of the underlying controlling genes would allow their individual identification and selection using standard high throughput molecular screens. We now have promising candidates for two of the resistance mechanisms/genes (anti-fungal biochemicals with Drs. Bostock and Crisosto, and cell wall breakdown enzymes/genes with Dr. Crisosto). Identification of the specific genes responsible for resistance would allow their direct selection even at the seedling stage, thus allowing a much greater efficiency and so breeding progress for brown rot resistance. This continuing integration of molecular techniques for applied disease resistance breeding highlights 4 crucial, though often unappreciated, requirements for success: 1) resistance has to be present, 2) resistance needs to be in advanced breeding lines of sufficiently good commercial quality to assess field value, 3) the mechanism of resistance needs to be sufficiently well-characterized Figure 6. Sampling of different resistance lines as well as resistance components. in identified candidate genes, and 4) the trait should be readily transmitted to progeny (i.e. controlled by 1 to 3 genes). These requirements have also been key to our recent joint efforts to exploit molecular markers to breed for greater resistant to fruit textural breakdown resulting from disease, 6 physical trauma, and postharvest storage. Breeding for improved fruit flesh integrity using molecular markers. Fruit flesh integrity defines the processing and fresh market peach industries. In order to achieve the durable fruit textures needed for bulk handling, the processing peach industry almost exclusively utilizes the nonmelting/clingstone peach rather then the melting/freestone peach used for fresh market. The clingstone trait results in a poor separation of the flesh from the stone, often leaving highly problematic lignified pit fragments in the processed fruit. While the stone adhesion and flesh-melting traits appear independent, clingstone is almost always inherited with non-melting flesh and freestone is always inherited with melting flesh, indicating that they are controlled by the same gene or by two genes which are so closely linked together as to behave as one unit in their segregation to progeny. Even in the more durable clingstone/non-melting peaches, textural integrity begins to collapse within days after full-ripe, requiring multiple harvests to ensure ripe fruit durable enough for transport and processing. Recognizing that the linkage between stone adhesion and fruit firmness had not been broken in efforts by a large number of long-term breeding programs using traditional peach germplasm, our processing peach breeding program began a program over 10 years ago to transfer novel non-melting flesh types from almond and related species. By the early 2000's we had developed a rather wide selection of novel freestone/non-melting to clingstone/longlasting fruit types, many of which were shown to be transferable to breeding progeny. In the early 2000's we had also generated large number of progeny from the cross between the Dr. Davis clingstone/non-melting peach and the Georgia Belle freestone/melting peach as part of an informal collaboration with Dr. Crisosto's lab at KAC to study the textural, biochemical and genetic nature of stone adhesion/flesh melting traits. This genetic material, having already been initially characterized morphologically and genetically (in terms of field inheritance) provided the foundation for our successful BioStar proposal to characterized these fruit textural differences at the molecular level and so allow a more precise understanding of the mechanisms involved as well as their manipulation in applied breeding. In the following sections, the chronology of this research is presented to demonstrate both the procedures and potential of molecular tools to applied to plant breeding. Collecting traditional and developing novel fruit firmness/pit adhesion types for morphological, genetic and molecular characterization. The two major peach fruit types in traditional varieties are the freestone/melting and clingstone/nonmelting types (Fig. 7). A third type, clingstone/melting, can be recovered in certain breeding lines but is almost always discarded as it appears to combine the worst commercial aspects of both types. A prototype of the ideal type, freestone/nonmelting, as been recovered in our breeding program from material introgressed from almond, as have ‘long-keeper’ processing peach selections showing a clingstone/nonmelting fruit type in which fruit softening after the full-ripe stage is dramatically reduced when compared even to clingstone-nonmelting types (Fig. 7). A comparison of fruit structure with softening patterns further shows the distinctiveness of these 4 fruit types (Fig. 7). The lower thinsectioned images were allowed to dry slightly, as it allows a clearer perception of the distinction between the inner fruit flesh (often visualized as straight strands radiating outwardly from the pit) from the outer flesh region adjacent to the skin (where radiating Figure 7. Different peach fruit stone adhesion and flesh melting types and graph showing puncture/shearing force for ripe fruit from skin (left) to pit cavity (right). 7 strands of tissue often branch into a more complex tangled mass of strands growing more perpendicular to the pit surface). The structural differences are also apparent in graphs showing the amount of force needed to penetrate from the skin into the central pit area (Fig. 7). In all fruit types the greatest force, and so most resistance to penetration, is seen at the epidermis (though only slightly so for freestone/melting). At full ripe, both the outer and inner tissue of freestone/melting is largely present as disintegrating strands, and so presenting minimal resistance to the force gauge. By comparison in our freestone/nonmelting type, the stone is clearly air-free but the firmness of both the outer and inner flesh is comparable to clingstone/nonmelting (at this full ripe stage). (As presented in previous reports, however, these types will soften to almost freestone levels by day 4 and 5). The clingstone/nonmelting image shows both the adhesion of a ' blanket' of flesh strands running across the pit surface as well as attached to the outwardly radiating strands which make up the inner and outer flesh. A high flesh firmness or integrity is apparent throughout the flesh at the full-ripe stage. Finally, in another novel type derived from almond germplasm, the fruit behaves similar to a clingstone/nonmelting at the full-ripe stage but, as has been documented in previous reports (as well as the 2007 regional testing report), the fruit flesh maintains its firmness or integrity for two weeks or more after the full ripe stage. The development of these breeding lines demonstrated both that unique genetic controls were involved and, further, that the inner and outer tissue showed different ripening patterns in different genetic backgrounds. EndoPG as a prime candidate gene controlling fruit softening. Because endopolygalacturonase (endoPG) was previously shown to be associated with softening in peach, the forms of this gene were studied in our novel breeding lines as well as standard freestone/melting varieties, canning clingstone/nonmelting varieties and progeny from the crosses between the two (Georgia Belle x Dr. Davis population). Results are summarized in Fig. 8, where ESTs (, i.e. expressed sequence tags or sequences of genes being actively expressed) of endoPG at fruit ripening are compared. The two distinct dark bands associated with freestone melting flesh fruit types (FMF) show that 2 separate but related genes of endoPG are being expressed (F and f) . In the clingstone/melting flesh (CMF) types only the upper band was present though with some individuals a closely paired, double-banding was apparent. The absence of the lower FMF band in all clingstone (CMF & CNMF) types demonstrated the presence of only one gene (‘f/f1’ in Fig. 8). Since peach is a diploid organism, (i.e. contains two forms of each gene; one from the seed parent and one from the pollen parent), the tight double banding in CMF fruit types indicates that different alleles or forms of the f-gene were inherited from each parent (f/f1). Other CMF types inherited the same f-allele from both parents (f/f or f1/f1). Significantly, in all processing-type clingstone/nonmelting flesh (CNMF) varieties, the freestone or F-gene was either absent or nonfunctional leaving only the f1 allele of the f-gene and in some cases no functional or expressed genes (designated nn or null). The finding of this close correlation between fruit type and the form of the endoPG gene appear to confirm our hypothesis that the endoPG gene is a major player in determining fruit softening. The finding that a different form of this endoPG gene appears to be responsible for the stone adhesion trait, was an added and very exciting bonus. (Since endoPG is known to be responsible for the breakdown of the intercellular matrix or glue that holds cells and cell strands together, its involvement 8 in fruit softening was intuitive. The possible control of the freestone/clingstone trait by endoPG was also anticipated as a possibility, since, as is apparent in Fig. 7, the freestone trait essentially involves a breakdown of the connections between the pitchannel strands and the pit-surface). As a final bonus, by knowing the sequence of the endoPG-genes, we were able to develop PCR-based molecular fingerprinting techniques which allowed the rapid and high-volume characterization of breeding lines and varieties. Rapid PCR-based molecular surveys of breeding lines and varieties. Using the very rapid and efficient PCR approach for molecular (DNA) fingerprinting we were able to evaluate over 100 varieties and breeding lines from the peach and almond breeding programs. Since over 70 of these items were commercially important processing and fresh market peach varieties, we believe the results (summarized in Fig. 9) represent an accurate general picture of the genetic control of fruit softening in current peach varieties. Results from the analysis of all commercial peach varieties were consistent with our hypothesis that the endoPG gene had duplicated itself early in the evolution of peach and almond in that one form had evolved to control stone adhesion while the other form evolved to control fruit softening at ripening. Because this duplication Figure 8. Gel banding patterns identifying different endoPG type genes (F & f) as well as different forms or alleles of the f-genes, and their associated fruit types. typically occurs as a copying error when the DNA is transcribed from mother to daughter cells, they are very closely linked together. This close linkage explains why freestone/melting and clingstone/nonmelting are always inherited together since the probability of a random DNA break/rearrangement to separate freestone from melting, for example, is highly unlikely within this very short distance. While both genes were 9 found to have multiple, often subtly different, forms or alleles, whenever any allele of the F-gene was present, the fruit was freestone and melting flesh. Clingstone fruit were only present when the F-gene was completely absent or nonfunctional allowing the f-gene, whose alleles further determine flesh melting characteristics, to be expressed. Different f-alleles determined different fruit types; if any f-alleles are present, then the clingstone flesh is melting. If all alleles are f1-alleles or null-alleles (absence of functional gene) than the flesh is clingstone/nonmelting. This advanced working hypothesis is also consistent with previous genetic studies were freestone/melting (F/{any f-form}) is dominant to (i.e. masks any expression of) clingstone/melting and clingstone/nonmelting. Consequently, the freestone/melting trait is rapidly purged out of any processing peach breeding program because 1) it is an undesirable commercial type and, 2) it hides or masks the expression of the commercially important f-gene. Similarly, because the f-allele, (which confers clingstone/melting and is commercially undesirable), masks the expression of the commercially important f1-allele, it is rapidly purged from processing peach breeding programs. Figure 9. Sumary of fruit types, firmness & molecular data supporting the working hypothesis of 2 separate multi-allelic genes controlling pit adhesion and fruit softening in California processing and fresh market varieties. 10 EndoPG expression and activity. We are presently comparing the levels of endoPG expression and activity in the different tissues of fruit types studied and the initial results support our working hypothesis and, in addition, suggest a strategy for developing improved processing peach types. Freestone/melting types consistently show very high endoPG gene expression and biochemical activity in both the inner and outer flesh. In nonmelting/clingstone types endoPG expression and activity are 50-fold lower and located predominately in the inner flesh adjacent to the pit. Interestingly, in nonmelting/clingstone genotypes completely lacking any functional alleles of the F-gene and the f-gene (i.e. null or an nn alleles), endoPG activity was almost completely absent and post-ripening fruit softening, while still occurring, was at the lowest level of any type measured. A high level of agreement between molecular fingerprinted genotypes, fruit flesh endoPG expression and activity, and final fruit type, further supports our working hypothesis. These molecular tools for fingerprinting (i.e. identifying specific forms of specific genes) provides the breeding program with both precise knowledge of the genetics involved as well as a more integrated knowledge of how these genes interact to determine different fruit types. Thus, these molecular approaches offer powerful tools to dissect or separate individual mechanisms conferring fruit firmness as well as allow a more efficient recombination of the most desirable genes to achieve commercial goals. While a powerful tool, however, this type of molecular dissection has certain limitations in applied breeding programs because, on a whole-plant level, individual genes frequently have multiple (and often unanticipated) consequences, and because different developmental processes such as fruit ripening, often have multiple pathways to achieve developmental goals. The final section of this report will document examples of such situations to highlight their potential barriers to achieving breeding goals. Dangers of over-dependence on molecular-based strategies for crop breeding. Based solely on our molecular findings, the development of null endoPG fruit types (i.e. complete absence of fruit flesh endoPG gene expression and biochemical activity) would be a highly desirable goal for processing nonmelting/clingstone peach breeding as it is associated with the lowest rate of post-ripening flesh softening. In fact, for the past 10 years our breeding program has been using the parent Fla9,20-C (a very lowchill clingstone/nonmelting fresh market peach developed at the University of Florida from Mexican germplasm), which has been identified as a double null. [It was the early analysis Figure 10. Fruit of Fla9,20-C showing the intense red-stained of this breeding selection which pit associated with the null condition for the endoPG gene. first identified the occurrence of null alleles for this gene]. Fig. 10 shows fruit samples of this selection collected 2004. 11 While conferring desirable fruit firmness, the trait is also associated with an intense red pigmentation in the pit cavity in both the parent as well as breeding progeny. We are presently using the molecular tools developed to target null genotypes in advanced breeding lines in which these undesirable consequences might be minimized. For example, we have recently identified another gene (designated high-liter) which appears to shut down all red anthocyanin pigment production in the flesh and so could eliminate the red-pit problem. (However, the presence of high-liter, itself, appears to be associated with a poorer gloss or luminosity of the processed fruit, which is presently perceived as one of the hallmarks of California processed peach quality). Another breeding line identified as having very low post-ripening fruit softening rates were the 'stony-hard' selections derived from the Chinese Yumyeong peach. Suppressed softening appears to be to due an independent mechanism here, possibly the shutting down of the ethylene trigger for fruit ripening. The descriptor stony-hard refers to the crisp, almost a pear-like texture of the ripe fruit. A number of stony-hard breeding lines were tested in our program in the early 1990s before being discarded because of the associated undesirable decline in processed fruit textural qualities (fruit were firm but ‘woody’ in texture). While the molecular identification/dissection of endoPG-based flesh softening has identified promising endoPG combinations and provided the tools to generate/monitor their precise recombination, this molecular approach has also proved important in identifying fruit firmness pathways independent and so possibly complementary to the endoPG activity. (Though it was only with the recent application of the endoPG molecular analysis that we became aware of the independence and so opportunities of these alternative pathways). An example is seen in the advanced 'long-keeper' ExtraLate breeding selections (Extra-Late#4 to#7) presently being placed on regional grower evaluations. Although possessing the f1-allele, associated with typical clingstone/nonmelting processing peaches, these selections appear to suppress endoPG softening of ripe fruit to levels comparable to the double null and stony-hard genotypes, yet without the undesirable characteristics (Figs. 7 & 11). (Because of the recent almondorigin of these lines, however, opportunities for undesirable linkages are relatively high, requiring thorough testing at both the field and molecular level Figure 11. Extra-Late#5: a fruit type that maintains high before consideration for varietal fruit firmness independent of the endoPG gene. release, (see discussion on Regional Testing report). Future research. Fruit softening via the endoPG and complementary pathways, while a major objective of our collaborative efforts with Carlos Crisosto's molecular group represents only one of the research avenues being evaluated within the larger fruit Fig. 12 12 quality context. With Cameron Peace and Ebenezer Ogundiwin, the molecular biologists directing the molecular testing, we are targeting a large number of traits of commercial importance to processing peach and almond. In addition, we have leveraged this research momentum to enlist the cooperation of a large number of molecular biologists doing similar research at other national and international programs. The combined efforts of these basic and applied researchers have resulted in the development of an ambitious 'roadmap' or plan for a more aggressive application of molecular tools to achieve applied breeding goals. Central to this progress has been the development of a relatively complete genetic map of peach and almond (Fig. 12) by combining the research results from these national and international programs. The resultant genetic linkage map shows the location Fig. 13.of a large number of different molecular markers (i.e. the specific DNA sequence as determined by different molecular approaches, PCR, SSR, etc.) as well as the location of known genes controlling important traits. The map can be used to evaluate the presence of desirable and/or undesirable linkages for candidate traits. For example in Fig. 12, the stick image labeled 'G 4' represents chromosome #4. Towards the bottom of the image, we can see the location of endoPG gene determined from our research. The location or locus for the 'Bi' gene controlling internal flesh bleeding is very close to endoPG indicating that in certain selections (as a hypothetical example, the red-stained double null selection Fla9,20-C described above) specific desirable forms of the endoPG allele (the null form in this case) may be physically linked and so almost always inherited with an undesirable forms of the Bi gene (conferring red anthocyanin bleeding in the pit area). It this association were confirmed, breeding programs could target the breakage of this linkage using molecular markers to select even at the seedling stage (and so eliminating the need to grow the very large numbers of plants required to field maturity). Alternatively, the breeder could target the CS (color around stone) gene known to be located in the central portion of chromosome 3 (G3 in Fig. 12) or the yet to be mapped high-liter gene, to suppress all red anthocyanin production in plants whose other genetic components would encourage it (i.e. shut down anthocyanin production upstream in the developmental pathway). 13 Finally, the high number of molecular markers on this peachalmond linkage map provide effective markers to identify and more thoroughly characterized additional traits of commercial value. For example, to characterize the previously described high-liter gene, we are mapping both molecular markers as well as high-liter trait expression in progeny from controlled crosses. Due to the high density of this map, it is likely that certain molecular marker are located very close to the high-liter locus and so will almost always segregate with it in breeding progeny. Once we have identified a molecular marker which co-segregates with the trait of interest, we have identified the general location of that trait as well. Once identified, the entire sequence of DNA within that region can be determined and individual embedded gene sequences compared against known sequences of similar functioning genes. (Recent advances in genomics and informatics have made accessible huge databases of known gene sequences and function, as well as computer software allowing the rapid and efficient comparison with hundreds of thousands of established sequences.) In addition, genetic and molecular mapping at the very high resolutions now possible, can identify the location (and through subsequent sequencing, the identity) of currently unknown genes. An example is seen in Fig. 13 where the level of association between specific molecular markers (here on a small segment of chromosome 5), and the trait of interest (flesh browning) was compared over multiple years in our Georgia Belle x Dr. Davis segregating population. The level of flesh-browning is normally difficult to evaluate because it can very greatly depending on environmental factors such as temperature and tissue stress. A multiyear, molecular mapping of this trait, however has identified a gene locus which is estimated to account for approximately 40% of all flesh browning observed in this population. In addition to identifying the presence of this gene, this 14 approach has also identified the specific location ( the qP-Brn5.1 region on chromosome 5) of the gene which, in turn, allows its sequence determination and ultimate identification using procedures described above. The sequencing of the flesh-browning locus will also allow the development of very precise molecular probes which can identify other forms of this gene which may be inactive (no flesh browning) in the breeding population (assuming such a gene exists). The sequence will also allow the development of transgenes which might effectively turn off this gene in future genetically engineered varieties. Equally important, using the huge genomic and informatics databases developed in ongoing national and international molecular research, the structure and putative function of the gene in question can often be predicted. Increasingly, this 'forward-looking' approach to determining gene function from DNA sequences can also be used in the reverse direction. For example, if the ongoing research by Rick Bostock's lab identifies a specific phenolic compound associated with resistance in processing peach, we might be able to predict a rough sequence for the controlling gene by examining gene sequences for that or similar phenolics from other plants (Arabidopsis, corn, tobacco, etc.) that are presently is listed in the genomic database. We could then use that rough sequence to develop molecular probes to specifically tag and efficiently identify the desired gene form even in seedling populations or in environments not conducive to disease screening. In summary, results from the first year of research have shown impressive increases in our genetic knowledge and so breeding potential. However, for proper application this knowledge needs to be tempered with field-based experience in the traits of interest, particularly concerning potentially undesirable associated effects. A second danger from the unbalanced use of molecular approaches to pursue breeding goals is the risk of canalizing breeding efforts (for example, targeting the null form of endoPG as the sole strategy to control fruit softening). Not only does such a narrow perspective increase the risk of ‘dead-end’ research when unacceptable negative consequences are recognized, but it eliminates the opportunities to identify independent genetic solutions which may avoid undesirable associations. We believe this project has achieved its goals of dramatically increasing molecular tools available to applied peach breeding (at no additional cost to industry sponsors) while concurrently allowing significant advances in our understanding of basic development pathways by exploiting the inherent genetic variability developed by applied breeding programs in their (typically) multifaceted approaches to targeted breeding goals. 15