CHANGES IN ATMOSPHERIC CARBON DIOXIDE (CO2) CONCENTRATIONS, SUMMER AND WINTER TEMPERATURES AND PRECIPITATION PATTERNS CAN POSITIVELY AND NEGATIVELY AFFECT FOREST PRODUCTIVITY. WHILE INCREASED CO2 IS LIKELY TO INCREASE PRODUCTIVITY, INCREASED TEMPERATURES AND DECREASED PRECIPITATION MAY NOT ONLY CAUSE DECREASED PRODUCTIVITY AND POSSIBLE DEATH DUE TO WATER DEFICIT STRESS BUT MAY ALSO LEAD TO INCREASED SEVERITY OF DISTURBANCES SUCH AS WILDFIRES, DISEASE AND INSECT OUTBREAKS. EFFECTS OF CLIMATE CHANGE HAVE BEEN OBSERVED IN MANY NORTH AMERICAN FORESTS. IN THIS PROJECT, WE FOCUS ON THE LOBLOLLY PINE FORESTS OF THE SOUTHEASTERN UNITED STATES, PARTICULARLY THE DRIER, WESTERN EDGE WHERE INCREASING ARIDITY THREATENS SURVIVAL AND GROWTH, HENCE DECREASING THE PRODUCTIVITY AND THE ECONOMIC VALUE OF LOBLOLLY PINE CULTIVATION.THE SOUTHEASTERN U.S. IS KNOWN AS THE WORLD'S WOOD BASKET, SUPPLYING 12% OF THE WORLD'S WOOD PRODUCTS AND 19% OF ITS PULP AND PAPER. THE FORESTS OF THE SOUTHEAST CONTAIN ONE-THIRD OF THE ENTIRE FOREST CARBON IN THE CONTIGUOUS U.S. THREE COOPERATIVE BREEDING PROGRAMS FOCUS ON IMPROVEMENT OF LOBLOLLY PINE, THE MOST ECONOMICALLY IMPORTANT SPECIES IN THE REGION. MUCH OF THE FOCUS OF TREE IMPROVEMENT PROGRAMS HAS BEEN ON IMPROVED GROWTH AND DISEASE RESISTANCE. AS TEMPERATURES INCREASE AND DROUGHTS BECOME MORE FREQUENT AND SEVERE, DROUGHT RESISTANCE AND WATER USE EFFICIENCY (WUE) HAVE BECOME IMPORTANT CHARACTERISTICS TO CONSIDER. AT THE SAME TIME HOWEVER, IT IS IMPORTANT TO MAINTAIN OR CONTINUE TO IMPROVE YIELD. MOLECULAR MARKERS ASSOCIATED WITH WUE, DROUGHT RESISTANCE, PHOTOSYNTHETIC EFFICIENCY AND GROWTH WOULD BE VALUABLE FOR FACILITATING BREEDING FOR MULTIPLE CHARACTERISTICS. IN OUR LABORATORIES, WE HAVE STARTED THE DEVELOPMENT OF TOOLS THAT WILL HELP BREEDERS SUCH AS THE WESTERN GULF FOREST TREE IMPROVEMENT PROGRAM (WGFTIP) TO SELECT FOR TREES BETTER ADAPTED TO HOTTER, MORE ARID ENVIRONMENTS.OUR LONG-TERM GOAL IS TO DEVELOP VARIETIES OF FAST-GROWINGLOBLOLLY PINES ADAPTED TO INCREASED DROUGHT AND HIGHER TEMPERATURES. THE OVERALL OBJECTIVE OF THIS PROJECT, WHICH IS AN IMPORTANT STEP TO MEET OUR LONG-TERM GOAL, IS TO DEVELOP BIOMARKERS AND MOLECULAR MARKERS THAT CAN BE USED BY COOPERATIVE TREE IMPROVEMENT PROGRAMS, TO ASSIST WITH BREEDING OF LOBLOLLY PINE WITH IMPROVED WUE, PHOTOSYNTHETIC EFFICIENCY, SURVIVAL AND GROWTH. IN TURN, THIS WILL INSURE A CONTINUED WOOD SUPPLY, EMPLOYMENT IN THE WOOD PRODUCTS INDUSTRY AND FORESTS FOR RECREATION AND HABITAT. SINCE FORESTS CAPTURE AND STORE CARBON THROUGH PHOTOSYNTHESIS, WELL-ADAPTED, FAST-GROWING LOBLOLLY PINES WILL PROVIDE GREAT VALUE IN OFFSETTING ATMOSPHERIC CARBON DIOXIDE AND MITIGATING LONG-LASTING CLIMATE CHANGES CAUSED BY GREENHOUSE GAS EMISSIONS.TWO FUNDAMENTAL BIOMARKERS WILL BE USED IN THIS PROJECT. THE STABLE ISOTOPE DISCRIMINATION BIOMARKER PROVIDES A MEASURE OF HOW MUCH WATER IS USED FOR METABOLIC PURPOSES AS OPPOSED TO THE WATER LOST THROUGHOUT THE LEAF STOMATA (EVAPOTRANSPIRATION), A RATIO KNOWN AS WATER USE EFFICIENCY. A SECOND BIOMARKER, THE A/CI CURVE, WILL SHOW HOW MUCH CARBON FROM THE ATMOSPHERIC CO2 IS INCORPORATED INTO SUGARS (GLUCOSE) IN THE LEAVES AND THUS REPRESENTS A MEASURE OF THE PHOTOSYNTHETIC EFFICIENCY. THE MOLECULAR MARKERS DERIVE FROM PREVIOUS ANALYSES OF THE DNA OF THOUSANDS OF LOBLOLLY GENES. THESE MARKERS, KNOWN AS SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS), ARE COMMON AMONG ORGANISMS. EACH INDIVIDUAL TREE HAS A UNIQUE SET OF SNPS, MOST OF WHICH ARE MATERNALLY AND PATERNALLY INHERITED IN SIMILAR PROPORTIONS. THE COLLECTION OF SNPS FROM INDIVIDUAL LOBLOLLY TREES REPRESENTS THE TREE GENOTYPE. MANY SNPS ARE ASSOCIATED WITH VARIATION IN TRAITS SUCH AS GROWTH RATE AND DROUGHT TOLERANCE. STUDYING SNPS ACROSS LARGE TREE POPULATIONS ALLOWS TO IDENTIFY THESE ASSOCIATIONS AND UTILIZE ASSOCIATED SNPS AS MARKERS FOR A TRAIT OF INTEREST IN OTHER TREES.THE CENTRAL HYPOTHESIS FOR THE PROPOSED RESEARCH IS THAT MARKERS ASSOCIATED WITH ISOTOPE DISCRIMINATION AND THE A/CI RESPONSE CAN BE USED FOR MARKER ASSISTED BREEDING OF LOBLOLLY PINES ADAPTED TO A WARMER, DRIER OR UNPREDICTABLE CLIMATE. IN ADDITION, CARBON ISOTOPE DISCRIMINATION (CID) AND A/CI WILL BE USEFUL BIOMARKERS TO IDENTIFY PARENTS IN BREEDING POPULATIONS WITH HIGH WUE AND PHOTOSYNTHETIC EFFICIENCY.WE PLAN TO TEST OUR CENTRAL HYPOTHESIS BY PURSUING THE FOLLOWING SPECIFIC AIMS.1) IDENTIFY GENETIC DIFFERENCES BETWEEN TREES, IN THIS CASE SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS), ASSOCIATED WITH WATER USE EFFICIENCY, PHOTOSYNTHETIC EFFICIENCY, AND GROWTH TRAITS.2) DETERMINE IF VARIATION IN STABLE ISOTOPE DISCRIMINATION (AN ESTIMATE OF WUE) AND A/CI CURVES (A MEASURE OF PHOTOSYNTHETIC EFFICIENCY) CAN BE USED TO PREDICT RELATIVE SURVIVAL AND GROWTH.3) DETERMINE IF MORE HETEROZYGOUS TREES FOR SPECIFIC SETS OF SNPS WILL PERFORM BETTER WITH REGARD TO AT LEAST ONE MEASURED TRAIT.TO ACHIEVE OUR GOALS, WE WILL WORK WITH THE WGFTIP AND USE A SET OF THREE PROGENY TESTS ESTABLISHED ALONG A LONGITUDINAL AND PRECIPITATION GRADIENT IN EAST TEXAS. THE SEEDLINGS IN THIS TEST ARE THE RESULT OF 68 FEMALE PARENTS BEING CROSSED TO A POLYMIX CONTAINING POLLEN FROM 14 MALE PARENTS. 40 INDIVIDUALS FROM EACH OF THE 68 FAMILIES WERE PLANTED AT EACH SITE. THE WGFTIP WILL PROVIDE SURVIVAL AND GROWTH MEASUREMENTS AFTER 3 AND 5 SEASONS OF GROWTH. WE WILL ANALYZE THE GENETICS OF THE PARENT TREES AND 600 PROGENY FROM ONE SITE (12 TREES FROM EACH OF 50 FAMILIES) USING TWO METHODS TO EXAMINE SNPS PRESENT IN GENES OF INTEREST. FIRST, WE WILL UTILIZE AN ARRAY THAT IS COMMERCIALLY AVAILABLE TO ANALYZE 50,000 LOBLOLLY PINE SNPS. HOWEVER, NOT ALL GENES OF INTEREST ARE REPRESENTED ON THE ARRAY. WE ARE ESPECIALLY INTERESTED IN GENES WITH ROLES IN DROUGHT RESPONSES AND WATER USE EFFICIENCY AND SNPS THAT MIGHT BE IMPORTANT IN THE WESTERN GULF REGION. ONE POSSIBLE METHOD TO ANALYZE VARIATION IN THESE GENES IS THROUGH SEQUENCE CAPTURE AND SEQUENCING. SMALL DNA SEQUENCES OF INTEREST (BAITS) ARE USED TO CAPTURE NEARLY IDENTICAL SEQUENCES FROM OUR DNA SAMPLES. WE WILL DESIGN THE BAITS TO CAPTURE GENES PREVIOUSLY DETERMINED TO BE OF INTEREST. THE CAPTURED DNA IS SEQUENCED REVEALING THE SNP DIFFERENCES BETWEEN INDIVIDUALS.WE WILL ESTIMATE WUE IN THE SAME 600 TREES USING CID. TO HELP MAKE THIS DATA MORE PRECISE, WE WILL ALSO MEASURE OXYGEN ISOTOPES AND DO AN ANALYSIS OF PHOTOSYNTHETIC EFFICIENCY, THE A/CI CURVE DETERMINATION. NEEDLE NITROGEN CONTENT WILL ALSO BE ASSESSED TO OBTAIN COMPLEMENTARY INFORMATION ON PHOTOSYNTHETIC POTENTIAL. USING THE SURVIVAL, GROWTH TRAIT, ISOTOPE AND A/CI CURVE DATA, WE WILL ATTEMPT TO IDENTIFY SNPS THAT CAN ACCOUNT FOR VARIATION IN THESE TRAITS USING ASSOCIATION GENETICS ANALYSES. THESE SNPS WILL BE VALUABLE FOR MARKER-ASSISTED BREEDING IN TREE IMPROVEMENT PROGRAMS. WE WILL ALSO DETERMINE PARENTAL BREEDING VALUES (OR GENERAL COMBINING ABILITY - HOW WELL A TREE PASSES ON GOOD TRAITS TO ITS OFFSPRING) FOR THE NITROGEN CONTENT, ISOTOPES AND A/CI CURVE POINTS. WE WILL DETERMINE IF WE CAN USE THESE TRAITS TO PREDICT GROWTH AND SURVIVAL IN THE OTHER POPULATIONS. IF SO, THESE BIOMARKERS MAY ALSO BE USEFUL TO TREE BREEDERS.WE WILL ALSO ANALYZE SPECIFIC SETS OF SNPS (SUCH AS THOSE WITH POSSIBLE ROLES IN WUE AND DROUGHT RESPONSE) TO SEE IF TREES THAT ARE MORE HETEROZYGOUS (TWO DIFFERENT SNPS AT A POSITION), ARE SUPERIOR FOR ANY OF THE TRAITS OF INTEREST. PREVIOUS STUDIES HAVE IDENTIFIED SOME CORRELATIONS BETWEEN HETEROZYGOSITY AND PHENOTYPIC TRAITS BUT HAVE USED TOO FEW OR TOO MANY MARKERS TO MAKE CONCLUSIONS.