Irwin L. Goldman

Quantitative trait loci influencing protein and starch concentration in the Illinois Long Term Selection maize strains.

A study was initiated to determine the number, chromosomal location, and magnitude of effect of QTL (quantitative trait loci or locus depending on context) controlling protein and starch concentration in the maize (Zea mays L.) kernel. Restriction fragment length polymorphism (RFLP) analysis was performed on 100 F3 families derived from a cross of two strains, Illinois High Protein (IHP), X Illinois Low Protein (ILP), which had been divergently selected for protein concentration for 76 generations as part of the Illinois Long Term Selection Experiment. These families were analyzed for kernel protein and starch in replicated field trials during 1990 and 1991. A series of 90 genomic and cDNA clones distributed throughout the maize genome were chosen for their ability to detect RFLP between IHP and ILP. These clones were hybridized with DNA extracted from the 100 F3 families, revealing 100 polymorphic loci. Single factor analysis of variance revealed significant QTL associations of many loci with both protein and starch concentration (P < 0.05 level). Twenty-two loci distributed on 10 chromosome arms were significantly associated with protein concentration, 19 loci on 9 chromosome arms were significantly associated with starch concentration. Sixteen of these loci were significant for both protein and starch concentration. Clusters of 3 or more significant loci were detected on chromosome arms 3L, 5S, and 7L for protein concentration, suggesting the presence of QTL with large effects at these locations. A QTL with large additive effects on protein and starch concentration was detected on chromosome arm 3L. RFLP alleles at this QTL were found to be linked with RFLP alleles at the Shrunken-2 (Sh2) locus, a structural gene encoding the major subunit of the starch synthetic enzyme ADP-glucose pyrophosphorylase. A multiple linear regression model consisting of 6 significant RFLP loci on different chromosomes explained over 64 % of the total variation for kernel protein concentration. Similar results were detected for starch concentration. Thus, several chromosomal regions with large effects may be responsible for a significant portion of the changes in kernel protein and starch concentration in the Illinois Long Term Selection Experiment.

Recombinant inbred lines for genetic mapping in tomato

A cross between the cultivated tomato Lycopersicon esculentum and a related wild species L. cheesmanii yielded 97 recombinant inbred lines (RILs) which were used to construct a genetic map consisting of 132 molecular markers. Significant deviation from the expected 1:1 ratio between the two homozygous classes was found in 73% of the markers. In 98% of the deviating markers, L. esculentum alleles were present in greater frequency than the L. cheesmanii alleles. For most of the markers with skewed segregation, the direction of the deviation was maintained from F2 to F7 generations. The average heterozygosity in the population was 15%. This value is significantly greater than the 1.5% heterozygosity expected for RILs in the F7 generation. On average, recombination between linked markers was twice as high in the RILs than in the F2 population used to derive them. The utility of RILs for the mapping of qualitative and quantitative traits is discussed.

The beet R locus encodes a new cytochrome P450 required for red betalain production

Anthocyanins are red and violet pigments that color flowers, fruits and epidermal tissues in virtually all flowering plants. A single order, Caryophyllales, contains families in which an unrelated family of pigments, the betalains, color tissues normally pigmented by anthocyanins. Here we show that CYP76AD1 encoding a novel cytochrome P450 is required to produce the red betacyanin pigments in beets. Gene silencing of CYP76AD1 results in loss of red pigment and production of only yellow betaxanthin pigment. Yellow betalain mutants are complemented by transgenic expression of CYP76AD1, and an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation. Finally, expression of CYP76AD1 in yeast verifies its position in the betalain biosynthetic pathway. Thus, this cytochrome P450 performs the biosynthetic step that provides the cyclo-DOPA moiety of all red betacyanins. This discovery will contribute to our ability to engineer this simple, nutritionally valuable pathway into heterologous species.

Quantitative trait locus analysis of a recombinant inbred line population derived from a Lycopersicon esculentum x Lycopersicon cheesmanii cross

Quantitative trait loci influencing fruit traits were identified by restriction fragment length polymorphism (RFLP) analysis in a population of recombinant inbred lines (RIL) derived from a cross of the cultivated tomato, Lycopersicon esculentum with a related wild species Lycopersicon cheesmanii. One hundred thirty-two polymorphic RFLP loci spaced throughout the tomato genome were scored for 97 F8 RIL families. Fruit weight and soluble solids were measured in replicated trials during 1991 and 1992. Seed weight was measured in 1992. Significant (P<0.01 level) quantitative trait locus (QTL) associations of marker loci were identified for each trait. A total of 73 significant marker locus-trait associations were detected for the three traits measured. Fifty-three of these associations were for fruit weight and soluble solids, many of which involved marker loci signficantly associated with both traits. QTL with large effects on all three traits were detected on chromosome 6. Greater homozygosity at many loci in the RIL population as compared to F2 populations and greater genomic coverage resulted in increased precision in the estimation of QTL effects, and large proportions of the total phenotypic variance were explained by marker class variation at significant marker loci for many traits. The RIL population was effective in detecting and discriminating among QTL for these traits previously identified in other investigations despite skewed segregation ratios at many marker loci. Large additive effects were measured at significant marker loci. Lower fruit weight, higher soluble solids, and lower seed weight were generally associated with RFLP alleles from theL. cheesmanii parent.

Genetic variation and selection response in model breeding populations of Brassica rapa following a diversity bottleneck.

Domestication and breeding share a common feature of population bottlenecks followed by significant genetic gain. To date, no crop models for investigating the evolution of genetic variance, selection response, and population diversity following bottlenecks have been developed. We developed a model artificial selection system in the laboratory using rapid-cycling Brassica rapa. Responses to 10 cycles of recurrent selection for cotyledon size were compared across a broad population founded with 200 individuals, three bottleneck populations initiated with two individuals each, and unselected controls. Additive genetic variance and heritability were significantly larger in the bottleneck populations prior to selection and this corresponded to a heightened response of bottleneck populations during the first three cycles. However, the overall response was ultimately greater and more sustained in the broad population. AFLP marker analyses revealed the pattern and extent of population subdivision were unaffected by a bottleneck even though the diversity retained in a selection population was significantly limited. Rapid gain in genetically more uniform bottlenecked populations, particularly in the short term, may offer an explanation for why domesticators and breeders have realized significant selection progress over relatively short time periods.

Genetic and phenological variation of tocochromanol (vitamin E) content in wild (Daucus carota L. var. carota) and domesticated carrot (D. carota L. var. sativa).

Carrot roots (Daucus carota L. var. sativa) produce tocochromanol compounds, collectively known as vitamin E. However, little is known about their types and amounts. Here we determined the range and variation in types and amounts of tocochromanols in a variety of cultivated carrot accessions throughout carrot postharvest storage and reproductive stages and in wild-type roots (Daucus carota L. var. carota). Of eight possible tocochromanol compounds, we detected and quantified α-, and the combined peak for β- and γ- forms of tocopherols and tocotrienols. Significant variation in amounts of tocochromanol compounds was observed across accessions and over time. Large increases in α-tocopherol were noted during both reproductive growth and the postharvest stages. The variation of tocochromanols in carrot root tissue provides useful information for future research seeking to understand the role of these compounds in carrot root tissue or to breed varieties with increased levels of these compounds.

Temporal aspects of onion-induced antiplatelet activity.

Organosulfur compounds in onion extracts are formed following thelysis of the S-alk(en)yl-L-cysteine sulfoxides by alliinase. Thesecompounds inhibit the aggregation of human blood platelets and offer thepotential for positive cardiovascular health benefits. An experiment wasdesigned to examine temporal and temperature effects on onion-inducedantiplatelet activity. Platelet aggregation is induced by various agonists,including ADP, collagen, and thrombin. Unexpectedly, all freshly-juicedonion extracts (ca. 5 minutes post-juicing) appeared to exhibit both anagonist-free aggregation peak (AFP) and a platelet inhibitory peak (PIP)characteristic of inhibition of platelet aggregation. The AFP was minimalby 30 minutes and dissipated in all treatments by 120 minutes, whilethe PIP increased as onion extracts aged and did not change after 30minutes at 25 °C. This finding confirms the observation that the in vitro platelet inhibitory activity of onion organosulfur compounds istime dependent. Freshly-prepared onion extracts were incubated with theADP scavenger enzyme apyrase (E.C. 3.6.1.5). AFPs were abolished inapyrase-treated extracts, suggesting that this response may have been dueto free ADP in onion extracts, although an amount of ADP required togenerate such a response would be unexpected in onion extracts. Inaddition, platelet aggregates were not observed in the AFP, suggesting thisresponse may be associated with changes in light transmission throughplatelet rich plasma that are not associated with platelet aggregation.Artifacts of analysis are, therefore, possible when assessing onion-inducedantiplatelet activity with freshly-juiced extracts. Temporal formation ofplatelet-inhibiting organosulfur compounds should be taken into accountduring both in vitro and in vivo assessment of onion-inducedantiplatelet activity.

Assessment of RAPD marker frequencies over cycles of recurrent selection for pigment concentration and percent solids in red beet (Beta vulgaris L.)

In the past twenty years, betalain pigments found in red beet (Beta vulgaris L.) have been adopted for use as natural red food colorings. Utility and extractability of these compounds is dependent upon native concentrations of both pigment and total dissolved solids. In an effort to develop red beet populations with elevated levels of betalain pigment, recurrent half-sib family selection for high pigment and both high and low solids was practiced for seven cycles. This scheme resulted in the development of a high pigment/high solids and a high pigment/low solids population. A total of 200 randomly chosen oligonucleotide decamer primers were used to amplify genomic DNA of individual plants in the selection scheme for repeatability and clarity of amplification products. Thirty-one primers were chosen on this basis and used to assess random-amplified polymorphic DNA (RAPD) marker frequencies on genomic DNA samples isolated from 47 randomly-chosen individual plants in each of cycles 1, 3, and 6 in both high pigment/high solids and high pigment/low solids. Number of scorable Polymerase Chain Reaction products ranged from 1–10, resulting in a total of 161 RAPD markers. Chi-square and regression analyses were performed to determine the presence/absence of a linear trend in marker frequencies during the selection scheme. Comparisons were made among cycles within high pigment/high solids and high pigment/low solids populations. Significant linear trends were detected in both cases for certain RAPD markers. Formulae were used to test whether observed linear marker frequency trends were due to the effects of selection or random genetic drift. Chi-square tests revealed a subset of markers which exhibited significant frequency changes across cycles changed due to selection. These data demonstrate changes in RAPD marker frequencies with recurrent selection and suggest linkage of RAPD markers to genes controlling pigment in red beet.

Linkage among the R, Y and BI loci in table beet

Abstract The primary pigments in table beet are the betalains, which are comprised of the red-violet betacyanins and the yellow betaxanthins. The presence of dominant alleles at two linked loci (R and Y) condition the qualitative production of betalain pigment in the beet plant. Red-pigmented roots are observed only in the presence of dominant alleles at both the R and Y loci, while white roots are conditioned by recessive alleles at the Y locus, and yellow roots by the genotype rrY-. A newly described gene ’blotchy’ (bl) conditions a blotchy or irregular pigment patterning in either red or yellow roots. The objective of the present investigation was to characterize the linkage relationships between the R and Y lociand the bl gene by evaluating segregating progenies developed from a series of matings of colored and white table beets. Due to epistatic interactions among the R, Y, and bl loci, algorithms for estimating linkage were developed using maximum-likelihood estimators for each cross. The two-point linkage estimate between the R and Y loci pooled over eight crosses was 7.4±1.7 cM. Segregation data indicated the bl gene is linked to the R and Y loci.The recombination fraction between R and bl was estimated from a pooled sample of four crosses at 16.7±10.8 cM. The most-likely gene order was R-Y-bl. These data demonstrate that the bl gene is a third locus conditioning betalain pigment production in table beet. The R-Y-bl genomic region is therefore important in the genetic control of betalain biosynthesis in table beet.

Molecular breeding of healthy vegetables

In September 2010, a group of carrot growers in the USA announced a US