Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6173. Adviser: Schuyler S. Korban. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

In the absence of whole genome sequencing, molecular markers are indispensable tools for the study of genome evolution, genetic diversity measurements, and genotype identification. We have used sequence-specific amplified polymorphism (S-SAP) markers that were derived from the BARE-1, an active retrotransposon of barley, to measure the contribution of this element to the evolution of barley genome among 103 cultivars that are commonly grown in Canada and the United States. The results were compared to the genome diversity measures obtained by the single nucleotide polymorphisms (SNP). The barley populations were divided into groups based on various agronomic traits such as end use, i.e., feed versus malting, and seed morphology, i.e., naked versus covered kernel. Analysis of the genetic structure in the population using analysis of molecular variance (AMOVA) for both S-SAP and SNP attributed the largest co-variance component (90%) to the genetic diversity among cultivars within groups. Co-variance component between groups was about 6% which indicated that there was no justification for population differentiation along the set based upon agronomic traits. Genetic relationships among cultivars was assessed by cluster analysis with UPGMA and found to vary substantially between S-SAP and SNP datasets. Quantitative analysis of BARE-1 retrotransposon with real-time PCR in a small group of cultivars showed significant differences in the copy number of the element among cultivars. Most of the BARE-1 elements were in the form of solo LTRs, indicating a high rate of homologous recombination between retrotransposon copies in the genome. Differences of up to 3000 BARE-1 copies per haploid genome were found among cultivars that have been developed and registered within the past three decades. Informative SNPs such as those with high polymorphic information content (PIC) values were used to generate identification keys to distinguish barley cultivars which were otherwise indistinguishable at the morphological and biochemical levels.

In the southwestern United States, rising costs and limited availability of water have lead to irrigation scheduling based on plant stress indicators in an effort to conserve water. This research was conducted to better define the recovery rates of transpiration, stomatal resistance, and leaf water potential in field grown extra long staple cotton (Gossypium barbadense cv. Pima S-6) following varied durations of water stress. Three water stress treatments were maintained by scheduling irrigations at different Crop Water Stress Index (CWSI) values. Plants irrigated at 0.19 and 0.68 CWSI units needed 72 hours for plant water potential to recover, while plants irrigated at 0.41 CWSI units needed only 24 hours. Water stress had a smaller effect on the recoveries of stomatal resistance and transpiration. Stomatal resistance recovered within 24 hours for all plants regardless of water treatment. Transpiration recovered within 24 hours for plants irrigated at 0.19 CWSI units, and within 48 hours for plants irrigated at 0.41 and 0.68 CWSI units.

The effects of subjection of 15-day-old plants of tomato (Lycopersicon esculentum L. 'Contessa') to low night temperature (LNT) of 4°C for up to 21 nights and pre-chilling application of 10% sucrose solution on vegetative, reproductive and yield responses were examined under greenhouse conditions. Chilled seedlings exhibited severe inhibition of growth, with the degree of inhibition increasing with length of chilling. While sucrose treatment (ST) reduced such inhibition and promoted subsequent recovery, it transiently inhibited growth of the unchilled controls. In the long-term, inhibition of growth was observed only for the 0%-sucrose-treated plants cooled for 21 nights. Although LNT lowered the position of the first inflorescence in a direct relation to duration of exposure, this effect was reduced by ST. LNT had no lasting effects on the reproductive and productivity responses of the first two inflorescence (FTI), earliness of flowering, or potential yield and quality. ST promoted flowering of only the 21-day-cooled plants as well as numbers of flowers, fruit set and large fruit responses of FTI. The results indicate that, although early chilling stunts seedling growth, plants may recover at a later favorable temperature without loss to their reproductive capacity or yielding potential.

Cultured tobacco mesophyll protoplasts were used as a model to studying the hormonal control of cell-cycle reactivation in plants. Tobacco mesophyll protoplasts were isolated from leaves in which cell division had ceased, and were then cultured. During the first three days in culture, the protoplasts dedifferentiate, resynthesize the cell wall, express wound-related transcripts, and reinitiate the cell cycle. Reinitiation depends on the hormones auxin and cytokinin. I examined the expression of one or two genes related to different aspects of protoplast development. The expression of following genes was examined: wound or stress response (extensin and PR-1a), cell-wall resynthesis (extensin), cytoskeletal reorganization (actin), and cell-cycle reactivation (cdc2, histones H3, and H4). I found that nuclear division began between 30 and 50 hrs of culture about 10-12 hours after the onset of DNA synthesis. By the third day, 70% of the protoplasts had divided. In freshly isolated protoplasts, histones H3 and H4, and cdc2 were not expressed. Expression of cdc2 was activated by 12 hrs of culture, histone expression was activated by 24 hrs concurrent with the initiation of DNA synthesis. Extensin showed a similar pattern of expression as the histones, but expression of PR-1 was activated more slowly. Actin was expressed in leaves and freshly isolated protoplasts, but its expression increased about 4-fold during protoplast culture. When either auxin or cytokinin was omitted from the media, the protoplasts did not divide and the histones and PR-1 were not expressed. Extensin and actin were expressed in the absence of hormones. Hormone delay studies suggested that auxin was required from the beginning of culture, whereas cytokinin was required before the cells enter S-phase. These results are consistent with auxin and cytokinin acting to induce a G$\sb0$ to G$\sb1$ / transition during protoplast development, possibly through initiation of transcription of cell-cycle required genes such as cdc2 and the histones. However, it appears that cdc2 and histone expression are not the primary target of the hormones because their expression is activated 12 hrs or more after the requirement for auxin has passed. Expression of genes associated with cell wall synthesis (extensin) and wounding (extensin and PR-1) do not appear to be required for reactivation of the cell cycle. / Source: Dissertation Abstracts International, Volume: 55-09, Section: B, page: 3665. / Major Professor: George W. Bates. / Thesis (Ph.D.)--The Florida State University, 1994.

A 712 bp partial cDNA clone (czbp- 1) of the citrus vascular zinc binding protein (CVZBP) was isolated using reverse transcriptase polymerase chain reaction (RT-PCR). The deduced amino acid sequence of czbp-1 was identical to the N-terminal amino acid sequence for the CVZBP. Czbp- 1 had a 549 bp open reading frame and two putative polyadenylation sites, +20 bp and +103 bp relative to the poly-A tail. The deduced amino acid sequence had identity with members of the Kunitz soybean proteinase inhibitor (KSPI) family. Many members of this family are present in high concentrations in storage organs such as seeds and tubers, increase in response to abiotic stress, and are considered defense or stress response proteins. The CVZBP did not appear to fit in this category. Unlike many members of the KSPI family CVZBP was not detected in citrus seeds and protein levels decreased in response to wounding. Transcript also decreased in response to osmotic stress; a similar result previously was reported for CVZBP protein levels. Accumulation of CVZBP and its transcript increased in Zn deficient citrus seedlings compared to those receiving sufficient levels of Zn, indicating that Zn nutrition can modulate CVZBP expression. Recombinant CVZBP was produced and used to determine the capacity of this protein to inhibit several types of proteinases. The CVZBP inhibited the cysteine proteinase, papain, but not the serine proteinases, trypsin and chymotrypsin. CVZBP protein was immunolocalized primarily to the xylem parenchyma in vascular tissue of citrus midribs. Based on these results it is possible that the CVZBP has a function in vascular differentiation. Cysteine proteinases were identified in developing tracheary elements in Zinnia cell cultures. Addition of inhibitors of cysteine proteinase to these cultures prior to secondary cell wall deposition prevents differentiation of the cells into tracheary elements. Perhaps cysteine proteinase inhibitors, such as the CVZBP, in the xylem, contribute to timing of tracheary element differentiation and determination.

Elongation factor 1A is one of the lysine-rich proteins increased in o2 mutants, and its concentration is highly predictive of the protein-bound lysine content of the endosperm. Understanding the biological basis of this relationship could help to explain the mechanisms of lysine accumulation in the endosperm, providing new insights for developing maize genotypes with better nutritional quality. Three different eEF1A isoforms were purified from developing endosperm and investigated in their accumulation, structural and functional activities. The accumulation of the isoforms appears to be developmentally regulated and independent of the o2 mutation. The purified proteins differed in their ability to bind F-actin in vitro, suggesting they are functionally distinct. The isoform that binds actin most effectively was the most predominant in high eEF1A genotypes, which may be related to enhanced cytoskeleton formation, and therefore increased synthesis of cytoskeleton-associated proteins in these genotypes. Tandem mass spectrometry revealed each isoform is composed of the four same gene products, which are modified post-translationally by methylation and phosphorylation. The chemical differences that account for their different actin binding activities could not be determined. Recombinant inbred lines varying in eEF1A content were developed from a cross between a high (Oh51Ao2) and a low (Oh545 o2) eEF1A inbred. The parental inbreds and RILs with the highest and lowest eEF1A content were used to investigate patterns of gene expression and protein synthesis. Transcript profiling with an endosperm EST microarray identified about 110 genes coordinately regulated with eEF1A. These genes encode proteins involved in several biological structures and processes, including the cytoskeleton, the endoplasmic reticulum and the protein synthesis apparatus. The content of alpha-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. Thus, higher levels of eEF1A may be related with a more extensive cytoskeletal network surrounding the rough ER and increased translation of mRNAs encoding cytoskeleton-associated proteins, all of which contribute significantly to the lysine content of the endosperm.

Endoreduplication is a process of genome duplication without mitosis. Although endoreduplication is common among plants and animals, the molecular mechanisms involved with this process are not fully understood. Two strategies were used to determine the genetic components involved with endoreduplication. One, based on QTL analysis and the other strategy involved identifying cell cycle candidate genes, and characterizing them in regard to endoreduplication. To map genes influencing endoreduplication, four backcross populations were created from crosses between a high (Sg18) and a low (Mo17) endoreduplication inbred and their F1 (Sg18 x Mo17) progeny. In all, fourteen quantitative trait loci were identified that affect the degree of endoreduplication in maize endosperm. Six QTLs were mapped with a triploid mode of inheritance in the endosperm. Two QTLs were mapped with parent-of-origin effect inheritance. Six QTLs were mapped using a model that considers genetic interaction between embryo and endosperm. Previous studies with maize endosperm showed that accumulation of a cyclin-dependent kinase (CDK) inhibitor is coincident with the onset of endoreduplication, but the identity of this inhibitor is unknown. We therefore tried to determine if cyclin-dependent kinase inhibitor (CKI) activity is required for the occurrence of endoreduplication in maize endosperm. The expression of two maize CKI genes, Zeama;CKI;1 and Zeama;CKI;2, were characterized in developing endosperm. The accumulation of Zeama;CKI;1 RNA is not developmentally regulated, and its expression encompasses the period in which endoreduplication takes place. In contrast, Zeama;CKI;2 gene expression is developmentally regulated in the endosperm, since its protein level decreases after 13 DAP. Both proteins were able to inhibit the maize Cdc2/CDK kinase activity associated with p13 Suc1. They also specifically inhibited cyclin A1;3/ and cyclin D5;1/-associated CDK activities, but not cyclin B1;3/CDK. Although Zeama;CKI;1 was found to be associated with the endosperm CKI activity, it did not account for all of the CDK inhibitor. Over-expression of ZeamaCKI1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein (RBR) function in the cell cycle, led to an additional round of DNA replication without nuclear division. However, a role for Zeama;CKI;1 in endoreduplication could not be demonstrated in maize endosperm.

The discharge of untreated aquaculture effluent can pollute receiving water bodies. I tested the feasibility of using salt-tolerant plants (halophytes) with potential as forage and oilseed crops, as biofilters to treat saline aquaculture effluent. Plants were grown in draining lysimeters in greenhouses and irrigated with effluent salinized with NaCl. Irrigation water came from a recirculating tilapia culture system. I measured yield potential, water use and capacity for nitrogen and phosphorus uptake. In Experiment 1, Suaeda esteroa, Salicornia bigelovii and Atriplex barclayana (Chenopodiaceae) were grown in sand in 0.02 m³ lysimeters. Plants were irrigated with effluent of 0.5 ppt, 10 ppt and 35 ppt salinity, to meet evapotranspiration demand and to allow 30% of the applied water to leach past the plant root zone. Despite the high leaching fraction and short residence time of water in the pots, the plant-soil system removed 98% and 94% of the applied total and inorganic nitrogen, respectively, and 99% and 97% of the applied total and soluble reactive phosphorus respectively. For all species, salt inhibited (P ≤ 0.05) the growth rate, nutrient removal, and volume of water the plants could process. The salt marsh species S. esteroa and S. bigelovii performed better than the desert saltbush, A. barclayana, at 35 ppt. In Experiment 2, Suaeda esteroa, was grown in lysimeters containing approximately 0.8 m³ sandy loam soil and irrigated three times per week with 31 ppt NaCl effluent. I used five irrigation treatments, ranging in volume from 50 to 250% of the potential evaporation rate. Plant biomass and water consumption increased significantly (P ≤ 0.05) with increasing irrigation volume. Nitrate concentrations in water draining from the lysimeters decreased during the experiment, and were significantly lower in the high-volume treatments than in the low-volume treatments. Phosphorus concentrations in the leachate water increased during the experiment as a function of increasing irrigation volume. Irrigating halophyte crops with aquaculture wastewater of seawater-salinity may be a viable strategy for disposal of effluent.

Collaborative plant breeding (CPB) is an approach to crop improvement incorporating close attention to local biophysical and sociocultural environments and interaction between farmers and plant breeders. CPB may have particular potential for improvement in highly stress-prone environments and for low-resource, traditionally based agricultural communities, situations where more conventional approaches have not been effective. However, CPB will require methodological adjustments or innovations relevant to the smaller scale of its target area and its participatory approach. This study investigated methodologies useful to CPB, working with maize farmers from two communities in the Central Valleys of Oaxaca, Mexico. A method for rapid estimation of broad sense heritability (H) was applied in farmers' maize fields. H estimates for morphophenological traits were compared with narrow sense heritability (h2) from half sib family analysis of five of the same populations and with published estimates. Absolute values of H were larger than h2 from this study and the literature, however trait ranking was the same as in the literature, but differed from h2 rankings from this study. With an understanding of their limitations, these rapid, economical estimations provide useful information for CPB work on-farm, where empirical information is frequently lacking. Collegial interaction based on the knowledge and skills of farmers and breeders will depend upon understanding those in terms relevant to each group. Methods from social and biological sciences were integrated to understand selection and its consequences from farmers' perspectives but based on concepts used by plant breeders. Information was elicited regarding farmers' perceptions of their maize populations, growing environments and expectations for response to selection. Farmers' decisions about varietal repertoires imply assessments of local genetic and environmental variation. Traits of high and low heritability are distinguished, as reflected in expected selection response. Farmers' selection practices were not always effective yet they understood the reasons for this and had no expectations for selection response in some traits given the methods available. Farmers' statements, practices and perceptions regarding selection and the genetic response of their maize populations to their selection indicate selection objectives different than may be typically assumed, suggesting a role for breeder and farmer collaboration.