Browsing by Author "Craig Yencho, Committee Co-Chair"

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    AFLP-based Genetic Diversity Assessment of Global Sweetpotato (Ipomoea batatas (L.) Lam.) Germplasm Resources: Progress Toward The Development of a Sweetpotato Core Collection.
    (2004-11-15) Bruckner, Adam Wesley; Jenny Xiang, Committee Member; Craig Yencho, Committee Co-Chair; Bryon Sosinski, Committee Co-Chair
    Sweetpotato (Ipomoea batatas (L.) Lam) is an extremely diverse and important crop ranking seventh in terms of global production. It is grown throughout the world as a staple crop, and is particularly important in tropical developing countries due to its wide adaptability as well as its enormous potential for preventing malnutrition. The International Potato Center (CIP) in Lima, Peru and the USDA-ARS Plant Genetic Resources Conservation unit (PGRCU) in Griffin, Georgia are the most important sweetpotato gene banks worldwide. Roughly 7,000 and 750 accessions are present in each, respectively. Both collections have been characterized for many phenotypic traits of economic importance, and they are very important in terms of germplasm conservation, as they represent a vital resource for breeders seeking future genetic improvement of sweetpotato. The molecular genetic diversity present in each collection, however, is poorly understood. In this study, 775 accessions from the PGRCU collected from various countries were sampled and fingerprinted using AFLP markers derived from 10 primer pair combinations. A total of 183 polymorphic and clearly scorable bands were generated. Using polymorphisms and phylogenetic software (PAUP and NTSysPC), we were able to clearly distinguish a different banding pattern for each of the 775 accessions. A Neighbor-Joining phylogram was constructed and revealed 6 base clades with no region specificity. Analysis of molecular variance (AMOVA) was carried out using the software program Arlequin. The within-region variation at 97.94% was the major source of molecular variance. Pairwise genetic distances between regions were calculated and the smallest distance was between the Caribbean and Central America and the greatest distance was between the Pacific Islands and South America. Principle coordinate analysis (PCO) was performed by NTSysPC and several clusters of germplasm were noted. This research indicates that there is an abundance of genetic diversity present in sweetpotato.
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    Development of a Genetic Linkage Map and QTL Analysis in Sweetpotato
    (2007-08-01) Cervantes-Flores, Jim Carlos; Bryon Sosinski, Committee Co-Chair; Craig Yencho, Committee Co-Chair; William Thompson, Committee Member; Zhao-Bang Zeng, Committee Member
    Despite its worldwide importance, sweetpotato [Ipomoea batatas (L.) Lam] genetic research lags behind that of other major crops. To address this issue, we developed a genetic linkage mapping population consisting of 240 individuals of a cross between 'Beauregard' (a leading orange-fleshed US sweetpotato cultivar) and 'Tanzania' (a cream-fleshed African landrace). A total of 1751 ('Beauregard') and 1944 ('Tanzania') AFLP markers were located and framework maps consisting of 726 and 947 single-dose AFLP markers were ordered into 90 and 86 linkage groups for 'Beauregard' and 'Tanzania', respectively. Multiple-dose markers were used to identify homologous groups, allowing the assignment of the linkage groups to 15 homologous groups for each parental map. Only few linkage groups remained unassigned. The mapping population was phenotyped and quantitative trait loci (QTL) identified for important traits using single-point and interval mapping procedures. Resistance to root-knot nematode [Meloidogyne incognita (Kofoid & White) Chitwood], a worldwide pest, in the progeny was assessed. QTL analyses revealed seven significant QTL in 'Tanzania' and two in 'Beauregard'. In 'Tanzania', QTL with negative and positive effects on resistance were observed, each explaining approximately 20% of the observed variation. In 'Beauregard', two QTL that increased resistance explained approximately 6% of the observed variation. Based on molecular and phenotypic data, RKN resistance is hypothesized to be conferred by several genes, but at least nine loci (7 from 'Tanzania' and 2 from 'Beauregard') had the biggest effect. Genetic analyses of dry-matter, starch, and beta-carotene content and storage root yield were also conducted. Several QTL ranging from 8 (beta-carotene) to 18 (yield) were identified that affected these traits, and their importance in their inheritance is discussed. Strong correlations were observed between carotene and starch content in the storage roots, and also between starch and dry-matter content. To our knowledge, this is the most complete genetic linkage map of sweetpotato available, and we report the first QTL mapping studies in sweetpotato for storage root quality traits. We anticipate that this research will have a great impact for the sweetpotato breeding community, providing a foundation for the use of molecular techniques in sweetpotato breeding.
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    Genetic, Genomic, and Transgenic Approaches to Understand Internal Heat Necrosis in Potato.
    (2009-08-12) McCord, Per Hilding; Craig Yencho, Committee Co-Chair
    MCCORD, PER HILDING. Genetic, Genomic, and Transgenic Approaches to Understand Internal Heat Necrosis in Potato. (Under the direction of G. Craig Yencho and Bryon R. Sosinski.) Internal heat necrosis (IHN) is a physiological disorder of potato tubers. A multifaceted approach was followed to better understand IHN. This approach entailed three major projects, involving genetic, genomic, and transgenic techniques. The genetics approach involved developing linkage maps of tetraploid potato using AFLP and SSR markers, and identifying QTL for IHN. Our mapping population consisted of 160 individuals from a cross between ‘Atlantic’, an IHN-susceptible cultivar, and B1829-5, an IHN-resistant breeding line. Phenotypic data indicated that the distribution of IHN is highly skewed toward resistance. Early foliage maturity was positively correlated with reduced IHN. QTL for resistance to IHN were located in multiple years on chromosomes IV, V, and group VII of ‘Atlantic’, and on group VII of B1829-5. The QTL explained between 6.7 and 40.9 percent of the variation for mean severity, and from 4.9 to 32.5 percent of the variation for percent IHN incidence. Two IHN-linked markers from chromosomes I and V were also associated with IHN in a secondary mapping population. The correlation between maturity and IHN may be partially explained by loosely linked QTL on chromosomes II and V. QTL were also detected in this population for tuber dry matter content, specific gravity, skin texture, yield, and flower color. The genomic approach entailed a microarray-based gene expression analysis of ‘Atlantic’ potatoes grown under normal and high temperatures in a growth chamber. Although overall levels of IHN were not significantly different between treatments, IHN symptoms increased more rapidly under high temperature. Mixed model analysis identified 17 genes whose expression was significantly different between high and normal temperatures at the first and later harvest dates. Expression levels of four of these genes were tested via quantitative RT-PCR (qRT-PCR). Confirmation of expression was obtained for one gene, encoding a heat shock protein, under all experimental conditions. Testing of these genes using field-grown potatoes showed no differences between clean and IHN-affected tubers; this may have been due to cold storage of the tubers prior to RNA extraction. Tuber calcium levels may be involved in the expression of IHN, and the transgenic project was designed to test this association. ‘Atlantic’ was transformed via Agrobacterium tumefaciens with a construct containing a maize calreticulin-derived calcium binding peptide (CBP) shown to elevate calcium levels in Arabidopsis. Plants were grown under mild heat stress conditions in walk-in growth chambers. Transgenic lines generally had higher yields than wild-type plants. Levels of IHN in transgenic tubers were equal to or higher than wild-type tubers. Quantitative RT-PCR of CBP showed no significant differences between transgenic lines, suggesting that differing protein abundance underlies phenotypic differences between transgenic lines. Two native calreticulin genes were assayed by qRT-PCR for evidence of silencing, but none was found. Nutrient analysis of calcium and 11 other nutrients was also performed. Calcium levels in leaves of two transgenic lines were higher than wild-type ‘Atlantic’. Leaf levels of magnesium, manganese, sulfur, and sodium were also higher than ‘Atlantic’ in at least one line. Most of these minerals are involved in photosynthesis; increased amounts in leaves could be responsible for the yield increase. In tubers, mineral levels were not significantly different. It is possible that expression of CBP in tubers is much lower than in leaves, which could explain the failure to detect any mineral differences. The increase in IHN could be due to the interruption of a signal from the leaves, or a subtle effect on tuber minerals that would require larger sample sizes to detect.
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    Sinks Above and Sinks Below - Physical Mapping of Fruit-derived ESTs in Peach, a Model Species for Prunus and Rosaceae Structural Genomics, and Identification of Amplified Fragment Length Polymorphism (AFLP) Markers Associated with Internal Heat Necrosis (IHN) in 4x-2x Solanum tuberosum x S. phureja-S. stenotomum hybrids
    (2005-07-26) McCord, Per Hilding; Craig Yencho, Committee Co-Chair; Bryon Sosinski, Committee Co-Chair; Rebecca Boston, Committee Member
    Peach (Prunus persica (L.) Batsch) has been proposed as a model organism for structural genomics in the family Rosaceae. Several resources have been or are currently being developed to study the structural genomics of the species. A highly saturated genetic linkage map has been developed from an interspecific 'Texas' almond X 'Earligold' peach cross (T X E), and two peach BAC libraries from the cultivars 'Nemared' and 'Lovell' have been generated for the creation of a physical mapping resource. A framework map has been generated by the hybridization of markers from the T X E and other Prunus maps to the 'Nemared' library. In addition, peach and almond unigenes are being hybridized to the library in order to generate an anchored transcript map, identify contigs for overall map construction, and provide information on genome organization. A subgroup of the Prunus unigene set comprising 942 ESTs derived from peach fruit mesocarp was hybridized to the 44,150-clone 'Nemared' library. Eight hundred ninety-nine of these ESTs successfully hybridized to the library, identifying 3475 BACs, of which 2725 were unique. Seventy-six of these ESTs are now anchored to the reference map by hybridization to genetically anchored BAC clones or contigs. Twenty-six of these ESTs mapped near loci for important agronomic traits, including flesh acidity, fruit skin pubescence, and almond shell hardness. Unanchored ESTs generated contigs of 2 to 44 clones, which are being fingerprinted and integrated into the growing peach physical map in preparation for sequencing of the peach genome. Internal heat necrosis (IHN) is a physiological disorder of potato tubers resulting in the discoloration of parenchymal tissue. Environmental conditions and the large acreage of the IHN-susceptible cultivar 'Atlantic' result in significant losses due to the disorder in the mid-Atlantic states and Florida. A combination of bulked segregant analysis and AFLP marker technology was used to search for molecular markers for IHN in a population of tetraploid 4x-2x S. tuberosum x S. phureja-S. stenotomum (tub x phu-stn) hybrids. These clones are being used to breed an IHN-resistant, high specific-gravity replacement for 'Atlantic'. One marker, potPCR13-HindIII-R, was identified in two small test populations and was strongly associated with resistance to IHN, explaining 69.9% and 64 % of the observed variation for IHN incidence and severity, respectively. This marker showed strong sequence homology to calcium-dependent protein kinases (CDPKs), reinforcing prior evidence of the importance of calcium to the manifestation of IHN. When tested on a different set of clones from a combining ability study for IHN, this marker was no longer significant. However, a second putative marker from the BSA-AFLP screen (potPCR31-A) that was not statistically significant in the small populations, was significantly associated with IHN susceptibility in this second population, and the regression models of potPCR-31-A on IHN severity and incidence explained 17.8% and 18.6% of the phenotypic variation, respectively. This marker showed homology to plastid terminal oxidases (PTOXs), which are involved in desaturation of carotenoids, and may have a role in protecting biomolecules under oxidative stress. The small sample sizes used in marker development and initial testing may have resulted in these markers being significant predictors only in certain populations. It is also possible that, given the quantitative nature of IHN, only a subset of all IHN-associated loci may be necessary for the disorder to be manifest. This research is the first molecular marker study of the genetics of IHN, and has set a foundation for future studies.