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Use of RAPD for the study of diversity within plant germplasm collections.

As part of the development of a molecular toolkit for the study of diversity within large plant germplasm collections, RAPD technology has been applied to accessions of rice (Oryza sativa) obtained from the major world collection held at IRRI (the International Rice Research Institute) which supplies germplasm to breeders. Methods for the speedy extraction of DNA representative of a rice accession, its amplification by PCR to reveal reproducible products, and the analysis of the banding data using numerical techniques have been established. The biological meaningfulness of RAPD data has also been demonstrated by reference to previous work on classification and crossability.

Base Sequence↗

Molecular mobility in the cytoplasm: an approach to describe and predict lifespan of dry germplasm.

Molecular mobility is increasingly considered a key factor influencing storage stability of biomolecular substances, because it is thought to control the rate of detrimental reactions responsible for reducing the shelf life of, for instance, pharmaceuticals, food, and germplasm. We investigated the relationship between aging rates of germplasm and the rotational motion of a polar spin probe in the cytoplasm under different storage conditions using saturation transfer electron paramagnetic resonance spectroscopy. Rotational motion of the spin probe in the cytoplasm of seed and pollen of various plant species changed as a function of moisture content and temperature in a manner similar to aging rates or longevity. A linear relationship was established between the logarithms of rotational motion and aging rates or longevity. This linearity suggests that detrimental aging rates are associated with molecular mobility in the cytoplasm. By measuring the rotational correlation times at low temperatures at which experimental determination of longevity is practically impossible, this linearity enabled us to predict vigor loss or longevity. At subzero temperatures, moisture contents for maximum life span were predicted to be higher than those hitherto used in genebanks, urging for a reexamination of seed storage protocols.

Cytoplasm↗

Neutral genetic markers and conservation genetics: simulated germplasm collections.

This study examines the use of neutral genetic markers to guide sampling from a large germplasm collection with the objective of establishing from it a smaller, but genetically representative sample. We simulated evolutionary change and germplasm sampling in a subdivided population of a diploid hermaphrodite annual plant to create an initially large collection. Several strategies of sampling from this collection were then compared. Our results show that a strategy based on information obtained from marker genes led to retention of the maximum number of neutral and nonneutral alleles in the smaller sample. This occurred when demes were composed of self-fertilizing individuals or when no migration occurred among demes, but not when demes of an outcrossing population were connected by high levels of migration.

Algorithms↗

Buffalograss germplasm resistance to Blissus occiduus (Hemiptera: Lygaeidae).

Plant germplasm collections may offer genetic variability useful in identifying insect resistance. The goal of this project was to evaluate buffalograss genotypes [Buchloë dactyloides (Nutt.) Engelm.] for resistance to the chinch bug, Blissus occiduus Barber (Hemiptera: Lygaeidae), and to relate resistance to ploidy level, chinch bug number, and pubescence. Forty-eight buffalograss genotypes from diverse geographic locations were evaluated in replicated studies under greenhouse conditions. Of the genotypes studied, four were highly resistant, 22 were moderately resistant, 19 were moderately susceptible, and three were highly susceptible to chinch bug damage. The mean number of chinch bugs was significantly different among the 48 genotypes. There was no significant correlation between chinch bug resistance and ploidy level or chinch bug resistance and pubescence. These results indicate the genetic source of resistance to chinch bugs exists in buffalograss germplasm. Highly resistant genotypes can be used in breeding programs to further improve buffalograss cultivars.

Animals↗

The National Animal Germplasm Program: challenges and opportunities for poultry genetic resources.

In the United States, poultry genetic resources have consolidated because of economic pressures. Such consolidations can potentially jeopardize the poultry industry and the ability of research communities to respond to future challenges. To address the loss of genetic resources for all livestock and aquatic species, USDA established the National Animal Germplasm Program (NAGP) in 1999. Since the initiation of NAGP, population surveys have been conducted on nonindustrial chicken and turkey breeds. These surveys not only provide insight into breed status, but also serve as a benchmark for future comparisons. The survey results revealed that 20 chicken breeds and 9 turkey breeds were in various stages of being lost. The NAGP has initiated an ex situ repository for cryopreserved germplasm and tissue that already contains 59 chicken lines and 2,915 tissue samples. As the NAGP, along with its industry and university partners, continues developing the ex situ collection, there are research opportunities in cryopreserved tissue utilization and studies of genetic diversity. For cryopreserved tissues, several key research areas include improving the cryopreservation protocols for rooster and tom semen by using cryoprotectants other than glycerol and utilizing embryonic cells. Although surveys have been conducted on public research lines and rare breeds, there is a void in understanding the level of genetic diversity present in U.S. poultry populations. Therefore, an opportunity exists to perform a series of genetic diversity studies using molecular- based approaches. Such an evaluation can help clarify population differences between research lines and rare breeds and, thereby, facilitate conservation strategies. There appears to be growing consumer interest in poultry products derived from heritage breeds and/or poultry raised in nonindustrial production systems. Although the depth of such market trends is unknown, such an interest may provide an important niche for rare poultry breeds and, thereby, strengthen the genetic base.

Agriculture↗

Variability, heritability and genetic advance in pheno-morphic and agronomic traits of tef [Eragrostis tef (Zucc.) Trotter] germplasm from eight regions of Ethiopia.

One hundred and twenty tef Eragrostis tef (Zucc.) Trotter, germplasm lines from eight regions of Ethiopia were field evaluated in two randomized complete blocks at Debre Zeit and Melkassa during the 1995/96, and at Debre Zeit and Alem Tena during the 1996/97 main seasons. The objectives were to assess the extent and pattern of their diversity with respect to 17 pheno-morphic and agronomic traits, and to obtain an insight into the broad sense heritability (H) and genetic advance (GA) expectations of the various traits. Discernible (p < or = 0.05) variability among regions and among genotypes within regions were observed in seven and nine of the traits, respectively. In the combined analysis of variance over environments, genotypes showed substantial (p < or = 0.05) variation in all traits, and genotypes and environments interacted significantly on nine of the traits. The phenotypic and genotypic coefficients of variation varied in that order from about 3% for days to maturity to 28% for grain yield/plant, and from less than 2% for days to maturity to 15% for number of fertile tillers/plant. Estimates of H were lowest for shoot phytomass/plant (17%) and highest for panicle length (75%). The minimum GA expectations were for days to maturity (2%) and the maximum was for number of fertile tillers/plant (23%). About 67% of the gross phenotypic diversity in the germplasm was explained on the basis of four principal components (PCs). Up to 28% of the variance extracted by the first PC was due mainly to variations in the second and first basal culm internode diameters, culm and panicle length, days to panicle emergence, and number of panicle branches and fertile tillers/plant. Largely variations in the length of the two basal culm internodes and grain yield/plant contributed to 19% of the whole variance accounted for by the second PC. Generally, the study showed that there is ample genetic variation in the genotypes evaluated and this presents a valuable base for use in the improvement work.

Agriculture↗

Simple sequence repeat analysis of a clonally propagated species: a tool for managing a grape germplasm collection.

The USDA germplasm repositories help to preserve the genetic variability of important crop species by collecting and maintaining representative cultivars and related germplasm. Simple sequence repeat markers with high allelic diversity were used to type 41 grapevines from 40 accessions. All vines were either seedless table grape cultivars or cultivars with names similar to table grape cultivars. The proportion of shared alleles was selected as the most appropriate statistical measure of genetic distance for this population. In conjunction with morphological traits, known synonyms were confirmed and a previously unknown synonym was discovered. An alleged synonym in the literature was disproved by the DNA data. The data were consistent with known parentage, where such data were available. Two mislabeled vines in the USDA collection were identified. UPGMA grouped the cultivars loosely into three groups: a group of nine mostly Middle Eastern cultivars, a group of 22 accessions mostly from Russia and Afghanistan that were morphologically similar to 'Thompson Seedless', and a third very loose group of 11 accessions consisting mostly of eastern European wine grape cultivars. The limitations and usefulness of this type of analysis are discussed.

Alleles↗

Genetic diversity in Bambara groundnut (Vigna subterranea L.) germplasm revealed by RAPD markers.

Random amplified polymorphic DNA (RAPD) markers were used to assess genetic diversity in Bambara groundnut (Vigna subterranea L.) germplasm using 25 African accessions from the collection in the International Institute for Tropical Agriculture, Ibadan, Nigeria. Fifty random decamer primers were screened to assess their ability to detect polymorphism in bambara; 17 of them were selected for this study. Considerable genetic diversity was found among the V. subterranea accessions studied. The relationships among the 25 accessions were studied by cluster analysis. The dendrograms showed two main groups of accessions mainly along the lines of their geographic origin. It is concluded that RAPD can be used for germplasm classification in bambara groundnut and hence for improving this crop.

Africa↗

Evaluation of AFLPs for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersicon esculentum L.).

Cultivated tomato (L. esculentum L.) germplasm exhibits limited genetic variation compared with wild Lycopersicon species. Amplified fragment length polymorphism (AFLP) markers were used to evaluate genetic variation among 74 cultivars, primarily from California, and to fingerprint germplasm to determine if cultivar-specific patterns could be obtained. All 74 cultivars were genotyped using 26 AFLP primer combinations; of the 1092 bands scored, 102 AFLP bands (9.3%) were polymorphic. Pair-wise genetic similarity coefficients (Jaccard and Nei-Li) were calculated. Jaccard coefficients varied from 0.16 to 0.98 among cultivar pairs, and 72% of pair-wise comparisons exceeded 0.5. UPGMA (unweighted pair-group method with arithmetic averaging) clustering and principle component analysis revealed four main clusters, I-IV; most modern hybrid cultivars grouped in II, whereas most vintage cultivars grouped in I. Clusters III and IV contained three and two cultivars, respectively. Some groups of cultivars closely related by pedigree exhibited high bootstrap values, but lower values (<50%) were obtained for cluster II and its four subgroups. Unique fingerprints for all 74 cultivars were obtained by a minimum of seven AFLP primer pairs, despite inclusion of some closely related cultivars. This study demonstrated that AFLP markers are effective for obtaining unique fingerprints of, and assessing genetic diversity among, tomato cultivars.

Base Sequence↗

Potential of trispecies bridge crosses and random amplified polymorphic DNA markers for introgression of Medicago daghestanica and M. pironae germplasm into alfalfa (M. sativa).

This report describes the production and cytology of the first interspecific hybrids between cultivated alfalfa (Medicago sativa L.) at the diploid level (2n = x = 16) and the diploid (2n = 2x = 16) perennial species M. daghestanica and M. pironae. An ovule-embryo culture technique was required to rescue hybrid embryos and all hybrids were diploid. Predominately bivalent chromosome pairing was observed at meiotic metaphase. All F1 hybrids were male and female sterile and no species backcross progeny could be produced. We discovered that trispecies hybrids could be efficiently recovered via crossing diploid F1 interspecific hybrids of M. sativa x M. rupestris with either M. daghestanica or M. pironae. Ovule-embryo culture was also required to recover these trispecies hybrids with recovery efficiency of trispecies hybrids about 10 times greater than for bispecies hybrids. Most chromosomes paired as bivalents in the trispecies hybrids. Importantly, progeny can be recovered from crossing the trispecies hybrids with M. sativa. Therefore, the M. sativa x M. rupestris hybrids provide a bridge cross to potential introgression of M. daghestanica or M. pironae germplasm. Analysis of randomly amplified polymorphic DNA (RAPD) markers in the trispecies hybrids indicates that RAPD markers offer considerable potential for assaying germplasm introgression following complex hybridization of the type reported here.

Base Sequence↗

Development of a low-coverage whole genome sequencing screen for apomixis using a diverse set of Malus germplasm.

In the past decade, plant biologists have made several major discoveries pertaining to the genetic basis of apomixis (clonal propagation by seed) that have shown promise in preserving high-value hybrid rice and sorghum genotypes. This progress was made possible by foundational gene discovery efforts in model species and natural apomicts, but pleiotropic obstacles still limit its broad agricultural adoption, especially in eudicots. Thus, it follows that investigations of novel apomicts should lead to the development of new molecular tools for plant breeding. The two most common ways to identify clonal seed production are flow-cytometry seed screens and genome sequencing to compare the DNA sequences of the maternal parent and progeny, traditionally using low-throughput markers. While flow-cytometry has been the dominant method for more than two decades, it provides indirect information on the genetics of a resulting embryo and can be ineffective in certain species. Here we developed a method using short-read whole-genome sequencing at moderately low coverage (averaging 3X and 6X) to screen diverse Malus genotypes maintained in a USDA germplasm collection for clonal seed production. In total, we sequenced 55 genotypes, 1,216 of their embryos, and identified 17 previously undescribed apomictic genotypes. Several more were detected with the flow cytometry seed screen, which helped resolve certain types of reproduction and sources of noise in low-coverage datasets. This low-pass screening-by-sequencing method is a relatively low-cost, rapid method for detecting apomictic genotypes in diverse plant germplasm and when used thoughtfully in conjunction with flow cytometry, provides a new way to visualize the genetic outcomes of sexual and asexual reproduction in plants.

Apomixis↗

Allele mining for stress tolerance genes in Oryza species and related germplasm.

Allele mining exploits the deoxyribonucleic acid (DNA) sequence of one genotype to isolate useful alleles from related genotypes. The international project to sequence the genome of Oryza sativa L cv. Nipponbare will make allele mining possible for all genes of rice and possibly related cereals. We used a rice calmodulin gene, a rice gene encoding a late embryogenesis-associated protein, and salt-inducible rice gene to optimize the polymerase chain reaction (PCR) for allele mining of stress tolerance genes on identified accessions of rice and related germplasm. Two sets of PCR primers were designed for each gene. Primers based on the 5' and 3' untranslated region of genes were found to be sufficiently conserved so as to be effective over the entire range of germplasm in rice for which the concept of allelism is applicable. However, the primers based on the adjacent amino (N) and carboxy (C) termini amplify additional loci.

3' Untranslated Regions↗

Germplasm Ownership: Related Corn Inbreds.

Corn (Zea mays L.) germplasm ownership methods are unwieldy and limit progress. Agronomic performance in hybrids better identifies inbreds for distinctness, i.e., independent varieties (IVs) or for dependency, i.e. essentially derived varieties (EDVs) than do molecular methods alone. Our objectives were to assess hybrid agronomic performance of some popular, closely related inbreds on the basis of molecular methods, and to offer thoughts on developing inbreds and on germplasm ownership. Significant agronomic differences were detected between the following: two hybrids in the same genetic group on the basis of 21 isozymic loci; two closely related inbreds (B73 and LH119) with 88.6% (62/70) RFLP (restriction fragment length polymorphism) similarity; and two closely related inbreds (Mo17 and LH51) with 88.2% (60/68) RFLP similarity. We suggest raising dependency standards for patenting inbreds to 90% or more, allowing the right to use and also paying a small royalty for only 5 yr on EDVs to the owner of the IV, and maintaining a research exemption to provide elite inbreds for developing new inbreds and experimental hybrids to all breeders. This will help maintain genetic gain for corn grain yield and serve the common good.

Journal Article↗

DNA fingerprinting of jute germplasm by RAPD.

The genotype characteristic of cultivars was investigated, along with varieties of both of the jute species, Corchorus olitorius and Corchorus capsularis, in the germplasm collection at the Bangladesh Jute Research Institute (BJRI). DNA fingerprinting was generated for 9 different varieties and 12 accessions of jute cultivars by using random amplified polymorphic DNA (RAPD). A total of 29 arbitrary oligonucleotide primers were screened. Seven primers gave polymorphism within the varieties, and 6 primers detected polymorphism within the accessions that were tested. A dendrogram was engendered from these data, and this gave a distinct clustering of the cultivated species of jute. Therefore, we generated RAPD markers, which are species-specific. These primers can distinguish between C. olitorius and C. capsularis. From the dendrogram that we generated between the various members of these two species, we found the existing genetic classification that agrees with our molecular marking data. A different dendrogram showed that jute accessions could be clustered into three groups. These data will be invaluable in the conservation and utilization of the genetic pool in the germplasm collection.

Base Sequence↗

Effective Population Size during Grass Germplasm Seed Regeneration.

Effective population size (N(e)) is the key parameter for predicting genetic drift associated with germplasm regeneration. A major factor reducing N(e) below the census population size (N(c)) is variation in seed production among plants in a given population. The objectives of this study were to estimate N(e)/N(c) associated with variation in seed production in three model wind pollinated, perennial grass species [Lolium perenne L., Festuca pratensis Huds., and Pseudoroegneria spicata (Pursh) Á. Löve] and to recommend cost effective sampling methodology to maximize N(e)/N(c) during seed regeneration. Three accessions of each species were grown at two field locations and variation in seed number among plants and mean seed production per plant used to estimate N(e)/N(c). Mean seeds per whole plant, standard deviations, and N(e)/N(c) differed among species, and accessions within species (P < 0.05). For whole plant samples, average N(e)/N(c) for each species differed with values of 0.42, 0.51, and 0.63 for L. perenne, F. pratensis, and P. spicata, respectively. However, average N(e)/N(c) based on two inflorescences per plant was 0.69, 0.88, and 0.86 for L. perenne, F. pratensis, and P. spicata, respectively, which was higher than that of whole plant samples. This higher N(e)/N(c) resulted from the elimination of the variation in inflorescence number per plant, a major source of variation in seed number among plants. The results showed the high potential for genetic drift in small regeneration populations. Increased plant populations and harvesting a constant number of inflorescences per plant are recommended as cost-effective methods to minimize genetic drift during regeneration of outcrossing grass germplasm.

Journal Article↗

Analysis of genetic diversity in Chinese sweetpotato [Ipomoea batatas (L.) Lam.] germplasm using DNA amplification fingerprinting.

The genetic diversity and evolutionary relationships in a representative sample of Chinese sweetpotato collection were assessed using the DNA amplification fingerprinting approach. DNA fingerprint profiles were developed for all 42 accessions tested. There were 19-26 bands for each accession and an average of 20.7 such bands were polymorphic. Chinese sweetpotato germplasm tested exhibited a high degree of genetic diversity. Phenetic analysis revealed five major clusters with the following components: (1) landraces from Guangdong Province, (2) landraces from Fujian province, (3) Chinese cultivars, (4) those closely related to Japanese sweetpotato cultivars, and (5) those closely related to the US sweetpotato cv. Nancy Hall. The genetic association observed between accessions was largely consistent with the known pedigree records. The DNA amplification fingerprinting may provide reliable insights into the domestication history of the sweetpotato crop and may be useful in germplasm enhancement.

Base Sequence↗

[Photosynthetic pigment contents of different germplasm of Rehmannia glutinosa and the relationship between pigments and leaf color].

OBJECTIVE: To evaluate the germplasm of Rehmannia glutinosa on the basis of photosynthetic pigment contents (PPC). METHOD: 20 cultivars were planted on the same condition. On Oct. 23 and Sept. 25, 3 leaves per cultivar were collected on different plants, and 80 mg mesophyll was collected among upper lateral veins and was ground in 96% alcohol, and the supernatant was subjected to measure on a spectrophotometer (Angilent 8453). RESULT: The PPCs among cultivars were significantly different at a P < or = 0.01 level. The results of the measurements were similar. Chlolophyll a was the most abundant pigment, but varied to a great extent among different cultivars. 20 cultivars were divided into 9 homogeneous groups according to the contents of chlorophyll a by Duncan's multiple range test at P < or = 0.05. In addition, the content of chlorophyll a was closely related to leaf color. The cultivars with higher chlolophyll a had deep green leaves, and those with lower had yellow green or pale green leaves. CONCLUSION: PPC was an inherent character and an important index for the germplasm evaluation of R. glutinosa.

Chlorophyll↗

[Isozymes variations among the germplasm resources of Houttuynia in Sichuan].

The peroxidase and esterase isozymes of the germplasm resources of Houttuynia in Sichuan were analyzed. The results showed that it existed plentiful isozymes variations among the germplasm resources of Houttuynia in Sichuan. The cultivated populations had the same isozymes band types as the wild ones. There were 6 different types of peroxidase isozymes bands and 10 different esterase isozymes band totally. Each peroxidase band type was composed of 4 to 6 bands, and esterase isozymes had 5 to 8 bands. There was no significant difference between Houttuynia cordata and Houttuynia emeiensis populations in their isozymes bands types. The results also showed that the number of peroxidase isozymes bands in northern areas was somewhat higher than that in southern areas. However, esterase isozymes band types had no geographic differences.

China↗