Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.     

Microsatellite marker-based genetic diversity assessment among exotic and native maize inbred lines of Bangladesh

Hybrid development is basically dependent on the variability among available genetic resources. Polymorphism among the maize inbreds is essentially needed for maize hybridization. This study aimed at the assessment of diversity among 22 maize inbreds by 18 microsatellite markers. The study identified 187 alleles at 18 SSR loci. The amplified allele frequency per microsatellite locus was 10.4 and the highest allele per locus was 17 in SSR primer pair phi026. SSR primer set p-umc1292, phi074 and phi090 showed the lowest 6 alleles per genotype per locus. The locus phi026 showed the highest degree of gene diversity (0.92), and the locus p-umc1292 had the lowest of gene diversity (0.77) with a mean value of 0.862 among the microsatellites. At each site, the most prevalent allele varied between 0.14 (bnlg371) and 0.36. (p-umc1292). At any given locus, an average of 0.22 out of the 22 selected maize inbred lines had a common major allele. The average value of the polymorphic information content (PIC) was 0.85, within the range of 0.74 at the lowest to 0.92 at the highest. The higher PIC values of phi026 and nc013 established them to be the best markers for maize inbred lines. The UPGMA clustering generated seven distinct groups having 12.5% of similarity coefficient. The results revealed that inbred lines E10, E27, E19, E34, E35, E4, E43, E28, E11, E21, E17, E38, E25, E34, E14, E16, E39 and E3 were more diversified. These lines are promising to be used as parent materials for hybrid maize development in the future.     

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties.

Genetic distances (GDs) based on molecular markers are important parameters for identifying essentially derived varieties (EDVs). In this context information about the variability of molecular markers within maize inbred lines is essential. Our objectives were to (1) determine the variation in the size of simple sequence repeat (SSR) fragments among different accessions of maize inbreds and doubled haploid (DH) lines, (2) attribute the observed variation to genetic and marker system-specific sources, and (3) investigate the effect of SSR fragment size differences within maize lines on the GD between maize lines and their consequences for the identification of essentially derived varieties. Two to five accessions from nine inbred lines and five DH lines were taken from different sources or drawn as independent samples from the same seed lot. Each accession was genotyped with 100 SSR markers that evenly covered the whole maize genome. In total, 437 SSR fragments were identified, with a mean of 4.4 alleles per locus. The average polymorphic information content (PIC) was 0.58. GD estimates between two accessions of the same genotype ranged from 0.00 to 0.12 with an average of 0.029 for inbred lines and 0.001 for DH lines. An average of 11.1 SSRs was polymorphic between accessions of the same inbred line due to non-amplification (8.1 SSRs), heterogeneity (4.0 SSRs) or unknown alleles (2.6 SSRs). In contrast to lab errors, heterogeneity contributed considerably to the observed variation for GD. In order to decrease the probability to be suited for infringing an EDV threshold by chance, we recommend to increase the level of homogeneity of inbred lines before applying for plant variety protection.     

Marker-assisted recurrent selection for cumulating additive and interactive QTLs in recombinant inbred lines

A computer program has been designed to manage marker information in recombinant inbred-line populations. The objective is to select pairs of inbred lines (either recombinant-inbred or doubled-haploid) to be intercrossed, in order to accumulate all or most favourable alleles, either with additive effects or with interactive effects. The population size required to have a 95% chance of obtaining the best line from a given cross is computed, taking into account the number of QTLs and the probability that no recombination event occurs in any of the QTL confidence intervals. It is shown that the accuracy of QTL location greatly affects selection efficiency and that a recurrent selection scheme is highly preferable for pyramiding many QTLs. An application to the bread-making quality improvement of wheat is presented. Received: 25 September 1998 / Accepted: 29 July 1999     

Biochemical polymorphic systems in inbred lines of chickens: A survey

Biochemical polymorphic gene frequency profiles from blood samples of two outbred and seven inbred lines of chickens were studied for hemoglobin, albumin, transferrin, alkaline phosphatase, esterase II, and leucine aminopeptidase, and from egg samples of these lines for ovoalbumen, ovoglobulin (G₂ and G₃), and conalbumen. Complete gene fixation was found for hemoglobin, albumin, transferrin, ovalbumin, ovoglobulin, and conalbumin. The same alleles were fixed in each system in each line. For four systems, a particular allele within a system predominated in seven populations; gene frequencies ranged from 0.60 to 0.98. For esterases I and II, the genes Es-I ^B and Es-II ^S ranged in frequency between 0.82 and 0.97, and between 0.64 and 0.93, respectively. For ovoglobulin, G ₂ B ranged between 0.75 and 1.00 with four lines fixed for this allele. The rather remarkable similarity of gene frequency profiles among lines, several of which are only remotely related, suggests that certain characteristic polymorphic frequencies for these biochemical polymorphisms possess higher adaptive values in an evolutionary sense.     

Tissue culture‐induced genomic alteration in maize (Zea mays) inbred lines and F1 hybrids

Genomic alteration is a common phenomenon associated with plant tissue culture, which often encompasses genetic changes and epigenetic modifications (e.g. cytosine methylation). Here, we studied genomic alteration in maize by assessing calli and regenerated plants derived from three inbred lines (M17, J7 and JC) and two pairs of reciprocal F1 hybrids (pair I: M17/J7 and J7/M17 and pair II: M17/JC and JC/M17). By employing two molecular markers, the amplified fragment length polymorphism and methylation‐sensitive amplified polymorphism, we found that both types of genomic alterations occurred in calli and regenerated plants of all the studied maize inbred lines and F1 hybrids, but the extent and pattern of changes varied substantially across the genotypes. Among the three inbred lines, M17 showed markedly higher frequencies of both genetic (from 2.1% to 3.8%) and methylation alterations (from 6.5% to 9.9%, by adding up the various patterns) than the other two lines which showed similar frequencies for both types of alterations (genetic: 0.5–1.8%, methylation: 2.1–3.7%). Of the two F1 hybrid pairs, while pair I showed genetic variation frequencies similar to that of the inbred parent with lower changing frequency and pair II was intermediate of those of the parents, both pairs showed frequencies of methylation alteration more or less intermediate of those of their inbred parental lines. Parent‐of‐origin effects in both genetic and methylation changes were detected in only one of the hybrid pairs (primarily pair II) for a given changing pattern. Statistical testing confirmed the genotypic difference in both genetic and methylation (hypomethylation) alterations among the regenerants. Taken together, it could be concluded that the frequency and pattern of both genetic and cytosine methylation alterations in maize tissue culture were largely genetic context‐dependent traits, but stochasticity also played an important part. F1 hybrids were not significantly more stable than their inbred parental lines under tissue culture conditions.     

Heterosis, sex and maternal interactions in crosses among inbred lines of mice.

All possible crosses and reciprocals were made among four inbred lines (F = 92%) developed from 12 generations of full-sib mating. All lines originated from a common outbred base population of ICR-albino mice. Data were obtained from 356 litter containing 2,734 mice to evaluate heterosis, reciprocal effects, sex effects and their interactions as they affect body weight and weight gain. Heterosis was significant for most of the postweaning traits (42- and 56-day weight and gain from 21 to 42 days). Nonadditive gene action may have included overdominance and epistasis since both reciprocal linecrosses were generally heavier than those of the better inbred lines. Although significant differences in reciprocals and inbred lines were not frequent, there were sufficient differences to indicate that lines varied in the fixation of loci during inbreeding. Sex-heterosis interactions were significant for 12 of 30 possible cases. However, eight of the 12 significant interactions occurred in crosses involving only one of the lines. The interactions were of the divergent type and arose from males exhibiting more heterosis than females. Overdominance in genes on the sex chromosomes modified by other loci (epistasis) was proposed as a possible explanation for these results. Some sex-linkage affecting growth was evident from the interaction of sex with reciprocal effects.     

Quality control genotyping for assessment of genetic identity and purity in diverse tropical maize inbred lines

Quality control (QC) genotyping is an important component in breeding, but to our knowledge there are not well established protocols for its implementation in practical breeding programs. The objectives of our study were to (a) ascertain genetic identity among 2–4 seed sources of the same inbred line, (b) evaluate the extent of genetic homogeneity within inbred lines, and (c) identify a subset of highly informative single-nucleotide polymorphism (SNP) markers for routine and low-cost QC genotyping and suggest guidelines for data interpretation. We used a total of 28 maize inbred lines to study genetic identity among different seed sources by genotyping them with 532 and 1,065 SNPs using the KASPar and GoldenGate platforms, respectively. An additional set of 544 inbred lines was used for studying genetic homogeneity. The proportion of alleles that differed between seed sources of the same inbred line varied from 0.1 to 42.3?%. Seed sources exhibiting high levels of genetic distance are mis-labeled, while those with lower levels of difference are contaminated or still segregating. Genetic homogeneity varied from 68.7 to 100?% with 71.3?% of the inbred lines considered to be homogenous. Based on the data sets obtained for a wide range of sample sizes and diverse genetic backgrounds, we recommended a subset of 50–100 SNPs for routine and low-cost QC genotyping, verified them in a different set of double haploid and inbred lines, and outlined a protocol that could be used to minimize errors in genetic analyses and breeding.     

Sequence variation between alleles reveals two types of copy correction at the 27-kDa zein locus of maize

In many inbred lines of maize, two 27-kDa storage protein (zein) genes are found within tandem duplications of 12 kb. Both genes of the duplicated allele from the maize inbred line A188 were sequenced and compared to a similar duplicated allele in another inbred line, W22, and to a single-copy allele in the inbred line W64A. The comparisons reveal interesting patterns in the distribution of sequence changes between these alleles. Differences between the two duplicated alleles that are conserved between the two genes of each allele are found exclusively in the 5' region. In contrast, differences between the individual genes of each allele in the 3' region are conserved between the two alleles. The first case is indicative of an intraallelic copy correction mechanism, whereas the second may result from interallelic copy correction. These may be mediated by gene conversion processes, as previously described for other multigene families.     

Genetic characterization of biodiversity in highly inbred chicken lines by microsatellite markers

Forty-two microsatellite loci were analysed in 23 highly inbred chicken lines derived from Leghorn, Jungle Fowl, Fayoumi and Spanish breeds. Line-specific alleles among breeds and lines were detected. The band-sharing (BS) values were calculated and the proportion of shared alleles distances (Dps) were estimated. The BS values and Dps between sets of MHC-congenic lines ranged from 0.74 to 0.96, and 0.05-0.35, respectively. The BS values between each pair of noncongenic Leghorn lines were 0.32-0.97, and between Leghorn and exotic (Jungle Fowl, Fayoumi and Spanish) breeds were 0.03-0.55. The Dps between Fayoumi lines and other lines were much larger (0.66-1.34) than within Leghorns, and the Jungle Fowl breed had the largest distances with other lines (1.12-5.38). The phylogenetic consensus tree that was constructed grouped these 23 inbred chicken lines into four different clusters. These results are in accordance with the origin and breeding history of these inbred lines, which indicates that the use of microsatellites for the study of genetic biodiversity is accurate and reliable. In addition, the significance and value of inbred chicken lines in molecular genetic research is discussed.     

Reciprocal translocation of small numbers of inbred individuals rescues immunogenetic diversity

Genetic rescue can reduce inbreeding depression and increase fitness of small populations, even when the donor populations are highly inbred. In a recent experiment involving two inbred island populations of the New Zealand South Island robin, Petroica australis, reciprocal translocations improved microsatellite diversity and individual fitness. While microsatellite loci may reflect patterns of genome‐wide diversity, they generally do not indicate the specific genetic regions responsible for increased fitness. We tested the effectiveness of this reciprocal translocation for rescuing diversity of two immunogenetic regions: Toll‐like receptor (TLR) and major histocompatibility complex (MHC) genes. We found that the relatively small number of migrants (seven and ten per island) effectively brought the characteristic TLR gene diversity of each source population into the recipient population. However, when migrants transmitted TLR alleles that were already present at high frequency in the recipient population, it was possible for offspring of mixed heritage to have decreased gene diversity compared to recipient population diversity prior to translocation. In contrast to TLRs, we did not observe substantial changes in MHC allelic diversity following translocation, with limited evidence of a decrease in differentiation, perhaps because most MHC alleles were observed at both sites prior to the translocation. Overall, we conclude that small numbers of migrants may successfully restore the diversity of immunogenetic loci with few alleles, but that translocating larger numbers of animals would provide additional opportunity for the genetic rescue of highly polymorphic immunity regions, such as the MHC, even when the source population is inbred.     

The major histocompatibility complex: the value of extended haplotypes in the analysis of associated immune diseases and disorders

Major histocompatibility complex antigens are critical to an animal's immune response. In most animals, the extreme polymorphism of MHC molecules complicates studies of the role of this complex in the immune response. In mice, however, MHC haplotype-homozygous inbred strains have been developed which are invaluable in the study of the immune system and the search for immune response genes. The human MHC bears many similarities to its murine equivalent with regard to antigen structure and polymorphism; furthermore, a number of combinations of specific MHC alleles between HLA-B and HLA-DR/DQ (extended haplotypes) are found in people more commonly than predicted by individual allele frequencies. Over 30 percent of Caucasian haplotypes are extended haplotypes, and over 55 percent of individuals have at least one extended haplotype. Examples of the same extended haplotype, even in unrelated individuals, should either all have or lack any gene within the MHC region. The value of considering extended haplotypes in searching for associations between the MHC and diseases, or immune response, is shown in three examples: congenital adrenal hyperplasia, hepatitis B immunization, and transfusion-associated graft-versus-host disease.     

Association of fertility, fitness and longevity with the murine Ah locus among (C57BL/6N) (C3H/HeN) recombinant inbred lines

The Ah locus encodes a cytosolic receptor which controls the induction of enzymes that metabolize drugs, chemical carcinogens, and other environmental pollutants. B6NXC3N recombinant inbred lines have been developed from the progenitors C57BL/6N and C3H/HeN inbred mouse strains. Ah phenotyping at each generation has resulted in the establishment of some lines containing high levels of the high-affinity Ah receptor; other lines, very low levels. A genetic model involving two unlinked loci is offered to explain the distribution of Ah receptor levels among (C57BL/6N) (C3H/HeN)F2 individuals. Between generations 7 and 13, individual females and males from the B6NXC3N recombinant inbred lines were crossed with DBA/2N males and females. Presence of high levels of the high-affinity Ah receptor in both female and male B6NXC3N mice was found to be associated with greater fertility, fitness, and longer life span. The data suggest that these parameters are correlated with the Ah locus or a closely segregating gene.     

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.     

Haplotype variation, recombination, and gene conversion within the turkey MHC-B locus

The major histocompatibility complex (MHC) is a gene dense region with profound effects on the disease phenotype. In many species, characterizations of MHC polymorphisms have focused on identifying allelic haplotypes of the highly polymorphic class I and class II loci through direct immunological approaches such as monoclonal antibodies specific for the major antigens or indirectly through DNA sequence-based approaches. Invariably, these studies fail to assess the broader range of variation at the other loci within the MHC. This study examines variation in the turkey MHC by resequencing 15 interspersed amplicons (∼14 kb) spaced across the MHC-B locus in a representative sampling of 52 commercial birds. Over 200 single nucleotide polymorphisms (SNPs) were identified with high levels of polymorphism (1 SNP/70 bp) and heterozygosity (average minor allele frequency of 0.15). SNP genotypes were used to identify the major haplotypes segregating in the commercial lines. Sequencing of the peptide binding region (PBR, exon 2) of the class IIB loci of select individuals identified 10 PBR alleles/isotypes among the major MHC haplotypes. Examination of pedigreed families provides direct evidence of gene conversion and recombination within the B locus. Results of this study demonstrate the MHC diversity available in commercial flocks and provide genomic resources for studying the effect of this diversity (alleles and/or haplotypes) on disease susceptibility and resistance.     

Study of possibility in raising maize inbred lines with two embryos

Summary Ears having 1 to 3 kernels with two embryos were found in a synthetic and local maize population at the Maize Research Institute, Beograd-Zemun, in 1963–1964. From this material, using the method of individual kernels, selection was initiated and inbred lines with two embryo kernels were obtained.The present paper gives the results of further breeding of maize lines having two embryo kernels, the frequency and variability of this occurrence within and among lines, and the results of some cytogenetic investigations of plants originating from two embryo kernels.The frequency of two embryo kernels in ears of 12 selected lines in 1973 varied between 2.1% (the line IT) and 25.3% (the line lab). The average for all lines was 11.8%. The best inbred lines have 8 times the number of kernels with two embryos found for the initial material (3.1%). Compared with normal kernels of the same lines, two-embryo kernels have a considerable increase in protein (4–6%), lysine g/l00 g of dry matter (38– 70.9%), lysine g/ l00 g of protein (21.3–34.0%) and oil (3.5–13.6%).The presence of univalent chromosomes at metaphase I is not relatively high and in most cases it occurs in approximately 10–20% meiocytes, indicating partial desynapsis. No obvious differences in the frequency of univalent chromosomes at metaphase I and lagging chromosomes at anaphase I were found between plants of various height originating from the same kernel.     

Analysis of genetic diversity and relationships among waxy maize inbred lines in Korea using SSR markers

Information regarding the genetic diversity and genetic relationships among elite inbred lines is necessary to improve new cultivars in maize breeding programs. In this study, genetic diversity and genetic relationships were investigated among 84 waxy maize inbred lines using 50 SSR markers. A total of 269 alleles were identified at all the loci with an average of 5.38 and a range between 2 and 13 alleles per locus. The gene diversity values varied from 0.383 to 0.923 with an average of 0.641. The cluster tree generated using the described SSR markers recognized two major groups at 32% genetic similarity. Group I included 33 inbred lines while group II included 51 inbred lines. The clustering patterns of most of the waxy maize inbred lines did not clearly agree with their source, pedigree or geographic location. The average GS among all inbred lines was 35.7 ± 10.8. Analysis of waxy maize inbred lines collected from Korea and China at 50 SSR loci revealed higher values of average number of alleles (4.9) and gene diversity (0.638) in Korean inbred lines as compared to Chinese inbred lines (3.5 and 0.563, respectively). The information obtained from the present studies would be very useful for maize breeding programs in Korea.     

Molecular marker-based genetic diversity assessment of Striga-resistant maize inbred lines

Striga-resistant maize inbred lines are of interest to maize breeding programs in the savannas of Africa where the parasitic weed is endemic and causes severe yield losses in tropical maize. Assessment of the genetic diversity of such inbred lines is useful for their systematic and efficient use in a breeding program. Diversity analysis of 41 Striga-resistant maize inbred lines was conducted using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers to examine the genetic relationships among these lines and to determine the level of genetic diversity that exists within and between their source populations. The two marker systems generated 262 and 101 polymorphic fragments, respectively. Genetic similarity (GS) values among all possible pairs of inbred lines varied from 0.45 to 0.95, with a mean of 0.61±0.002 for AFLPs, and from 0.21 to 0.92, with a mean of 0.48±0.003, for SSRs. The inbred lines from each source population exhibited a broad range of GS values with the two types of markers. Both AFLPs and SSRs revealed similar levels of within population genetic variation for all source populations. Cluster and principal component analysis of GS estimates with the two markers revealed clear differentiation of the Striga-resistant inbred lines into groups according to their source populations. There was clear separation between early- and late-maturing Striga-resistant inbred lines. Considering the paucity of germplasm with good levels of resistance to Striga in maize, the broad genetic diversity detected within and among source populations demonstrates the genetic potential that exists to improve maize for resistance to Striga.     

Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression patterns in the F1 hybrid

Microarray analysis of gene expression patterns in immature ear, seedling, and embryo tissues from the maize inbred lines B73 and Mo17 identified numerous genes with variable expression. Some genes had detectable expression in only one of the two inbreds; most of these genes were detected in the genomic DNA of both inbreds, indicating that the expression differences are likely caused by differential regulation rather than by differences in gene content. Gene expression was also monitored in the reciprocal F1 hybrids B73xMo17 and Mo17xB73. The reciprocal F1 hybrid lines did not display parental effects on gene expression levels. Approximately 80% of the differentially expressed genes displayed additive expression patterns in the hybrids relative to the inbred parents. The approximately 20% of genes that display nonadditive expression patterns tend to be expressed at levels within the parental range, with minimal evidence for novel expression levels greater than the high parent or less than the low parent. Analysis of allele-specific expression patterns in the hybrid suggested that intraspecific variation in gene expression levels is largely attributable to cis-regulatory variation in maize. Collectively, our data suggest that allelic cis-regulatory variation between B73 and Mo17 dictates maintenance of inbred allelic expression levels in the F1 hybrid, resulting in additive expression patterns.