The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression

In the chicken, the nucleolus organizer regions, or sites of the genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA), map to one pair of microchromosomes that can be identified by silver nitrate cytochemistry. This nucleolar organizer chromosome also contains the major histocompatibility complex. Chickens aneuploid for this chromosome have been identified and reproduced for over seven generations. Crossing two trisomic parents results in the production of viable disomic, trisomic, and tetrasomic progeny, showing two, three, and four nucleoli and nucleolar organizers per cell, respectively. A molecular analysis of rRNA genes was undertaken to establish the gene copy numbers in the aneuploid genotypes, and to determine if elevated numbers of rRNA genes are stably maintained and inherited over multiple generations. Gene copy numbers were determined using hybridization analysis of erythrocyte DNA obtained from individuals comprising a family which segregated disomic, trisomic, and tetrasomic genotypes. The values obtained were 290, 420, and 570 rDNA repeats per cell for disomic, trisomic, and tetrasomic animals, respectively. These results provide molecular confirmation of the two aneuploid states and show that elevated gene copy numbers have been maintained over multiple generations. Fibroblasts derived from disomic and tetrasomic embryos were found to grow at similar rates in culture, and mature rRNA levels in chicken embryo fibroblasts from disomic, trisomic and tetrasomic embryos were also found to have similar levels of mature rRNA. Therefore, despite the increase in rDNA content, the level of rRNA is regulated to diploid amounts in aneuploid fibroblasts.     

MHC-chromosome dosage effects: evidence for increased expression of Ia glycoprotein and alteration of B cell subpopulations in neonatal aneuploid chickens

Quantitative variation in the expression of MHC-encoded class II (Ia) glycoproteins has been associated with stages of lymphocyte development and a number of disease conditions. We have used an avian MHC dosage model to study the regulation of Ia expression and the effects of quantitative variation in membrane Ia on B-cell development. Lymphocyte membrane expression of Ia glycoprotein molecules and the frequency of small-versus-large lymphocytes were examined in trisomic line chickens containing either two (disomic), three (trisomic), or four (tetrasomic) copies of the microchromosome encoding the MHC. This was accomplished by quantitative laser flow cytometry analysis of bursa-resident B lymphocytes from neonatal trisomic line chickens. The aneuploids (trisomics and tetrasomics) expressed more cell surface Ia than did normal disomic birds. Furthermore, the aneuploids exhibited a greater frequency of small B lymphocytes as compared to disomic chickens. Dual parameter analysis of Ia. quantity and cell size was undertaken to study B lymphocyte subpopulations in these birds. It was observed that the aneuploids had altered frequencies of two distinct subpopulations of cells: (1) an increased percentage of small cells which express high levels of Ia antigen and (2) a decreased percentage of large cells which express medium levels of Ia antigen. These findings support the view that MHC class II genes are regulated and expressed in a dosage-dependent manner. Therefore, increases in the number of MHC copies per cell result in the increased expression of Ia glycoprotein on bursa-resident B cells. The stepwise increase in membrane Ia on trisomic and tetrasomic B cells is correlated, and perhaps casually linked, with progressive degrees of alteration of developing B cell subpopulations in the bursa of aneuploid chicks. These events may ultimately alter the humoral immunity of the aneuploid animals.     

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.     

Ultrastructural studies of the nucleoli in diploid and trisomic chickens

Chickens having a trisomic complement for the nucleolar organizer region (NOR) serve as a model for investigations on gene expression, molecular regulation, and physiological alterations resulting from extra genetic material. Of fundamental importance is the question of whether the extra set of ribosomal cistrons in trisomic chickens generates a morphologically and biochemically complete nucleolus. Ultrastructural and cytochemical studies of diploid and trisomic embryonic and adult cells were undertaken. All structures identified in trisomic cells as nucleoli by gross morphology were shown to be RNA rich by acridine orange fluorescence. At the ultrastructural level, the expected granular and fibrillar components were identified in all nucleoli of trisomic cells. Fibrillar centers, indicative of NOR's, were seen in all cases. These and other results from silver staining suggest full gene activity for each of the three chromosomes in the trisomic cell at all stages of development.     

The effect of trisomy on meiotic behaviour of interchange complexes in pearl millet,Pennisetum americanum (L.) leeke

Summary In the selfed progeny of a spontaneously produced triploid interchange heterozygote four different double trisomic plants were observed. In all the plants the frequency of alternate orientation of multivalents was lower compared to their respective types in the sib single trisomic plants. The frequency of alternate co-orientation of the interchange complex in these trisomics was also reduced compared to that of parental euploid disomic interchange heterozygotes. It is suggested that the presence of extra chromosomes influences the orientation behaviour of higher associations in different trisomics.     

Chromosomal localisation of a recessive gene tp controlling the pleiotropic character topiary in Solanum

Summary Seven out of twelve possible types of primary trisomies of dihaploid S. tuberosum were crossed as females with a disomic recessive mutant for topiary (tp tp) identified in S. infundibuliforme. All primary trisomics used proved to be homozygous dominant. Trisomic plants from the seven F1's were crossed with a disomic heterozygous F1 plant (supposed genotype Tp tp). In the half sib progeny of each trisomic type the mutant plants could be easily identified by the presence of typical lateral shoots, particularly at the cotyledonary nodes. The observed segregation ratios for normal to mutant were tested against the expected non-critical ratio 3 1 and against various critical ratios. It is concluded that the gene tp is located on chromosome 3 of the potato.     

Secondary Trisomics and Telotrisomics of Rice: Origin, Characterization, and Use in Determining the Orientation of Chromosome Map

Secondary trisomics and telotrisomics representing the 12 chromosomes of rice were isolated from the progenies of primary trisomics. A large population of each primary trisomic was grown. Plants showing variation in gross morphology compared to the primary trisomics and disomic sibs were selected and analyzed cytologically at diakinesis and pachytene. Secondary trisomics for both arms of chromosomes 1, 2, 6, 7 and 11 and for one arm of chromosomes 4, 5, 8, 9 and 12 were identified. Telotrisomics for short arm of chromosomes 1, 8, 9 and 10 and for long arms of chromosomes 2, 3 and 5 were isolated. These secondary and telotrisomics were characterized morphologically and for breeding behavior. Secondary trisomics 2n + 1S.1S, 2n + 1L.1L, 2n + 2S.2S, 2n + 2L.2L, 2n + 6S.6S, 2n + 6L.6L and 2n + 7L.7L are highly sterile, and 2n + 1L.1L, 2n + 2L.2L and 2n + 7L.7L do not set any seed even upon backcrossing. Telotrisomics are fertile and vigorous. Genetic segregation of 43 marker genes was studied in the F(2) or backcross progenies. On the basis of segregation data, these genes were delimited to specific chromosome arms. Correct orientation of 10 linkage groups was determined and centromere positions on nine linkage groups were approximated. A revised linkage map of rice is presented.     

Association of the yellow leaf (y10) mutant to soybean chromosome 3

At least 19 single recessive gene yellow leaf mutants and one duplicate recessive gene mutant have been described in soybean. This study was conducted to associate a yellow leaf mutant, y10, with a specific soybean chromosome by using primary trisomics (2n = 41). Seven soybean primary trisomics were hybridized as female parent with genetic stock strain, T161, carrying y10. F(1) disomic and primary trisomic plants were identified cytologically. One disomic (control) and all primary trisomic plants were allowed to self-pollinate and F(2) populations were classified for green versus yellow leaf mutant. The F(2) population of Triplo 3 segregated in a 17:1 ratio, while a disomic (3:1) ratio was observed with Triplo 8-, 17-, 18-, and 20-derived F(2) populations, suggesting that the y10 locus is on chromosome 3. The y10 locus was examined with four simple sequence repeat (SSR) markers (Satt584, Sat_033, Satt387, and Satt022) from molecular linkage group (MLG) N and y10 was found linked with Satt022. Therefore we confirmed the association of MLG N with chromosome 3. The possible association of y10 with Triplo 16 and Triplo 19 are discussed.     

Chromosomal localization of four isozyme loci by trisomic analysis in rice (Oryza sativa L.)

Summary Four genes coding for isozymes in rice (Oryza sativa L.), were located to respective chromosmes through trisomic analysis. Twelve primary trisomics in IR36 background were crossed with 2 lines having contrasting alleles at four loci. For each gene, all 12 disomic and trisomic F₁ hybrids were screened for allele dosage effects. Either F₂ or BC1 populations of all cross combinations were assessed for gene segregtion. Evidence from both sources indicated the following locations: Pgi-1 on chromosome 4, Sdh-1 on chromosome 6, Est-8 on chromosome 7 and Adh-1 on chromosome 11. The location of Sdh-1 was further confirmed through the production of triallelic heterozygotes with trisomic 6.     

Trisomics from triploid-diploid crosses in self-incompatible Lycopersicum peruvianum

Summary An attempt was carried out to produce trisomics of the wild tomato L. peruvianum, to define their essential features, and to detect relationships between trisomy and the expression of self-compatibility.Triploid-diploid crosses in L. peruvianum yielded nearly 40% aneuploids. Of these, 18% were single trisomics, and the rest had 2, 3 and 4 extra chromosomes. Almost all the trisomics occurred in crosses where the triploid was used as female parent. Vigour and fertility of trisomics were not much different from those of disomics, and morphologically they were very similar.The extra chromosome was identified in three self-compatible trisomic plants through somatic and pachytene chromosome morphology. One of these plants was trisomic for chromosome 1, while the other two were trisomic for chromosome 3. In these trisomics a positive correlation was found between chromosome length and trivalent formation, but no relationship between chromosome length and frequency of laggards was observed.A series of test-crosses revealed that the capacity of the trisomics to produce seed upon selfing always resulted from alterations of the incompatibility phenotype of the style and not from competitive interaction in the pollen. Progeny analyses showed that the self-compatibility features of the trisomics were not transmitted from one generation to the next. The implications of these findings are discussed.This work has been supported by a contract between the European Communities and the CNEN. This publication is contribution no. 1458 from the Biology Division of the European Communities and contribution no. 472 from the Divisione Applicazioni delle Radiazioni del CNEN.     

Five primary trisomics from translocation heterozygote progenies in common bean,Phaseolus vulgaris L.

Summary Twelve distinct phenotypic groups of plants were isolated from nondisjunction progenies of 11 translocation heterozygote stocks. All the plants in these phenotypic groups originated in the light weight seed class. Five of the 12 phenotypic groups of plants have been verified as primary trisomics. They are all phenotypically distinguishable from each other and from disomics. One of the five primary trisomic groups, puckered leaf, was directly recovered as a primary trisomic from the original translocation heterozygote progenies. Three of the five trisomics — weak stem, dark green leaf, and convex leaf — originated first as tertiary trisomics. The related primary trisomics were isolated later from progenies of selfed tertiary trisomics. The fifth group, chlorotic leaf, originated at a low frequency among the progenies of three other trisomics: puckered leaf, convex leaf, and dark green leaf. The chlorotic leaf did not set seed under field conditions. The remaining four groups — puckered leaf, dark green leaf, convex leaf, and weak stem — are fertile, though sensitive to high temperature conditions. The transmission rate of the extra chromosome on selfing ranges from 28% to 41%. Physical identification of the extra chromosome has not been achieved for any of the five trisomic groups. Two trisomic groups, dark green leaf and convex leaf, have produced tetrasomics at low frequency. The phenotypes of these two tetrasomics are similar to the corresponding trisomics but more exaggerated.Fla. Agr. Expt. Stn. Journal Series No. 7137     

Location of the recessive gene ym (yellow margin) on chromosome 12 of diploid Solatium tuberosum by means of trisomic analysis

Summary Ten out of twelve primary trisomics of dip-loid S. tuberosum were crossed as females with a recessive mutant for yellow margin (ym ym) obtained from S. phureja. All primary trisomics used proved to be homozygous dominant. Trisomic plants from all ten F₁'s were backcrossed with the mutant and trisomics from eight F₁'s were crossed also with a disomic heterozygous f₁ plant from triple 10 X mutant.In both BC₁ and half sib progeny of each trisomic type the mutant plants were easily identified because of their typical small roundish leaflets with yellow or reddish margins. The observed segregation ratios for normal to mutant were tested against the expected non-critical ratios and against various expected critical ratios.From the results of these tests it is concluded that the gene ym is located on chromosome 12 of the potato. A hypothesis of linkage between ym and a gene l _x for lethality is put forward. It is concluded that l _x is not identical with a previously detected recessive gene l ₂ which is responsible for yellow cotyledons and lethality.     

Meiotic disjunction and embryonic lethality in trisomies derived from an X-linked translocation in the onion fly,Hylemya antiqua (Meigen)

Translocation- and tertiary trisomies (for the X-chromosomes) were obtained after testcrossing translocation heterozygous females of an X-linked “simple” translocation stock. Meiotic disjunction as judged from segregations at M II (males) and in young eggs of testcrosses (males and females) in translocation trisomics was studied. No progeny of tertiary trisomic males and females was found, but male M II could be studied. Six different orientation types appeared in translocation trisomie (2n + 1) males and these were present in equal frequencies. No adjacent II configurations were found. The small X- and Y-chromosomes and the large translocated X-chromosome of the translocation complex disjoin at random (n and n + 1 gametes) in both translocation- and tertiary trisomic males. In translocation trisomic females four different orientation types appeared. From the high frequency of two of these (together, 94.5%) it is concluded that the two normal X-chromosomes show preferential pairing and disjunction, while the translocated X-chromosome moves to either one of the two poles at random. Primary trisomic (for the X-chromosome) males (XXY) and females (XXX) were obtained from testerossed translocation trisomics. Cytological analysis of adult male progeny of testerossed XXY males showed that no random orientation for the X-, X- and Y-chromosomes occurred because half of the sons was disomic (XY) and half of them trisomic (XXY). A possible mechanism is discussed. Analysis of young eggs of testerossed XXX females indicated a segregation of 2X∶1X=1∶1. The level of “semi”-sterility as scored from testcrosses of translocation trisomies appeared to be as in translocation heterozygotes. Here again a close relation exists between “semi”-sterility and deficiencies in eggs for a large chromosomal segment. The possible use of this translocation for genetic control of insect pests is discussed.     

Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens

Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.     

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.     

Tertiary trisomics in the garden pea as a model of B chromosome evolution in plants

It is hypothesized that, in plants, genetically empty B chromosomes may originate from the extra chromosome (E) of tertiary trisomics if (i) the region of basic chromosomes homologous to the E (H-region) harbors a sporophytic lethal covered by the wild-type allele in E, and (ii) crossing-over between E and the H-region is suppressed. Under these conditions, most loss-of-function mutations occurring in the H-region are deleterious for haploid gametophytes, whereas those occurring in E are neutral or advantageous for hyperploid (n+1) gametophytes. As a result, natural selection at the gametophyte level can lead to the degeneration of E, leaving the H-region intact. Using Hammarlund translocation T(3-6)a, we synthesized two trisomic lines of the garden pea (Pisum sativum L.), where E was composed of the short arms of chromosomes 3 and 6 and the H-region carried recessive markers. In the trisomic line TRIS, we found few crossovers between E and the H-region. In the trisomic line TRUST, obtained after a change of basic chromosome constitution, recombination in this region was completely suppressed. After induction in the H-region of TRUST of a recessive sporophytic mutation rmv, two 15-chromosome lines of stable trisomics were established. One of them passed 11 generations, having produced more than 6000 individuals, all of them trisomic, and E remained present as a single element with no pairing partners. No tetrasomics were detected in these lines. If such trisomics occurred in nature, their extra chromosomes are likely to become a B chromosome.     

A PCR method for typing B-LβII family (class II MHC) alleles in broiler chickens

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.     

Biochemical characterization of six trisomics of grain sorghum,Sorghum bicolor (L.) Moench

To determine protein differences of grain sorghum disomics and trisomics, we analyzed leaf extracts from six trisomics and a disomic control by disc gel, gel isoelectric focusing, and SDS gel electrophoresis. Based on the number and position of protein bands revealed by Commassie blue staining, the disomic control could be differentiated from the trisomics, and trisomics could be shown to differ among themselves in most cases. SDS gels revealed the most protein bands, followed by isoelectric focusing and disc gel. However, disc gel electrophoresis was the simplest technique of the three and was just as effective in identifying trisomics and differentiating trisomics from the disomic control.Contribution 1596-j, Department of Agronomy, and 182-j, Department of Biochemistry, Kansas State University, Manhattan, Kansas.     

Production of n + 1 plants and tetrasomics by means of anther culture of trisomic plants in rice (Oryza sativa L.)

Summary Eleven primary trisomics of rice, variety Nipponbare, were subjected to anther culture. The 12th trisomic did not produce normal anthers. A total of 3,734 plants were obtained, which were examined morphologically at the seedling stage in the greenhouse. A number of plants appeared in the progenies of ten trisomics which had unique morphological features. The frequency of these variant types differed among different progenies. Cytological observations revealed that 43 variant plants in the progenies of nine trisomics had 13 chromosomes (n + 1), and 56 were tetrasomics (2n = 26). The tetrasomic plants in the progeny of a trisomic were morphologically identical. Similarly, n + 1 plants in the progeny of a trisomic were also identical. Plants with 23, 25, 36, 39, and 73 chromosomes were also obtained. Results show that valuable aneuploids such as n + 1 and 2n + 2 can be obtained in the anther-culture-derived progenies of trisomics.