Detection of individual human chromosomes by chromosome in situ suppression hybridization using PCR-amplified bacteriophage library probes

We used the polymerase chain reaction (PCR) to prepare chromosome-specific probes from the bacteriophage λ library LA01NS01, prepared at the Los Alamos National Laboratory from flow sorted human chromosome 1. By using oligonucleotide primers flanking the EcoRI insertion site of the Charon 21A vector, we were able to amplify the human sequences preferentially in the library up to 9.1 kb (maximum insert size). The product of the PCR reaction was nick translated with incorporation of biotinylated residues and used with fluorescence in situ hybridization to observe metaphase chromosomes by fluorescence microscopy. This technique allows for a relatively easy method for preparation of chromosome-specific library probes for “chromosome painting.” The quality of the results obtained by this method compares favorably to those obtained by using bulk-purified library inserts. This method offers potential advantages in terms of cost and east of use.     

Cotton chromosome substitution lines crossed with cultivars: genetic model evaluation and seed trait analyses

Seed from upland cotton, Gossypium hirsutum L., provides a desirable and important nutrition profile. In this study, several seed traits (protein content, oil content, seed hull fiber content, seed index, seed volume, embryo percentage) for F₃ hybrids of 13 cotton chromosome substitution lines crossed with five elite cultivars over four environments were evaluated. Oil and protein were expressed both as percentage of total seed weight and as an index which is the grams of product/100 seeds. An additive and dominance (AD) genetic model with cytoplasmic effects was designed, assessed by simulations, and employed to analyze these seed traits. Simulated results showed that this model was sufficient for analyzing the data structure with F₃ and parents in multiple environments without replications. Significant cytoplasmic effects were detected for seed oil content, oil index, seed index, seed volume, and seed embryo percentage. Additive effects were significant for protein content, fiber content, protein index, oil index, fiber index, seed index, seed volume, and embryo percentage. Dominance effects were significant for oil content, oil index, seed index, and seed volume. Cytoplasmic and additive effects for parents and dominance effects in homozygous and heterozygous forms were predicted. Favorable genetic effects were predicted in this study and the results provided evidence that these seed traits can be genetically improved. In addition, chromosome associations with AD effects were detected and discussed in this study.     

Genetic compensation abilities of Aegilops speltoides chromosomes for homoeologous B-genome chromosomes of polyploid wheat in disomic S(B) chromosome substitution lines

The S genome of Aegilops speltoides is closely related to the B and G genomes of polyploid wheats. However, little work has been reported on the genetic relationships between the S-genome and B-genome chromosomes of polyploid wheat. Here, we report the isolation of a set of disomic substitutions (DS) of S-genome chromosomes for the B-genome chromosomes and their effects on gametophytic and sporophytic development. Ae. speltoides chromosomes were identified by their distinct C-banding and fluorescence in situ hybridization patterns with the Ae. speltoides-derived clone pGc1R-1. Although no large structural differences between S-genome and B-genome chromosomes exist, significant differences in gametophytic compensation were observed for chromosomes 1S, 3S, 5S and 6S. Similarly, chromosomes 1S, 2S, 4S, 5S and 6S affected certain aspects of sporophytic development in relation to spike morphology, fertility and meiotic pairing. The DS5S(5B) had disturbed meiosis with univalents/multivalents and suffered chromosome elimination in the germ tissues leading to haploid spikes in 50% of the plants. The effect of the Ph1 gene on meiosis is well known, and these results provide evidence for the role of Ph1 in the maintenance of polyploid genome integrity. These and other data are discussed in relation to the structural and functional differentiation of S- and B-genome chromosomes and the practical utility of the stocks in wheat improvement.     

陆地棉背景的Pima棉染色体片段置换系创制

染色体片段置换系(CSSLs,chromosome segment substitution lines)基因组内只有1个或少数几个纯合的供体亲本染色体片段,而其余部分与受体亲本相同,是进行QTL分析的理想材料。本研究以陆地棉中棉所8号(CCRI8)为受体亲本,海岛棉Piam90-53为供体亲本在BC3F1-3借助分子标记辅助选择培育了一套182个株系构成的染色体片段置换系。这套置换系置换片段平均长度21.0 c M,总长度19957.8 c M,是棉花基因组总长度4168.7 c M的4.7倍。每个株系内置换片段长度不一,最短为0.7 c M,最长是83.2 c M,导入片段数量为1~11个。CSSLs在纤维品质性状上的分布表现为相对连续的正态分布,部分株系较CCRI8有了明显提高。本研究为进一步开展棉花纤维品质QTL定位以及陆地棉纤维品质育种研究提供了新材料。     

Genetic analysis of anther culture response in wheat using aneuploid,chromosome substitution and translocation lines

Summary Marked effects of genotype on wheat anther culture response have been observed. Genetic factors have been recognised to be one of the major contributors to in vitro responses of cultured wheat tissues. In wheat anther culture, embryo induction, plant regeneration and albina/green ratio have been determined to be heritable traits. Using Chinese Spring (CS) monosomic 1D, single chromosome substitution lines of chromosome 5B or chromosome arm 5BL from Chinese Spring into six varieties, and F₁ hybrids heterozygous for the 1B chromosome structure (1BL-1BS/1BL-1RS), the anther culture response was studied: genes on CS1D chromosome and 5BL chromosome arm increases the embryo frequency; gene(s) involved in regeneration ability are located on the 1RS chromosome arm; a gene increasing albina frequency is located on Chinese Spring 5B chromosome. Our results support the fact that without gametic selection, a differential development occurred from the particular classes of microspores carrying genes for higher regeneration ability. Moreover, in some crosses, a few genes with major effects were involved in determination of anther culture response.     

早熟禾品种间遗传多样性分析

草地早熟禾（Poa pratensis L.）是一种重要的冷季型草坪草,抗逆性强,适应性广,株型低矮,绿色期长,并且在良好的管理条件下,特别适合用作运动场草坪。近几年来在我国的园林绿化和建植运动场中得到了广泛的应用,现在已经引进的品种多达上百种。选用28个随机引物对15个草地早熟禾品种和1个加拿大早熟禾品种进行RAPD分析,25个引物共扩增出218条带,多态条带比率为89.91％。并且对每个品种的几项坪用特性进行了观察。聚类分析结果表明草地早熟禾品种之间的遗传多样性较低, 相似性集中在60.76％～98.52％。加拿大早熟禾的一个品种单成一支,与其他草地早熟禾的品种相似性较低。品种之间的聚类关系与表型特征呈现不完全相关性。     

Polymorphism of heterochromatin C-blocks of rye genome chromosomes in rye-wheat amphidiploids and their chromosome substitution lines

Polymorphism of heterochromatin C-blocks in chromosomes of rye genome has been studied in the F1BC1 hybrids and the D/A substitution lines of rye-wheat amphidiploids (Verasen x L374, Novosibirskaya x x L246)--secalotriticum for revealing cytogenetic markers of rye chromosomes. An increase in polymorphism for the presence and value of heterochromatin blocks in chromosomes of rye genome was shown in the F1BC1 hybrids ((Verasen x L374) x L145 x L145) and unstable genotypes of the F6BC1 chromosome substitution lines (line 118 (Novosibirskaya x L246) x Reso x Reso), line 104 ((Verasen x x L374) x Garmoniya x Garmoniya) that was related to activation of mobile genetic elements present in cereals. Heterochromatin markers of all seven chromosomes in rye genome were revealed. The polymorphism system of heterochromatin blocks may serve as a marker for specificity of the linear chromosome structure during reconstruction of synthetic cereal genomes, and as a test for cytological and morphogenetic stability of hybrid polygenomes in a series of generations.     

Genetic dissection of chromosome substitution lines of cotton to discover novel Gossypium barbadense L. alleles for improvement of agronomic traits

We recently released a set of 17 chromosome substitution (CS-B) lines (2n = 52) that contain Gossypium barbadense L. doubled-haploid line ‘3-79’ germplasm systematically introgressed into the Upland inbred ‘TM-1’ of G. hirsutum (L.). TM-1 yields much more than 3-79, but cotton from the latter has superior fiber properties. To explore the use of these quasi-isogenic lines in studying gene interactions, we created a partial diallel among six CS-B lines and the inbred TM-1, and characterized their descendents for lint percentage, boll weight, seedcotton yield and lint yield across four environments. Phenotypic data on the traits were analyzed according to the ADAA genetic model to detect significant additive, dominance, and additive-by-additive epistasis effects at the chromosome and chromosome-by-chromosome levels of CS-B lines. For example, line 3-79 had the lowest boll weight, seedcotton yield and lint yield, but CS-B22Lo homozygous dominance genetic effects on seedcotton and lint yield were nearly four times those of TM-1, and its hybrids with TM-1 had the highest additive-by-additive epistatic effects on seedcotton and lint yield. CS-B14sh, 17, 22Lo and 25 produced positive homozygous dominance effects on lint yield, whereas doubly heterozygous combinations of CS-B14sh with CS-B17, 22Lo and 25 produced negative dominance effects, suggesting that epistatic effects between genes in these chromosomes strongly affect lint yield. The results underscore the opportunities to systematically identify genomic regions harboring genes that impart agronomically significant effects via epistatic interactions. The chromosome-by-chromosome approach significantly complements other strategies to detect and quantify epistatic interaction effects, and the quasi-isogenic nature of families and lines from CS-B intermatings will facilitate high-resolution localization, development of markers for selection and map-assisted identification of genes involved in strong epistatic effects.     

Interspecific chromosomal effects on agronomic traits in Gossypium hirsutum by AD analysis using intermated G. barbadense chromosome substitution lines

The untapped potential of the beneficial alleles from Gossypium barbadense L. has not been well utilized in G. hirsutum L. (often referred to as Upland cotton) breeding programs. This is primarily due to genomic incompatibility and technical challenges associated with conventional methods of interspecific introgression. In this study, we used a hypoaneuploid-based chromosome substitution line as a means for systematically introgressing G. barbadense doubled-haploid line ‘3-79’ germplasm into a common Upland genetic background, inbred ‘Texas marker-1’ (‘TM-1’). We reported on the chromosomal effects, lint percentage, boll weight, seedcotton yield and lint yield in chromosome substitution CS-B (G. barbadense L.) lines. Using an additive-dominance genetic model, we studied the interaction of alleles located on two alien substituted chromosomes versus one alien substituted chromosome using a partial diallel mating design of selected CS-B lines (CS-B05sh, CS-B06, CS-B09, CS-B10, CS-B12, CS-B17 and CS-B18). Among these parents, CS-B09 and CS-B10 were reported for the first time. The donor parent 3-79, had the lowest additive effect for all of the agronomic traits. All of the CS-B lines had significant additive effects with boll weight and lint percentage. CS-B10 had the highest additive effects for lint percentage, and seedcotton and lint yield among all of the lines showing a transgressive genetic mode of inheritance for these traits. CS-B09 had greater additive genetic effects on lint yield, while CS-B06, CS-B10 and CS-B17 had superior additive genetic effects on both lint and seedcotton yield compared to TM-1 parent. The 3-79 line had the highest dominance effects for boll weight (0.513 g) and CS-B10 had the lowest dominance effect for boll weight (?0.702). Some major antagonistic genetic effects for the agronomic traits were present with most of the substituted chromosomes and chromosome arms, a finding suggested their recalcitrance to conventional breeding efforts. The results revealed that the substituted chromosomes and arms of 3-79 carried some cryptic beneficial alleles with potential to improve agronomic traits including yield, whose effects were masked at the whole genome level in 3-79.     

QTL mapping of protein content in rice using single chromosome segment substitution lines

Protein content (PC) is an important component of rice nutritional quality. In order to better understand the genetic basis of this trait and increase related breeding efficiency, 21 single chromosome segment substitution (SCSS) lines grown in four sites over two growing seasons (regarded as eight environments) were used to associate PC with particular chromosome segments. Segments from 15 chromosomes were found to contain quantitative trait loci (QTLs) for PC in at least one environment. These included segments from chromosome 3 and 8, in which QTLs for PC had not previously been identified. The segment of chromosome 8 in CSSL-48 had the largest positive effect across all environments. The interaction between substitution and environment was highly significant. Some substitutions had large effects in one environment, but no effect in another (i.e. CSSL-08 and CSSL-17), while some substitutions significantly increased PC in one environment but decreased it in another (i.e. CSSL-41 and CSSL-43). By biplot and clustering analysis, the eight environments were grouped into two contrasting environment types, that is, Hainan and Jiangsu. The segment of chromosome 8 in CSSL-48 had PC-enhancing QTLs in both of the environment types. The segments in CSSL-34 had QTLs which increase PC in the Jiangsu environment but have no effect in the Hainan environment. For enhancing PC, CSSL-48 could be explored in breeding for wide adaptation across all environments, while CSSL-12, CSSL-14, CSSL-17, CSSL-41 and CSSL-43, and that in CSSL-34 could be explored in breeding for specific adaptation to the Hainan and Jiangsu environments, respectively. Near isogenic lines are under development to validate the QTLs with large effects in a range of genetic backgrounds relevant to Jiangsu and Hainan breeding programs. Secondary mapping populations are also being developed for further localising the responsible QTLs in CSSL-14, CSSL-34 and CSSL-48.     

Orientation of interphase chromosomes as detected by Giemsa C-bands

The orientation of Giemsa C-bands has been studied in mitotic and interphase cells of Allium cepa, A. sativum and of Aloe vera. The C-bands in these three species are located at the telomeres, secondary constriction region of the nucleolar chromosomes and the centromeric regions, respectively. Observations in A. cepa and Aloe indicate clearly that the interphase chromosomes are non-random in their orientation and possibly maintain their telophase configuration through the attachment of telomeres and perhaps of kinetochores with the nuclear membrane. Electron micrographs of onion cells also reveal that certain heterochromatic segments are associated with the nuclear membrane. — The nucleolar interstitial C-bands in A. sativum remain free in the nucleoplasm and may come close to each other due to heterochromatic attraction. Such a heterochromatic attraction is also evident between telomeric regions and between centromeres. However, a two by two attachment could not be noticed. A diagrammatic representation of the orientation of interphase chromosomes has been presented.The major part of this work was presented at the First International Congress on Cell Biology, Boston, Sept. 5–10, 1976 (Platform Session 36, J. Cell Biol. 70, 418a (1976)     

Mapping and characterization of seed dormancy QTLs using chromosome segment substitution lines in rice

Seed dormancy—the temporary failure of a viable seed to germinate under favorable conditions—is a complex characteristic influenced by many genes and environmental factors. To detect the genetic factors associated with seed dormancy in rice, we conducted a QTL analysis using chromosome segment substitution lines (CSSLs) derived from a cross between Nona Bokra (strong dormancy) and Koshihikari (weak dormancy). Comparison of the levels of seed dormancy of the CSSLs and their recurrent parent Koshihikari revealed that two chromosomal regions—on the short arms of chromosomes 1 and 6—were involved in the variation in seed dormancy. Further genetic analyses using an F₂ population derived from crosses between the CSSLs and Koshihikari confirmed the allelic differences and the chromosomal locations of three putative QTLs: Sdr6 on chromosome 1 and Sdr9 and Sdr10 on chromosome 6. The Nona Bokra alleles of the three QTLs were associated with decreased germination rate. We discuss the physiological features of the CSSLs and speculate on the possible mechanisms of dormancy in light of the newly detected QTLs.     

An additive-dominance model to determine chromosomal effects in chromosome substitution lines and other gemplasms

When using chromosome substitution (CS) lines in a crop breeding improvement program, one needs to separate the effects of the substituted chromosome from the remaining chromosomes. This cannot be done with the traditional additive-dominance (AD) model where CS lines, recurrent parent, and their hybrids are used. In this study, we develop a new genetic model and software, called a modified AD model with genotype × environment interactions, which can predict additive and dominance genetic effects attributed to a substituted alien chromosome in a CS line as well as the overall genetic effects of the non-substituted chromosomes. In addition, this model will predict the additive and dominance effects of the same chromosome of interest (i.e. chromosome 25 of cotton in this study) in an inbred line, as well as the effects of the remaining chromosomes in the inbred line. The model requires a CS line, its recurrent parent and their F₁ and/or F₂ hybrids between the substitution lines and several inbred lines. Monte Carlo simulation results showed that genetic variance components were estimated with no or slight bias when we considered this modified AD model as random. The correlation coefficient between predicted effects and true effects due to the chromosomes of interest varied from zero to greater than 0.90 and it was positively relative to the difference between the CS line and the recurrent line. To illustrate the use of this new genetic model, an upland cotton, Gossypium hirsusum L, CS line (CS-B25), TM-1 (the recurrent parent), five elite cultivars, and the F₂ hybrids from test-crossing these two lines with the five elite cultivars were grown in two environments in Mississippi. Agronomic and fiber data were collected and analyzed. The results showed that the CS line, CS-B25, which has chromosome 25 from line 3 to 79, Gossypium barbadense substituted into TM-1, had positive genetic associations with several fiber traits. We also determined that Chromosome 25 from FiberMax 966 had significantly positive associations with fiber length and strength; whereas, chromosome 25 from TM-1 and SureGrow 747 had detectable negative genetic effects on fiber strength. The new model will be useful to determine effects of the chromosomes of interest in various inbred lines in any diploid or amphidiploid crop for which CS lines are available.     

Increasing the physical markers of wheat chromosomes using SSRs as FISH probes

In plants the marker sequences used to identify chromosomes are mainly repetitive DNA probes. Simple sequence repeats (SSRs) are major components of many plant genomes and could be good markers for chromosome identification. In a previous work, we reported the physical distribution of 4 oligonucleotides, (AG)12, (CAT)5, (AAC)5, and (AAG)5, on Triticum aestivum L. chromosomes. The distinctive distribution pattern found suggested that SSR in situ hybridization is useful as a diagnostic tool in wheat cytogenetics. To check whether that finding is generally applicable, we analyzed the chromosomal distribution of the rest of the 14 possible classes of di- and tri-nucleotide repeats by FISH. A detailed knowledge of the sequence content of hexaploid wheat chromatin was acquired based on the hybridization signals, which also provide a rich set of chromosome markers for chromosome identification. Except for (AT)10 and (GC)10, for which the chromosomal distribution could not be accurately determined, and (AC)8 and (GCC)5, which were found dispersed throughout the chromosomes, the remaining repeats were observed as clusters on specific chromosome sites. (AGG)5, (CAC)5, (ACG)5, (AAT)5, and (CAG)5 exhibited a preferential distribution in the pericentromeric regions of the B genome chromosomes. The richest patterns of intercalary signals on several A and B genome chromosomes were produced by (ACT)5. A karyotype based on the SSR probes providing the best FISH patterns was constructed for T. aestivum 'Chinese Spring'.     

Genetic variation in wheat endosperm proteins: an analysis by two-dimensional electrophoresis using intervarietal chromosomal substitution lines

Summary The major endosperm proteins in a range of genotypes of hexaploid wheat have been fractionated by two-dimensional electrophoresis. The genotypes included nine varieties and forty four intervarietal substitution lines in which chromosomes 1A, 1B, 1D, 6A, 6B or 6D from eight of the varieties have been introduced one at a time into a common genetic background. The appearance of different protein subunits was often correlated with a chromosome substitution. This showed that many of the genes for the high molecular weight protein subunits (molecular weight range 55,000 to 140,000 determined by SDS polyacrylamide gel electrophoresis) are specified by chromosomes 1A, 1B and 1D while many of the lower molecular weight subunits (molecular weight range 30,000 to 45,000) are specified by chromosomes 6A, 6B and 6D. The different protein subunits correlated with chromosome substitution could not always be recognised in the varietal source of the substituted chromosome. The different subunits specified by homologous chromosomes in different wheat varieties may differ in isoelectric point and/or molecular weight.     

Genetic dissection and pyramiding of quantitative traits for panicle architecture by using chromosomal segment substitution lines in rice

To understand the genetic basis of yield-related traits of rice, we developed 39 chromosome segment substitution lines (CSSLs) from a cross between an average-yielding japonica cultivar, Sasanishiki, as the recurrent parent and a high-yielding indica cultivar, Habataki, as the donor. Five morphological components of panicle architecture in the CSSLs were evaluated in 2 years, and 38 quantitative trait loci (QTLs) distributed on 11 chromosomes were detected. The additive effect of each QTL was relatively small, suggesting that none of the QTLs could explain much of the phenotypic difference in sink size between Sasanishiki and Habataki. We developed nearly isogenic lines for two major QTLs, qSBN1 (for secondary branch number on chromosome 1) and qPBN6 (for primary branch number on chromosome 6), and a line containing both. Phenotypic analysis of these lines revealed that qSBN1 and qPBN6 contributed independently to sink size and that the combined line produced more spikelets. This suggests that the cumulative effects of QTLs distributed throughout the genome form the major genetic basis of panicle architecture in rice. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Ando and T. Yamamoto contributed equally to this work.