Genetic distances estimated from DNA fingerprints in crosses of White Plymouth Rock chickens

Crosses were produced between two lines of White Plymouth Rock chickens, one of which had been selected for low 8-week body weight for 31 generations (L) and the other of which was a bantam population (B). The parental lines, reciprocal F1s, reciprocal F2s and all possible back-crosses to each parental line (total of 16 populations) were available for study. Blood was obtained from 10 females within each population. DNA was extracted from blood mixes (equal amounts of blood from each individual) for each population, and from blood samples of each individual in the two parental lines. Fourteen line-specific DNA fingerprint (DFP) bands (those bands present in one parental population, but not in the other parental population) were analysed (eight from line L and six from line B). Regression analyses were conducted to compare the known proportion of genomic contribution from each parental population with values based on relative band intensity obtained with a scanning densitometer. The resulting regression coefficient of 1.004 demonstrated that DFP analysis of relative band intensity is an effective method of estimating the relative proportion of genome contributed by parental populations.     

Prediction of Heterosis from DNA Fingerprints in Chickens

To assess the value of DNA fingerprints for the prediction of heterosis in chickens, retrospective analyses of data from three crossbreeding experiments and DNA fingerprints (DFP) of parental strains were conducted using two minisatellite and one middle-repetitive DNA probes. DFP bands were assessed on pooled DNA samples of 10-15 individuals per parental genetic group. The number of DFP bands evaluated in the experiments ranged from 81 to 139. The probes varied in their predictive value, but predictability of heterosis generally increased with multiple probes. Highly significant correlations (0.68-0.87) between band sharing ratios (SH) and heterosis were found in 25 crosses of White Leghorns in the first egg production cycle for age at sexual maturity, egg production, and mature body weight: traits with heterosis of 10% or more of the means. Regressions of SH explained 78.4% of the variation in heterosis in age at sexual maturity, 60.2% in egg production and 46.4% in mature body weight. For ``broiler' traits with heterosis of <1%, none of the correlations, based on 13 crosses, were significant. It was concluded that multilocus probe DFP of pooled DNA samples show promise as predictors of heterosis.     

Genetic markers linked to quantitative traits in poultry

This study utilized DNA fingerprints and crosses of two genetically distinct lines of layer-type chickens to identify genetic markers linked to quantitative trait loci (QTL). In phase I, back-cross (BC¹) hens were separately ranked for each of eight traits and then blood pools were produced in groups along each phenotypic distribution. The DNA was isolated from the blood pools and used in a gradient analysis to screen for DNA fingerprint bands that exhibited intensity gradients associated with the phenotypic traits. To identify linkage of bands with QTL and to estimate band effects, F₂ progeny were produced in phase II from the phase I BC, population. A single-trait animal model was used for analysis of associations of all individual DNA fingerprint bands of sires and their progeny phenotypic performance. Twenty fingerprint bands, only two of which had shown trait-associated gradients in phase I, were identified by the animal model analysis of the progeny test as QTL linked (P≤005) to specific traits of growth, reproduction and egg quality. These 20 bands warrant further study as potentially valuable molecular markers for QTL.     

Genetic characterization of highly inbred chicken lines by two DNA methods: DNA fingerprinting and polymerase chain reaction using arbitrary primers

Thirteen highly inbred chicken lines were analysed at the DNA level by DNA fingerprinting (DFP) and by polymerase chain reaction (PCR) using random primers. In general, the DFP patterns of individuals within a line were identical. The DFP band-sharing (BS) values among lines from different breeds (Leghorn, Fayoumi, Spanish) ranged from 0.10 to 0.20. The DFP BS values among Leghorn lines from different genetic backgrounds ranged from 0.42 to 0.79. The DFP BS values among lines selected for different major histocompatibility complex serotypes from a common genetic background ranged from 0.70 to 0.95. Some randomly amplified polymorphic DNA (RAPD) PCR products were specific to a single line, some to all lines from the same genetic base, and some to all lines from the same breed. The RAPD-PCR band-sharing values ranged from 0.66 to 0.99 for all between-line comparisons. Thus, the ability to detect biodiversity at the DNA level was greater in this study for DFP than for RAPD-PCR. The possible origin of line-specific bands, relative advantages of detecting biodiversity by using different molecular screening techniques and uses of highly inbred chicken lines in molecular analysis are discussed.     

利用回交结合Wx基因分子标记培育部分糯性小麦

颗粒结合型淀粉合成酶Ⅰ(GBSSI,Waxy蛋白)是小麦胚乳中直链淀粉合成的关键酶。小麦基因组中存在3个Waxy基因(Wx-A1、Wx-B1、Wx-D1)。在白火麦中Wx-D1位点的突变(Wx-D1b)引起Wx-D蛋白的缺失,导致直链淀粉含量下降,其面粉表现出部分糯性。与目前生产上推广品种相比,白火麦的农艺性状较差,产量非常低。为了培育农艺性状优良的部分糯性小麦,我们将白火麦与具有优良农艺性状的小麦品种PH85-16、济南17和烟农15(轮回亲本)分别进行杂交,后代分别与相应的三个轮回亲本回交五代,在每代中均选择农艺性状与轮回亲本相近并含有Wx-D1b的后代。在第六代自交后选择具有Wx-D1b的纯合体,选出的单株连续自交三代。获得了六个农艺性状与轮回亲本相似的品系,它们均携带纯合的Wx-D1b位点。研究表明采用回交的方法并结合基于Wx基因序列的分子标记技术,是培育优良部分糯性小麦的一种非常有效的方法。本研究培育出的部分糯性小麦品系可以直接用于大田生产。     

Development of a STS marker linked to a major locus controlling flour colour in wheat (Triticum aestivum L.)

Flour colour is an important quality trait in the production of bread, noodles and other related end products. Current screening for flour colour in breeding programs requires several grams of flour to be milled. In order to screen large numbers of plants, a rapid PCR-based assay is required. We report here the conversion of a codominant AFLP marker linked to a major locus controlling flour colour in hexaploid wheat, to a sequence tagged site (STS) marker for use in marker-assisted selection (MAS). The two-allelic AFLP bands were cloned and sequenced to allow specific primers to be designed. The primers amplified bands of the expected size in the parental varieties and co-segregated with the original AFLP marker in the mapping population. The primers also amplified alleles of the expected size from the DNA of parental lines of two other related mapping populations. Cultivars that contributed to the pedigree of the original parent `Schomburgk' used to generate the mapping population were also screened to determine the origin of the `yellow' allele.     

不同作图群体对显隐性分子标记位点的作图效率

根据位点组合和位点的有效性,发展了一种对使用３种不同的作用图群体作图显隐性分子标记的作图效率评价方法,应用该方法所评价的结果是,双单倍体（ＤＨ）群体的作图效率最高,自交群体（Ｆ２群体）与回交群体的作图效率相同,因此使用双单倍体群体作图不仅所用费用低,而且作图速度快,但只有在极少数植物中能获得双单倍体群体,对于那些不能获得双单倍体的动植物物种而言,可使用自交群体或回交群体作图。如果作高密度的分子标记     

DNA fingerprints of chickens selected for high and low body weight for 31 generations

Two lines of White Plymouth Rock chickens that have been divergently selected for 8-week body weight for 31 generations were compared for patterns of DNA fingerprints (DFP). Digestion of DNA with HinfI and hybridization to Jeffreys' minisatellite probe 33.6 resulted in DFPs that were relatively similar within lines (bandsharing = 0.50) and less similar between lines (bandsharing = 0.22). Analyses of scorable DFP bands produced by mixing DNA from individuals within lines indicated that 48% were line-specific. Causes for the differences in DFP patterns between lines and for occurrence of line-specific bands for the two lines divergently selected for body weight are discussed.     

水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Ｘａ－４紧密连锁的分子标记Ｍ５５的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片ＤＮＡ、半粒种子提取物及Ｘａ－４基因的杂合体ＤＮＡ的ＰＣＲ特异扩增,初步建立了Ｘａ－４的ＰＣＲ标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Ｘａ－４在这些材料中的分布情况。     

IRAP,REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species

Retrotransposon (RTN)-based markers, such as the inter-retrotransposon amplified polymorphism (IRAP) and the retrotransposon-microsatellite amplified polymorphism (REMAP), are highly informative, multilocus, and reveal insertion polymorphisms among individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, and detection of genetic rearrangements induced by allopolyploidization. The hexaploid tritordeum (H^chH^chAABB; 2n?=?6x?=?42) is an allopolyploid produced from crosses between wild barley (Hordeum chilense Roem. et Schultz.) (H^chH^ch; 2n?=?2x?=?14) and durum wheat (Triticum turgidum L. conv. durum) (AABB; 2n?=?4x?=?28). With this study, we carried out the DNA fingerprinting of two newly formed hexaploid tritordeum lines (HT22 and HT27) and their respective parents, line H1 of H. chilense and line T81 of durum wheat, based on IRAPs, REMAPs and inter-simple sequence repeats (ISSRs), in order to detect potential rearrangements in tritordeum derived from polyploidization. The amphiploid nature of the HT22 and HT27 individuals was successfully confirmed after fluorescence in situ hybridization (FISH), which was performed on their mitotic chromosome spreads with genomic DNA from H. chilense and 45S ribosomal DNA (rDNA), simultaneously, as probes. Six combinations of LTR (long terminal repeat) primers and seven combinations of one LTR and one SSR (simple sequence repeat) primers successfully produced IRAPs and REMAPs, respectively, in both tritordeum lines, and their respective parents. ISSRs were produced with three SSR primers (8081, 8082, and 8564). The analysis of the presence/absence of bands among the tritordeum lines and the respective parents allowed the detection of polymorphic bands: (1) shared by tritordeum and one of the parents; (2) exclusively amplified in tritordeum; and (3) exclusively present in one of the parents. Once no polymorphism was detected among the individuals of each parental species, the polymorphic bands that fit into the second and third cases probably constituted rearrangements in the newly formed tritordeums that arose in response to allopolyploidization, which resulted from the loss of parental bands or, conversely, from the appearance of novel bands not seen in the parental species. Most of the polymorphic IRAPs in tritordeum were shared with the female parent (H. chilense), while most of the polymorphic REMAPs and ISSRs were common to the male parent (durum wheat), but globally, most of the bands inherited by tritordeum had a wheat origin. In conclusion, these dominant markers were successful for DNA fingerprinting and detection of rearrangements in newly formed tritordeum derived from responses to allopolyploidization.     

Identification and validation of QTLs for salt tolerance during vegetative growth in tomato by selective genotyping.

The purpose of this study was to identify quantitative trait loci (QTLs) for salt tolerance (ST) during vegetative growth (VG) in tomato by distributional extreme analysis and compare them with the QTLs previously identified for this trait. A BC1 population (N = 792) of a cross between a moderately salt-sensitive Lycopersicon esculentum Mill. breeding line (NC84173, maternal and recurrent parent) and a salt-tolerant L. pimpinellifolium (Jusl.) Mill. accession (LA722) was evaluated for ST in solution cultures containing 700 mM NaCl + 70 mM CaCl2 (electrical conductivity, EC = 64 dS/m and phiw approximately -35.2 bars). Thirty-seven BC1 plants (4.7% of the total) that exhibited the highest ST were selected (referred to as the selected population), grown to maturity in greenhouse pots and self-pollinated to produce BC1S1 progeny seeds. The 37 selected BC1S1 progeny families were evaluated for ST and their average performance was compared with that of the parental BC1 population before selection. A realized heritability of 0.50 was obtained for ST in this population. The 37 selected BC1 plants were subjected to restriction fragment length polymorphism (RFLP) analysis using 115 markers, and marker allele frequencies were determined. Allele frequencies for the same markers were also determined in an unselected BC1 population (N = 119) of the same cross. A trait-based marker analysis (TBA), which measures differences in marker allele frequencies between selected and unselected populations, was used to identify marker-linked QTLs. Five genomic regions were detected on chromosomes 1, 3, 5, 6, and 11 bearing significant QTLs for ST. Except for the QTL on chromosome 3, all QTLs had positive alleles contributed from the salt tolerant parent LA722. Of the five QTLs, three (those on chromosomes 1, 3, and 5) were previously identified for this trait in another study, and thus were validated here. Only one of the major QTLs that was identified in our previous study was not detected here. This high level of conformity between the results of the two studies indicates the genuine nature of the identified QTLs and their potential usefulness for ST breeding using marker-assisted selection (MAS). A few BC1S1 families were identified with most or all of the QTLs and with a ST comparable to that of LA722. These families should be useful for the development of salt tolerant tomato lines via MAS.     

Genetics of drought tolerance during seed germination in tomato: inheritance and QTL mapping.

A BC1 population (N = 1000) of an F1 hybrid between a stress-sensitive Lycopersicon esculentum breeding line (NC84173; maternal and recurrent parent) and a germination stress-tolerant Lycopersicon pimpinellifolium accession (LA722) was evaluated for seed germination rate under drought stress (DS) (14% w/v polyethyleneglycol-8000, water potential approximately -680 kPa), and the most rapidly germinating seeds (first 3% to germinate) were selected. The 30 selected BC1 seedlings were grown to maturity and self pollinated to produce BC1S1 progeny seeds. Twenty of the 30 selected BC1S1 progeny families were evaluated for germination rate under DS and their average performance was compared with that of a "nonselected" BC1S1 population of the same cross. Results indicated that selection for rapid germination under DS significantly improved progeny germination rate under DS (selection gain = 19.6%), suggesting a realized heritability of 0.47 for rate of germination under DS in this population. The 30 selected BC1 plants were subjected to restriction fragment length polymorphism (RFLP) analysis, and marker allele frequencies for 119 RFLP markers which spanned 1153 cM of the 12 tomato chromosomes were determined. A distributional extreme marker analysis, which measures statistical differences in marker allele frequencies between a selected and a nonselected population, detected four quantitative trait loci (QTLs) for rate of germination under DS in this population. Of these, two QTLs, located on chromosomes 1 and 9, were contributed by the L. pimpinellifolium donor parent and had larger effects than the other two QTLs, located on chromosomes 8 and 12, which were contributed by the L. esculentum recurrent parent. A few BC1S1 families were identified with all or most of the identified QTLs and with germination rates comparable with that of LA722. These families should be useful for the development of germination drought-tolerant tomato lines using marker-assisted selection (MAS). The overall results indicate that drought tolerance during seed germination in tomato is genetically controlled and potentially could be improved by directional phenotypic selection or MAS.     

Hybrid rice (Oryza sativa L.): identification and parentage determination by RAPD fingerprinting

Summary DNA from three families of rice plants selected in Northern China (each comprising the male sterile, the restorer, the hybrid F1 and the maintainer lines) has been extracted and amplified by PCR with different random DNA primers (RAPD analysis). Then, DNA has been analysed by agarose gel electrophoresis and DNA bands scored as present or absent. The generated matrices are reproducible and amenable for identification of each single plant line. Thus, RAPD fingerprinting of the inbred parental lines and of the resulting hybrid is proposed as a convenient tool for the identification, protection and parentage determination of plant hybrids. Furthermore, by offering a molecular tool to verify the degree of dissimilarity between the parental lines, the RAPD analysis may also be used to search for new parental combinations.     

The origin of ecological divergence in Helianthus paradoxus (Asteraceae): selection on transgressive characters in a novel hybrid habitat

Diploid hybrid speciation in plants is often accompanied by rapid ecological divergence between incipient neospecies and their parental taxa. One plausible means by which novel adaptation in hybrid lineages may arise is transgressive segregation, that is, the generation of extreme phenotypes that exceed those of the parental lines. Early generation (BC2) hybrids between two wild, annual sunflowers, Helianthus annuus and Helianthus petiolaris, were used to study directional selection on transgressive characters associated with the origin of Helianthus paradoxus, a diploid hybrid species adapted to extremely saline marshes. The BC2 plants descended from a single F1 hybrid backcrossed toward H. petiolaris. The strength of selection on candidate adaptive traits in the interspecific BC2 was measured in natural H. paradoxus salt marsh habitat. Positive directional selection was detected for leaf succulence and Ca uptake, two traits that are known to be important in salt stress response in plants. Strong negative directional selection operated on uptake of Na and correlated elements. A significant decrease in trait correlations over time was observed in the BC2 population for Na and Ca content, suggesting an adaptive role for increased Ca uptake coupled with increased net exclusion of Na from leaves. Patterns of directional selection in BC2 hybrids were concordant with character expression in the natural hybrid species, H. paradoxus, transplanted into the wild. Moreover, the necessary variation for generating the H. paradoxus phenotype existed only in the BC2 population, but not in samples of the two parental species, H. annuus and H. petiolaris. These results are consistent with the hypothesis that transgressive segregation of elemental uptake and leaf succulence contributed to the origin of salt adaptation in the diploid hybrid species H. paradoxus.     

A molecular marker associated with the H21 Hessian fly resistance gene in wheat

Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat (Triticum aestivum L.) associated with the H21 gene conferring resistance to biotype L of Hessian fly [Mayetiola destructor (Say)] larvae. Near-isogenic lines were developed by backcross introgression BC3F3:4 (Coker 797 * 4 / Hamlet) and differed by the presence or absence of H21 (on 2RL) derived from Chaupon rye (Secale cereale L.). Bulked DNA samples were prepared from near-isogenic lines and BC3F2 population individuals segregating for reaction to Hessian fly biotype L and screened for random amplified polymorphic DNA markers using 46 10mer primers. Random-amplified polymorphic DNA markers from resistant and susceptible individuals and parental lines were scored and these data were used to identify a 3 kb DNA fragment that was related to the occurrence of H21. This fragment was amplified from DNA isolated from Hamlet, a near-isogenic line carrying 2RL, and bulked DNA from resistant BC3F2 individuals, but not from the recurrent parent Coker 797 or DNA bulks from susceptible BC3F2 plants. Analysis of 111 BC3F2 segregating individuals and BC3F2:3 segregants confirmed the co-segregation of the 3 kb DNA marker with the H21 resistance gene to Hessian fly. Use of this marker could facilitate more rapid screening of plant populations for Hessian fly resistance and monitoring the introgression of H21.     

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.     

Evaluation of quantitative trait loci affecting intramuscular fat and reproductive traits in pigs using marker‐assisted introgression

We investigated the effects of previously identified quantitative trait loci (QTL) in an experimental backcross (BC) between Chinese Meishan pigs and commercial Duroc pigs. We performed marker‐assisted introgression of two QTL for intramuscular fat (IMF) content (IMF population) and three QTL for reproductive traits (reproduction population) from a donor Meishan pig into a recipient Duroc pig. At the fourth BC generation of the IMF population and third BC generation of the reproduction population, carrier animals were selected for the production of animals homozygous for the QTL. Our previous studies have shown that the presence of a Meishan allele on the IMF QTL is associated with low IMF values, and the Meishan allele on the reproductive QTL is associated with large litters. In this study, the presence of a Duroc allele at the IMF QTL on SSC9 resulted in a 0.27% increase in IMF (additive effect = 0.27 ± 0.08), whereas the presence of a Meishan allele at the IMF QTL on SSC7 resulted in a 0.34% increase in IMF (additive effect = ?0.34 ± 0.09). The presence of the Meishan allele at the IMF QTL on SSC7 thus had the opposite effect to our previous studies, that is, increased IMF. In the reproduction population, we observed no differences between the genotypes of the three QTL in regard to number of corpora lutea or litter size. Marker‐assisted introgression at these QTL is thus unlikely to result in an associated increase in litter size. These results show that it is possible to introgress alleles from other breeds into a selection population using molecular markers; any unexpected results might be associated with the genetic background.     

Use of a novel outbred by inbred F1 cross to detect genetic markers for growth

A unique outbred by inbred F1 resource population was established. The population structure facilitated the unique opportunity of examining gene by genetic background interaction through crossing two modern broiler sires with dams from two unrelated inbred lines, with no selection for growth rate, to produce about 600 F1 chicks. Pools of DNA were generated from the phenotypic extremes (20% high and low) for 8-week body weight for each of the four combinations of sire and dam line. For one sire family, pools were also separately generated for each sex. The pools were genoyped with 25 informative (segregating) microsatellites. This unique F1 cross between outbred and inbred populations allowed use of the inbred alleles as an 'internal control' for polymerase chain reaction amplification quality in DNA pools. Ten microsatellites showed marked differences (P < 0.05) in allele frequencies between high and low pools, suggesting an association between marker and quantitative trait loci (QTL). These differences were verified using selective genotyping. For many markers, differences in allele frequencies between the high and the low pools, or marker effect, varied between the two dam lines and the two sexes, suggesting an interaction between some genes and the genetic background as represented by different dam lines or sexes. The suggestive marker-QTL associations identified in this F1 population demonstrate the efficiency of this population design while different QTL effects in different genetic line crosses and sexes highlight the importance of gene by genetic background interaction in QTL detection.     

Identification of Hybrids of Painted and Milky Storks Using FTA Card-Collected Blood, Molecular Markers, and Morphologies

Suspicious hybrids of painted storks and milky storks were found in a Malaysian zoo. Blood of these birds was sampled on FTA cards for DNA fingerprinting. Of 44 optimized primers, 6 produced diagnostic markers that could identify hybrids. The markers were based on simple, direct PCR-generated multilocus banding patterns that provided two sets of genetic data, one for each of the two stork species and another for the hybrids. It also revealed that large DNA fragments (3,000 bp) could be amplified from blood collected on FTA cards. When the results of each individual bird’s DNA fingerprint were compared with plumage characters, the hybrids were found to express a range of intermediate phenotypic traits of the pure breeds with no dominant plumage characteristic from either parental species.     

Identification of the BrRHP1 locus that confers resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis) and development of linked molecular markers

Inheritance of resistance to downy mildew (Hyaloperonospora parasitica) in Chinese cabbage (Brassica rapa ssp. pekinensis) was studied using inbred parental lines RS1 and SS1 that display strong resistance and severe susceptibility, respectively. F(1), F(2), and BC(1)F(1) populations were evaluated for their responses to downy mildew infection. Resistance to downy mildew was conditioned by a single dominant locus designated BrRHP1. A random amplified polymorphic DNA (RAPD) marker linked to BrRHP1 was identified using bulked segregant analysis and two molecular markers designated BrPERK15A and BrPERK15B were developed. BrPERK15B was polymorphic between the parental lines used to construct the reference linkage map of B. rapa, allowing the mapping of the BrRHP1 locus to the A1 linkage group. Using bacterial artificial chromosome clone sequences anchored to the A1 linkage group, six simple polymerase chain reaction (PCR) markers were developed for use in marker-assisted breeding of downy mildew resistance in Chinese cabbage. Four simple PCR markers flanking the BrRHP1 locus were shown to be collinear with the long-arm region of Arabidopsis chromosome 3. The two closely linked flanking markers delimit the BrRHP1 locus within a 2.2-Mb interval of this Arabidopsis syntenic region.