Segregant Analysis Method Research Articles

Penetrance of a rare familial mutation predisposing to papillary thyroid cancer.

Familial non-medullary thyroid cancer (FNMTC) is clinically defined as two or more first-degree relatives with NMTC and appears to follow an autosomal dominant inheritance pattern. Approximately 5-7% of NMTC is hereditary and affects multiple generations with a young age of onset. The primary aim of this study was to determine the age-specific penetrance of NMTC in individuals from a large family with FNMTC with a previously identified private mutation at 4q32, with a secondary aim to determine the penetrance for benign thyroid disease in this family. We present a large family with NMTC in which we had previously described a culpable mutation. Participants provided their personal medical history and family history. The germline 4q32 A > C mutation was detected in 34 of 68 tested individuals. Age-specific penetrance of thyroid cancer and benign thyroid disease was determined using the inverted Kaplan-Meier method of segregation analysis. Individuals who tested positive for the 4q32 mutation have a 68.9% (95% CI 46.5-88.7) risk of developing thyroid cancer by age 70 and a 65.3% (95% CI 46.0-83.8) risk of developing benign thyroid disease by age 70. The 4q32 A > C mutation significantly increases the risk to develop thyroid cancer but not benign thyroid disease in members of this family. The female:male sex ratio of 1.33 that we observed in affected mutation carriers differs greatly from the ratio of approximately 3:1 observed in PTC, supporting a central role of the mutation. Early thyroid surveillance with annual ultrasound is recommended to individuals testing positive for this private familial mutation.

Interval mapping for red/green skin color in Asian pears using a modified QTL-seq method

Pears with red skin are attractive to consumers and provide additional health benefits. Identification of the gene(s) responsible for skin coloration can benefit cultivar selection and breeding. The use of QTL-seq, a bulked segregant analysis method, can be problematic when heterozygous parents are involved. The present study modified the QTL-seq method by introducing a |Δ(SNP-index)| parameter to improve the accuracy of mapping the red skin trait in a group of highly heterozygous Asian pears. The analyses were based on mixed DNA pools composed of 28 red-skinned and 27 green-skinned pear lines derived from a cross between the ‘Mantianhong’ and ‘Hongxiangsu’ red-skinned cultivars. The ‘Dangshansuli’ cultivar genome was used as reference for sequence alignment. An average single-nucleotide polymorphism (SNP) index was calculated using a sliding window approach (200-kb windows, 20-kb increments). Nine scaffolds within the candidate QTL interval were in the fifth linkage group from 111.9 to 177.1 cM. There was a significant linkage between the insertions/deletions and simple sequence repeat markers designed from the candidate intervals and the red/green skin (R/G) locus, which was in a 582.5-kb candidate interval that contained 81 predicted protein-coding gene models and was composed of two subintervals at the bottom of the fifth chromosome. The ZFRI 130-16, In2130-12 and In2130-16 markers located near the R/G locus could potentially be used to identify the red skin trait in Asian pear populations. This study provides new insights into the genetics controlling the red skin phenotype in this fruit.

Rapid mapping and cloning of the virescent-1 gene in cotton by bulked segregant analysis-next generation sequencing and virus-induced gene silencing strategies.

Map-based gene cloning is a vital strategy for the identification of the quantitative trait loci or genes underlying important agronomic traits. The conventional map-based cloning method is powerful but generally time-consuming and labor-intensive. In this context, we introduce an improved bulked segregant analysis method in combination with a virus-induced gene silencing (VIGS) strategy for rapid and reliable gene mapping, identification and functional verification. This method was applied to a multiple recessive marker line of upland cotton, Texas 582 (T582), and identified unique genomic positions harboring mutant loci, showing the reliability and efficacy of this method. The v1 locus was further fine-mapped. Only one gene, GhCHLI, which encodes one of the subunits of Mg chelatase, was differentially down-regulated in T582 compared with TM-1. A point mutation occurred in the AAA+ conserved region of GhCHLI and led to an amino acid substitution. Suppression of its expression by VIGS in TM-1 resulted in a yellow blade phenotype that was similar to T582. This integrated approach provides a paradigm for the rapid mapping and identification of the candidate genes underlying the genetic traits in plants with large and complex genomes in the future.

Assessing the pathogenicity of RYR1 variants in malignant hyperthermia

Background.Missense variants in the ryanodine receptor 1 gene (RYR1) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. MethodsWe identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. ResultsWe identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. ConclusionsSegregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.

Investigation and genetic mapping of a Glomerella leaf spot resistance locus in apple

AbstractApple Glomerella leaf spot (GLS) is a severe fungal disease that damages apple leaves during the summer in China. Breeding new apple varieties that are resistant to the disease is considered the best way of controlling GLS. Fine mapping and tightly linked marker are critically essential for the preselection of resistant seedlings. In this study, a population of 207 F1 individuals derived from a cross between ‘Golden Delicious’ and ‘Fuji’ was used to construct a fine simple sequence repeat (SSR)‐based genetic linkage map. The position of Rgls, a locus responsible for resistance to GLS, was identified on apple linkage group (LG) 15 using SSR markers CH05g05 and CH01d08, which was adapted from a published set of 300 SSR markers that were developed using the bulked segregant analysis (BSA) method. These two SSR markers flanked the gene, and its recombination rate was 8.7% and 23.2%, respectively. A total of 276 newly developed SSR markers around the target region and designed from the genome apple assembly contig of LG15 were screened. Only nine of these were determined to be linked to the Rgls locus. Thus, a total of 11 SSR markers were in linkage with Rgls, and mapped at distances ranging from 0.5 to 33.8 cM. The closest marker to the Rgls locus was S0405127, which showed a genetic distance of approximately 0.5 cM. The first mapping of the gene Rgls was constructed, and the locations of the 11 effective primers in the ‘Golden Delicious’ apple genome sequence were anchored. This result facilitates better understanding of the molecular mechanisms underlying the trait of resistance to GLS and could be used in improving the breeding efficiency of GLS‐resistant apple varieties.

Genetic Analysis and Mapping of the Purple Gene in Purple Heading Chinese Cabbage

To analyze genetic linkage and map purple gene (BrPur) controlling purple inner leaves trait of Chinese cabbage, a F2 population was constructed by selfing a F1 plant from homozygous purple heading line ‘14S839’ and homozygous orange heading line ‘14S162’. The phenotype investigation of head color showed that the segregation ratio of purple to non-purple individuals was consistent with the expected ratio of 3:1, which indicated that purple inner leaves trait is controlled by a single dominant gene. A total of 297 SSRs in whole genome of Chinese cabbage were tested by modified Bulked Segregant Analysis (BSA) method. Two linked markers flanking BrPur, A710 and A714, were obtained and BrPur was mapped on linkage group A07 based on the sequence of these two markers. Subsequently, two new markers flanking BrPur, CL-12 and B214-87, were developed based on two known anthocyanins-related genes, Br4CL3 and Bra004214. Genetic mapping of all markers in the F2 mapping population showed CL-12 and B214-87 linked to BrPur with the genetic distance of 3.1 cM and 3.5 cM, respectively.

Thermal tolerance evaluation and related microsatellite marker screening and identification in the large yellow croaker Larimichthys crocea

Thermal tolerance to high temperature was evaluated in the large yellow croaker Larimichthys crocea. The survival thermal maximum for L. crocea was 33.0°C, the 50% critical thermal maximum (50% CTMax) was 35.5°C, and the critical thermal maximum (CTMax) was 36.0°C. Three microsatellite markers (LYC0148, LYC0200 and LYC0435), associated with thermal tolerance were screened and identified using a Bulked Segregation Analysis (BSA) method. These markers have six amplified fragments in which four are related to thermal tolerance. These fragments were cloned and sequenced, and the results showed the core motif were all “AC” repeats. For LYC0148 and LYC0200, the lengths of fragments are 181 bp and 197 bp, respectively. For LYC0435, which has two fragments, the fragment lengths are 112 bp and 100 bp. The results provide useful molecular markers for thermal-tolerance breeding of large yellow croaker in the near future.

Genetics and Molecular Marker Identification of a Resistance to Glomerella Leaf Spot in Apple

Apple Glomerella leaf spot (GLS) is a destructive fungal disease that damages apple leaves during the summer in China. Breeding new disease-resistant varieties is considered to be the best way of controlling GLS. A genetic study of resistance to Glomerella leaf spot (GLS) in apple was conducted by using four F1 hybrid groups (‘Fuji’ × ‘Golden Delicious’, ‘Golden Delicious’ × ‘Fuji’, ‘Gala’ × ‘Fuji’, and ‘Fuji’ × ‘QF-2’) generated from two highly resistant varieties or selections, ‘Fuji' and ‘QF-2’, and two highly susceptible varieties, ‘Golden Delicious’ and ‘Gala’. The results showed that the separation ratios of resistant plants to the susceptible ones in the four F1 hybrid groups were statistically consistent with the theoretical ratios of 1:1, 1:1, 0:1, and 1:0. Comprehensive analysis enabled us to generate the following conclusions: GLS resistance in apple may be controlled by a single recessive gene. The genotype of the resistant plants was rr, whereas the genotypes of the susceptible ones were RR and Rr. By using ‘Golden Delicious’ × ‘Fuji’ F1 hybrid groups and the bulked segregation analysis (BSA) method, the marker S0506206-243bp associated with disease resistance character to GLS was identified through screening 500 SSR primers encompassing the entire apple genome with even coverage, and the genetic distance between the marker and the GLS resistance gene was 9.8 cM.

Preliminary studies on the molecular identification of sex in Taxus baccata L.

Abstract The scientific objective of this research was to screen random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) primers in order to find the molecular markers enabling the distinction between male and female individuals of the European yew. This is an initial step toward understanding the mechanisms of sex determination in this species. The study was conducted on European yew originating from two sites in Poland: the Zadni Gaj nature reserve near Cieszyn and the yew collection from the Botanical Garden of the Jagiellonian University in Krakow. In the present study, 716 random primers (696 RAPD primers: OPA-OPAI_1-16 and 20 ISSR primers marked as UBC) were tested to identify the sex in European yew by means of a modified bulked segregant analysis (BSA) method. The work was conducted in three stages, gradually limiting the number of primers through the elimination of primers that either did not exhibit any differences between the examined groups or did not provide amplification products. Among the tested primers, no ideal markers that would be present in all individuals of one sex but absent in the individuals of the other sex were found. However, some markers were found (A07_954, H13_729, J08_660, L12_390, U01_457, V14_527, AE03_941, AE03_1014) to occur with greater frequency in one sex. Using these, we further examined 13 band combinations (profiles) that were observed to occur only in male individuals and another 13 combinations that were observed only in female individuals, which could be used in the practical identification of sex. This is the first report to ascertain the sex of Taxus baccata trees, and it may help to determine the sex at an early stage of development.

Bulk Segregant Analysis Reveals the Genetic Basis of a Natural Trait Variation in Fission Yeast

Although the fission yeast Schizosaccharomyces pombe is a well-established model organism, studies of natural trait variations in this species remain limited. To assess the feasibility of segregant-pool-based mapping of phenotype-causing genes in natural strains of fission yeast, we investigated the cause of a maltose utilization defect (Mal-) of the S. pombe strain CBS5557 (originally known as Schizosaccharomyces malidevorans). Analyzing the genome sequence of CBS5557 revealed 955 nonconservative missense substitutions, and 61 potential loss-of-function variants including 47 frameshift indels, 13 early stop codons, and 1 splice site mutation. As a side benefit, our analysis confirmed 146 sequence errors in the reference genome and improved annotations of 27 genes. We applied bulk segregant analysis to map the causal locus of the Mal- phenotype. Through sequencing the segregant pools derived from a cross between CBS5557 and the laboratory strain, we located the locus to within a 2.23-Mb chromosome I inversion found in most S. pombe isolates including CBS5557. To map genes within the inversion region that occupies 18% of the genome, we created a laboratory strain containing the same inversion. Analyzing segregants from a cross between CBS5557 and the inversion-containing laboratory strain narrowed down the locus to a 200-kb interval and led us to identify agl1, which suffers a 5-bp deletion in CBS5557, as the causal gene. Interestingly, loss of agl1 through a 34-kb deletion underlies the Mal- phenotype of another S. pombe strain CGMCC2.1628. This work adapts and validates the bulk segregant analysis method for uncovering trait-gene relationship in natural fission yeast strains.

Molecular mapping and candidate gene analysis for yellow fruit flesh in cucumber

Vitamin A deficiency is a major health problem in many countries around the world. Efforts to alleviate this have included adding supplements to wheat and maize flour. China produces over 70 % of the world production of cucumbers, and any attribute that could increase the β-carotene content of the fruit has the potential to have a major impact on world health. Cucumbers with yellow flesh contain larger amounts of β-carotene than those with white and green flesh. In this study, yellow fruit flesh cucumber inbred line PI200815 and white fruit flesh inbred line 931 were used as parents to construct a population for genetic analysis of cucumber fruit flesh color. The F2 segregating population was analyzed by the bulked segregant analysis method and SSR analysis with 2,112 pairs of SSR primers to build a genetic map using JoinMap 4.0 to map the yellow fruit flesh gene. The results showed that the yellow fruit flesh trait of PI200815 was controlled by a single recessive gene, namely yf. A total of 12 SSR primers and five Indel markers were used to build a molecular marker linkage group. The yf gene was mapped to cucumber chromosome 7 (Chr. 7). The closest flanking markers linked to yf were yfSSR108 and yfIndel29 with genetic distances of 0.6 and 0.3 cM, respectively. The physical distance for the region harboring yf was 149.0 kb with 21 predicted candidate genes. The accuracy of marker-assisted selection breeding using the molecular markers, yfSSR108 and yfIndel29, was 92.3 and 84.6 %, respectively.

Development of AFLP markers associated with zucchini yellow mosaicvirus resistance in cucumber (Cucumis sativus L.)

Zucchini yellow mosaic virus (ZYMV) is one of the most important pathogens that cause significant yield losses in many cucurbit crops including cucumber (Cucumis sativus L). ZYMV resistance in cucumber is inherited by a single recessive gene. The purpose of this study was to identify molecular markers linked to the gene conferring ZYMV resistance in cucumber. We developed a population of 188 F2 plants derived from inbred cucumber lines. Individual F2 plants were self-pollinated to generate F3 populations. Ten randomly selected plants from each F3 population were tested for ZYMV resistance. We used a bulk segregant analysis method to identify putative molecular markers linked to ZYMV resistance. Using bulked DNA samples with parental lines and F1, a total of 170 sequence-related amplified polymorphism (SRAP), 586 simple sequence repeat (SSR), and 308 amplified fragment length polymorphism (AFLP) primer combinations were screened. Neither polymorphic SRAP nor SSR markers were linked with ZYMV resistance. Among the 308 AFLP primer combinations tested, an AFLP marker in the E-ACA/MCA primer combination showed significant association among parental lines, F1, and resistant and susceptible plants. The combination of E-ACA/M-CA was achieved on parental lines, F1, and 188 F2 individuals for confirmation of the marker segregation on the F2 population. We found that the combination of E-ACA/M-CA was linked to the zym locus with 6.91 cM.

Screening of molecular markers linked to dwarf trait in crape myrtle by bulked segregant analysis.

Plant height is one of the most important traits of plant architecture as it modulates both economic and ornamental values. Crape myrtle (Lagerstroemia indica L.) is a popular ornamental woody plant because of its long-lasting mid-summer bloom, rich colors, and diversified plant architecture. These traits also make it an ideal model of woody species for genetic analysis of many ornamental traits. To understand the inheritance of plant height and screen for genes modulating plant height in Lagerstroemia, segregation of the plant height trait was analyzed using the F1 population of L. fauriei (standard) x L. indica 'Pocomoke' (dwarf) with 96 seedlings, while dwarf genes were screened using the bulked segregant analysis method, combined with 28 amplified fragment length polymorphism primers and 41 simple sequence repeat primers. The results showed that the dwarf trait of crape myrtle was controlled by a major gene and modified by minor genes. An amplified fragment length polymorphism marker, M53E39-92, which was 23.33 cM from the loci controlling the dwarf trait, was screened. These results provide basic information for marker-assisted selection in Lagerstromia and cloning of dwarf genes in future studies.

Identification and characterization of Mini1, a gene regulating rice shoot development.

The aerial parts of higher plants are generated from the shoot apical meristem (SAM). In this study, we isolated a small rice (Oryza sativa L.) mutant that showed premature termination of shoot development and was named mini rice 1 (mini1). The mutant was first isolated from a japonica cultivar Zhonghua11 (ZH11) subjected to ethyl methanesulfonate (EMS) treatment. With bulked segregant analysis (BSA) and map-based cloning method, Mini1 gene was finally fine-mapped to an interval of 48.6 kb on chromosome 9. Sequence analyses revealed a single base substitution from G to A was found in the region, which resulted in an amino acid change from Gly to Asp. The candidate gene Os09g0363900 was predicted to encode a putative adhesion of calyx edges protein ACE (putative HOTHEAD precursor) and genetic complementation experiment confirmed the identity of Mini1. Os09g0363900 contains glucose-methanol-choline (GMC) oxidoreductase and NAD(P)-binding Rossmann-like domain, and exhibits high similarity to Arabidopsis HOTHEAD (HTH). Expression analysis indicated Mini1 was highly expressed in young shoots but lowly in roots and the expression level of most genes involved in auxin biosynthesis and signal transduction were reduced in mutant. We conclude that Mini1 plays an important role in maintaining SAM activity and promoting shoot development in rice.

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

AbstractIn this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F2‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F2 population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.

High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as ‘mite growth inhibitors’, and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a Tetranychus urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea (BPU) compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs.

Methods of segregation analysis applied to simulated multicomponent multiphase microstructures

Abstract Advanced microstructure simulation models can predict solute segregation in 2D and 3D space, which often leads to outputs of large arrays of concentration values with a multitude of detailed information that hinders straightforward evaluations. The target of this study is to establish a common evaluation method for both simulation results and experimental data as a standard for quantitative comparison and validation. For this purpose, a methodology is adopted from experimental segregation analysis to transform the multi-dimensional data into meaningful 1D segregation profiles, which can easily be plotted and discussed. As an application example, a directional solidification experiment on an AZ31 magnesium alloy is selected and the solidification process is simulated using the phase-field method. The subsequently obtained 1D segregation profiles are compared to measured segregation profiles. As part of the study, two common sorting methods are evaluated with respect to their applicability to recover the general segregation behavior and the solidification path, as well as to handle numerical noise.

Identification of sequence-related amplified polymorphism and insertion-deletion markers linked to the male fertility restorer gene of pol-like CMS06J45 in heading Chinese cabbage (Brassica rapa subsp pekinensis).

In order to map the restorer gene BrRfp of the polima (pol)-like cytoplasmic male sterility (CMS) 06J45 line in heading Chinese cabbage, an F2 segregating population with 258 individuals of CMS06J45 and the restorer line 01S325 were tested by sequence-related amplified polymorphism (SRAP) and insertion-deletion (InDel) technologies combined with the bulked segregant analysis method. As a result, two SRAP markers, me3em3.366 and pm88bg5.263, that were linked with the BrRfp gene were identified from 463 SRAP primer pairs. By cloning, sequencing, and basic local alignment search tool analysis, the two markers were targeted to the BGIScaffold000053 of Brassica rapa in the Brassica database. Using the BGIScaffold000053 sequence, four InDel primer pairs were designed and identified to be linked with the BrRfp gene in this population. Linkage analysis showed that these markers were distributed on both sides of the BrRfp gene, the linkage distances of two nearest markers InDel878.1125 and InDel920.713 were 0.82 and 0.46 cM, respectively, and the BrRfp gene was restricted to a 243-kb genomic region of B. rapa. These specific markers provided basic information for map-based cloning of the BrRfp gene and will be very valuable for the marker-assisted selection of a new restorer line in heading Chinese cabbage.

栽培菊花与菊属-近缘属属间杂种杂交后代耐盐性的遗传分析

为了解菊花近缘种属植物耐盐性的遗传规律,对栽培菊花与菊属-近缘属属间杂种杂交后代耐盐性进行了遗传分析。以栽培菊花'韩2’为母本,大岛野路菊×芙蓉菊属间杂种为父本进行杂交,以盐害指数作为指标,通过水培法对获得的F₁群体进行耐盐性鉴定,并应用植物数量性状主基因+多基因混合遗传模型,采用单个F₂世代的分离分析方法对F₁群体耐盐性进行混合遗传分析。结果发现:F₁群体的耐盐性出现广泛分离,变异系数达53.63%,盐害指数的变异范围为3.33%-96.67%;中亲优势为2.47,未达到显著水平;将后代的耐盐性分为5个级别,其中高耐的占14.52%,耐盐的占38.70%,中耐的占30.65%,敏盐的占9.68%,高敏的占6.45%。F₁群体的耐盐性符合B-2模型,由两个主效基因控制,加性效应均表现正向增效,分别为18.06和19.13,显性效应表现负向效应,分别为-17.99和-1.44,主基因遗传率为61.14%,属高度遗传力。综合分析表明:菊花近缘种属植物耐盐性可通过杂交导入栽培菊花,实现栽培菊花耐盐性遗传改良;菊花近缘种属植物盐害指数受两对主基因的控制,主基因在F₁群体的遗传率属高度遗传力,耐盐性选育可在早期世代进行。;Chrysanthemum (<em>Chrysanthemum grandiflorum</em>, sym. <em>Chrysanthemum morifolium</em>) is a leading ornamental species in garden and cut flower.The aims of this study were to find the genetic mechanism of salt tolerance of relative genera species of chrysanthemum, which will provide an experimental basis for choosing and breeding salt tolerant germplasm. In allied genera of chrysanthemum, many wild species possess elite attributes such as resistance to disease, insect, virus and abiotic stresses, <em>Crossostephium chinense</em> is one of them. But it is usually difficult to obtain hybrids between<em> Crossostephium chinense</em> and chrysanthemum cultivars. Bridge parent is an effective way to overcome the barriers of wild hybridization and transfer useful genetic variation to elite germplasm. The F1 progeny of <em>Chrysanthemum crassum </em>(Kitam. Kitam.×<em>Crossostephium chinense</em> (L.) Makino, as bridge parent, was crossed with <em>Chrysanthemum morifolium</em> ‘Han 2', then the progenies were obtained successfully. The salt tolerance of plants is a complex physiological process, but morphological changes is the most direct reflection of the stress, so salt harm index is often used as an important indicator of tolerance identification. Based on the salt harm index, salt tolerance inheritance of F₁population was investigated by single generation segregation analysis method of the mixed major gene plus polygene mixed inheritance model of quantitative traits under the treatment by the concentration of NaCl 120 mmol/L. The results showed that the transgressive segregation of salt tolerance commonly existed in F₁ progenies; the salt harm index ranged from 3.33% to 96.67%, the phenotypic coefficient of variability was 53.63%, mid-parent heterosis was 2.47, did not reach a significant level. According to the data, F₁population could be divided into high salt tolerant, salt tolerant, middle tolerant, sensitivity, and high sensitivity grade, respectively, in which 14.52% are high salt tolerant, 38.70% are salt tolerant, 30.65% are middle tolerant, 9.68% are sensitive, 6.45% are high sensitive. Based on the vaules of AIC and the tests for goodness _ of-fit under different models, salt tolerance of relative genera species of chrysanthemum was accorded with B-2 model with additive-dominant effect, additive and dominant effect of the first major gene were 18.06,-17.99; additive and dominant effect of the second major gene were 19.13,-1.44. The heritability of major genes for salt tolerance was 61.4%. These data indicated that the F1 progeny of the intergeneric, as bridge parent, crossed with chrysanthemum to innovate salt tolerant chrysanthemum germplasm is practicable. Two major genes with dominantly additive gene effects were detected for salt tolerance in relative genera species of chrysanthemum. The heritability of the major genes was high, so the salt tolerance can be screened in the earlier generation. This study just takes a single generation genetic analysis for the salt tolerance in relative genera species of chrysanthemum and failed to detect the presence of multiple genes control or estimate the impact of environmental factors on salt tolerance, but detection of these major genes controlling the salt tolerance traits would provide a theoretical basis for the further study of QTL analysis and molecular marker assisted breeding program for salt tolerance traits in chrysanthemum.

Development of Molecular Marker Linked to Cf-10 Gene Using SSR and AFLP Method in Tomato

The leaf mould resistance gene Cf-10 on tomato confered resistant or immune to all prevalent physiological races of Cladosporium fulvum presented in three northeastern provinces of China in inoculation test. In order to better utilize Cf-10 gene in a marker-assisted selection program and to permit the pyramiding of one or several resistance genes in a cultivar, tightly linked SSR and AFLP markers were obtained by the bulked segregant analysis method. One SSR marker and three AFLP markers were identified linked to Cf-10 gene, with the distance of 9.73, 5.8, 8.5, and 10.6 cM, respectively. These markers will facilitate the selection of resistant tomato germplasm containing Cf-10 gene.