玉米相对饱和遗传连锁图谱构建与一种新的AFLPs共显性分析方法探讨

利用一个F2作图群体(X178×B73),首先构建了一个含有130个SSRs的玉米连锁框架图,然后用119个AFLPs位点增加图谱密度,得到一个全长1659·3cM,标记间平均间距6·66cM的玉米相对饱和连锁图。同时,对SSRs和AFLPs的一些遗传特性进行了分析,探讨了AFLP标记进行共显性分析的一种新方法。分析表明SSRs和AFLPs分子标记具有多态性和可靠性高等特点,是构建高密度分子标记遗传连锁图的有效技术。加密的玉米遗传连锁图谱为比较基因组研究、数量性状位点(quantitativetraitloci,QTLs)克隆、杂种优势机理研究以及标记辅助选择等提供了技术基础。     

Molecular mapping of the male-sterile, female-sterile mutant gene (st8) in soybean

Soybean male-sterile, female-sterile mutant genes have been identified by genetic and cytological studies. The St8 gene has been identified as an asynaptic mutation resulting in male and female sterility. This mutant gene was derived from a gene-tagging study using the soybean w4-mutable line. In this report we identified the genetic map position of st8 via restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers. The St8 gene mutation was located between RFLP marker E107 and SSR markers Satt132, Sct_065, and Satt414 on molecular linkage group J and linked to each by 7.8 cM and 3.4 cM, respectively.     

Linkage Mapping of DNA Markers Generated with Specific and Non-Specific Gene Primers in Soybean

Polymerase chain reaction (PCR) has been used extensively in the construction of linkage maps for many cultivated crops including soybean, [Glycine max (L.) Merr]. In this study, four sets of oligonucleotide primer pairs of known genes (pearl millet Adh 1, nodule specific proline-rich protein, Drosophila homeobox, heat shock protein), several different combinations from kits A, D, E, and J of arbitrary primers and five primer pairs of soybean simple sequence repeats of varying length (Satt 9, Satt 20, Satt 42, Satt 64, and Satt 30) were utilized in PCR to identify molecular markers which were then used to construct a genetic linkage map. DNA for the PCR reactions was isolated from 65 recombinant inbred soybean lines resulting from crossing PI 290,136 and BARC-2 (Rj ₄ ), followed by self-pollination for seven generations without selection. Mapmaker 3.0, a computer package, was used for construction of the linkage map. A total of 43 polymorphic markers were identified; 30 markers were linked and distributed among 5 linkage groups while 13 markers were unlinked. Arbitrary primers revealed more polymorphisms than specific primers. A combination of arbitrary primers A5 and A18 revealed the maximum number of polymorphic bands. Five observed linkage groups can be expanded in future soybean research by using additional markers.     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

Expression of fasciation mutation in apical meristems of soybean, Glycine max (Leguminosae)

The phenotype of the apical meristem was used to examine the effect of fasciation mutation at the f locus in different genetic backgrounds in soybean Glycine max (L.) Merr. Comparisons of meristem development in fasciation mutant and wild type were conducted with scanning electron microscope (SEM) on isogenic lines BARC-11-11-ff and BARC-11-11-FF at postgermination and early vegetative stages. Studies of apical meristems of three independently originated fasciation mutants, PI 83945-4, PI 243541, and T173, were carried out at vegetative and early floral transition stages. Corolla Fasciation, the extreme mutant phenotype, was used for comparison of meristem development. Enlargement of the apical meristem and shortened plastochron were observed in the mutant lines 2 d after germination. Similar to Corolla Fasciation, in PI 83945-4, PI 243541, and T173, enlargement of the apical meristem was followed by growth along one axis at the V3 stage and establishment of a ridge-like meristem at the V4 stage. Influence of pedigree on the expression of the fasciation phenotype was demonstrated by different growth patterns (subangular vs. ridge-like) of the apical meristem in BARC-11-11-ff and PI 243541 with the same f gene. During transition of the apical meristem from vegetative to reproductive stage in all mutant lines further production of leaf primordia ceased. The developmental pattern of the apical meristems suggests that the f locus may have the same allele in fasciation mutants of independent origin in soybean.     

Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes

 Simple sequence repeats (SSRs) are versatile DNA markers that are readily assayed and highly informative. Unfortunately, non-targeted approaches to SSR development often leave large genomic regions without SSR markers. In some cases these same genomic regions are already populated by other types of DNA markers, especially restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and amplified fragment length polymorphisms (AFLPs). To identify SSR markers in such regions, bacterial artificial chromosome (BAC) clones can be used as intermediaries. First, one or more BAC clones in a region of interest are identified through the use of an existing DNA marker. BAC clones uncovered in this initial step are then used to create a small insert DNA library that can be screened for the presence of SSR-containing clones. Because BAC inserts are often 100-kb pairs or more in size, most contain one or more SSRs. This strategy was applied to two regions of the soybean genome near genes that condition resistance to the soybean cyst nematode on molecular linkage groups G and A2. This targeted approach to identifying new DNA markers can readily be extended to other types of DNA markers, including single nucleotide polymorphisms. Received: 13 August 1998 / Accepted: 13 October 1998     

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.     

Genetic identification of a female partial-sterile mutant in soybean.

We report here the genetic identification of a female partial-sterile mutant derived from soybean mutant L67-3483. L67-3483, which originated from the cultivar Clark after X-ray irradiation, is male and female fertile. All F1 plants in reciprocal pollinations of L67-3483 with 'Clark', 'Minsoy', or 'BSR 101' were female partial sterile. Partial sterility is expressed in the heterozygous condition at a single locus and upon self-pollination this locus exhibits a 1:1 segregation pattern. This locus is located on the terminus of the soybean molecular linkage group D1b+W, between simple sequence repeat (SSR) markers Satt157 and Satt266, and is linked to each by 5.3 and 1.2 cM, respectively. This gene is transmitted through both female and male gametes and there was no segregation distortion of SSR markers linked to this gene. We concluded that this female partial-sterile gene is a new mutation class, and differs from the previously reported mutation classes in soybean, i.e., sporophytic mutation, gametophytic female-specific mutation, and general gametophytic mutation. Restriction of recombination around the mutant gene suggested that this gene is located near or within (a) small inversion(s) or adjacent to (a) chromosomal deletion(s).     

A consolidated linkage map for rainbow trout (Oncorhynchus mykiss)

Androgenetic doubled haploid progeny produced from a cross between the Oregon State University and Arlee clonal rainbow trout (Oncorhynchus mykiss) lines, used for a previous published rainbow trout map, were used to update the map with the addition of more amplified fragment length polymorphic (AFLP) markers, microsatellites, type I and allozyme markers. We have added more than 900 markers, bringing the total number to 1359 genetic markers and the sex phenotype including 799 EcoRI AFLPs, 174 PstI AFLPs, 226 microsatellites, 72 VNTR, 38 SINE markers, 29 known genes, 12 minisatellites, five RAPDs, and four allozymes. Thirty major linkage groups were identified. Synteny of linkage groups in our map with the outcrossed microsatellite map has been established for all except one linkage group in this doubled haploid cross. Putative homeologous relationships among linkage groups, resulting from the autotetraploid nature of the salmonid genome, have been revealed based on the placement of duplicated microsatellites and type I loci.     

Duplicate chlorophyll-deficient loci in soybean.

Three lethal-yellow mutants have been identified in soybean (Glycine max (L.) Merr.), and assigned genetic type collection numbers T218H, T225H, and T362H. Previous genetic evaluation of T362H indicated allelism with T218H and T225H and duplicate-factor inheritance. Our objectives were to confirm the inheritance and allelism of T218H and T225H and to molecularly map the locus and (or) loci conditioning the lethal-yellow phenotype. The inheritance of T218H and T225H was 3 green : 1 lethal yellow in their original parental source germplasm of Glycine max 'Illini' and Glycine max 'Lincoln', respectively. In crosses to unrelated germplasm, a 15 green : 1 lethal yellow was observed. Allelism tests indicated that T218H and T225H were allelic. The molecular mapping population was Glycine max 'Minsoy' x T225H and simple sequence repeat (SSR) markers were used. The first locus, designated y18-1, was located on soybean molecular linkage group B2, between SSR markers Satt474 and Satt534, and linked to each by 4.4 and 13.4 cM, respectively. The second locus, designated y18-2, was located on soybean molecular linkage group D2, between SSR markers Satt543 and Sat-001, and linked to each by 2.2 and 4.4 cM, respectively.     

Mapping the <Emphasis Type="Italic">Fas</Emphasis> locus controlling stearic acid content in soybean

Increasing the stearic acid content to improve soybean [ Glycine max (L) Merr] oil quality is a desirable breeding objective for food-processing applications. Although a saturated fatty acid, stearic acid has been shown to reduce total levels of blood cholesterol and offers the potential for the production of solid fat products (such as margarine) without hydrogenation. This would result in the reduction of the level of trans fat in food products and alleviate some current health concerns. A segregating F(2) population was developed from the cross between Dare, a normal stearic acid content cultivar, and FAM94-41, a high stearic acid content line. This population was used to assess linkage between the Fas locus and simple sequence repeat (SSR) markers. Three SSR markers, Satt070, Satt474 and Satt556, were identified to be associated with stearic acid (P < 0.0001, r(2) > 0.61). A linkage map consisting of the three SSR markers and the Fas locus was then constructed in map order, Fas, Satt070, Satt474 and Satt556, with a LOD score of 3.0. Identification of these markers may be useful in molecular marker-assisted breeding programs targeting modifications in soybean fatty acids.     

Integrated map of AFLP, SSLP and RFLP markers using a recombinant inbred population of rice (Oryza sativa L.)

 A molecular map of rice consisting of 231 amplified fragment length polymorphisms (AFLPs), 212 restriction fragment length polymorphisms (RFLPs), 86 simple-sequence length polymorphisms (SSLPs), five isozyme loci, and two morphological mutant loci [phenol staining of grain (Ph), semi-dwarf habit (sd-1)] has been constructed using an F₁₁ recombinant inbred (RI) population. The mapping population consisted of 164 RI lines and was developed via single-seed descent from an intercross between the genetically divergent parents Milyang 23 (M) (tongil type) and Gihobyeo (G) ( japonica type). A subset of previously mapped RFLP and SSLP markers were used to construct the map framework. The AFLP markers were derived from ten EcoRI(+2) and MseI(+3) primer combinations. All marker types were well distributed throughout the 12 chromosomes. The integrated map covered 1814 cM, with an average interval size of 3.4 cM. The MG map is a cornerstone of the Korean Rice Genome Research Program (KRGRP) and is being continuously refined through the addition of partially sequenced cDNA markers derived from an immature-seed cDNA library developed in Korea, and microsatellite markers developed at Cornell. The population is also being used for quantitative trait locus (QTL) analysis and as the basis for marker-assisted variety development. Received: 24 June 1997 / Accepted: 25 November 1997     

A linkage map of hexaploid oat based on grass anchor DNA clones and its relationship to other oat maps.

A cultivated oat linkage map was developed using a recombinant inbred population of 136 F6:7 lines from the cross 'Ogle' x 'TAM O-301'. A total of 441 marker loci, including 355 restriction fragment length polymorphism (RFLP) markers, 40 amplified fragment length polymorphisms (AFLPs), 22 random amplified polymorphic DNAs (RAPDs), 7 sequence-tagged sites (STSs), 1 simple sequence repeat (SSR), 12 isozyme loci, and 4 discrete morphological traits, was mapped. Fifteen loci remained unlinked, and 426 loci produced 34 linkage groups (with 2-43 loci each) spanning 2049 cM of the oat genome (from 4.2 to 174.0 cM per group). Comparisons with other Avena maps revealed 35 genome regions syntenic between hexaploid maps and 16-34 regions conserved between diploid and hexaploid maps. Those portions of hexaploid oat maps that could be compared were completely conserved. Considerable conservation of diploid genome regions on the hexaploid map also was observed (89-95%); however, at the whole-chromosome level, colinearity was much lower. Comparisons among linkage groups, both within and among Avena mapping populations, revealed several putative homoeologous linkage group sets as well as some linkage groups composed of segments from different homoeologous groups. The relationships between many Avena linkage groups remain uncertain, however, due to incomplete coverage by comparative markers and to complications introduced by genomic duplications and rearrangements.     

A genetic linkage map for the ectomycorrhizal fungus Laccaria bicolor and its alignment to the whole-genome sequence assemblies

A genetic linkage map for the ectomycorrhizal basidiomycete Laccaria bicolor was constructed from 45 sib-homokaryotic haploid mycelial lines derived from the parental S238N strain progeny. For map construction, 294 simple sequence repeats (SSRs), single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers were employed to identify and assay loci that segregated in backcross configuration. Using SNP, RAPD and SSR sequences, the L. bicolor whole-genome sequence (WGS) assemblies were aligned onto the linkage groups. A total of 37.36 Mbp of the assembled sequences was aligned to 13 linkage groups. Most mapped genetic markers used in alignment were colinear with the sequence assemblies, indicating that both the genetic map and sequence assemblies achieved high fidelity. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 13 linkage groups spanning 812 centiMorgans (cM) at an average distance of 2.76 cM between markers (range 1.9-17 cM). The WGS and the present linkage map represent an initial step towards the identification and cloning of quantitative trait loci associated with development and functioning of the ectomycorrhizal symbiosis.     

A genetic linkage map for azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi]

To make progress in genome analysis of azuki bean (Vigna angularis) a genetic linkage map was constructed from a backcross population of (V. nepalensis x V. angularis) x V.angularis consisting of 187 individuals. A total of 486 markers—205 simple sequence repeats (SSRs), 187 amplified fragment length polymorphisms (AFLPs) and 94 restriction fragment length polymorphisms (RFLPs) —were mapped onto 11 linkage groups corresponding to the haploid chromosome number of azuki bean. This map spans a total length of 832.1 cM with an average marker distance of 1.85 cM and is the most saturated map for a Vigna species to date. In addition, RFLP markers from other legumes facilitated finding several orthologous linkage groups based on previously published RFLP linkage maps. Most SSR primers that have been developed from SSR-enriched libraries detected a single locus. The SSR loci identified are distributed throughout the azuki bean genome. This moderately dense linkage map equipped with many SSR markers will be useful for mapping a range of useful traits such as those related to domestication and stress resistance. The mapping population will be used to develop advanced backcross lines for high resolution QTL mapping of these traits. O.K. Han, A. Kaga, T. Isemura have contributed equally to this paper.     

A genetic map of Gibberella zeae (Fusarium graminearum)

We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is approximately 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.     

An unusual polymorphic locus useful for tagging Rps1 resistance alleles in soybean

The Phytophthora root and stem resistance locus Rps1 has been mapped to linkage group N of the USDA-ARS soybean molecular map, approximately 2 cM from locus A071-1. To determine if A071-1 polymorphisms exist that distinguish and tag different Rps1 alleles, germplasms containing the seven Rps1 alleles were screened with eight enzymes for pA071-detectable polymorphisms. Six enzymes revealed at least one polymorphic fragment. All six detected a polymorphism at A071-1 as determined by restriction fragment length polymorphism mapping, comparison to an EMBL3 clone containing locus A071-1, and Southern hybridization with probes specific for locus A071-1. Screening of the Rps1 donors and 24 Rps1-and 15 Rps1-containing U.S. soybean varieties showed that locus A071-1 exhibited three polymorphisms with each enzyme. The polymorphisms detected by one enyme did not always correlate with those detected by the other four, suggesting that multiple mutation events may be responsible for the different A071-1 polymorphisms. Although no combination of alleles distinguished Rps1-and Rps1-containing genotypes, polymorphism at A071-1 made it possible to distinguish five groups of soybean germplasms. Thus, the unusual polymorphism of locus A071-1 should useful for following Rps1 inheritance in many breeding programs.Joint contribution of North Central Region, USDA-ARS and Journal Paper no. 15383 of the Iowa Agricultural and Home Economics Experiment Station, Ames, IA 50011. Project no. 2974     

Allelism and molecular mapping of soybean necrotic root mutants.

Mutability of the w4 flower color locus in soybean, Glycine max (L.) Merr., is conditioned by an allele designated w4-m. Germinal revertants recovered among self-pollinated progeny of mutable plants have been associated with the generation of necrotic root mutations, chlorophyll-deficiency mutations, and sterility mutations. A total of 24 necrotic root mutant lines were generated from a total of 24 independent reversion events at the w4-m locus. The initial mutable population included 4 mutable categories for w4-m, designated (1) low frequency of early excisions, (2) low frequency of late excisions, (3) high frequency of early excisions, and (4) high frequency of late excisions. These mutable categories were based upon flower phenotype, i.e., somatic tissue. A total of 22 of 24 necrotic root mutations occurred from germinal reversions classified in the high frequency of excision categories. Of these 22 mutants, 14 came from early excisions and 8 came from late excisions. These necrotic root mutants were allelic to 6 previously identified necrotic root mutants derived from the study of germinal revertants, i.e., gene tagging studies, chemical mutagenesis, and "spontaneous" occurrences from genetic crosses. Thus, all 30 necrotic root mutants in soybean are allelic. An F2 mapping population from the cross of Minsoy (Rn1 Rn1) x T328 (rn1 rn1) was used to map the Rn1 locus using simple sequence repeat (SSR) markers. The Rn1 locus was located between Satt288 and Satt612 on molecular linkage group G.     

Constructing a Genetic Linkage Map Using an F<Subscript>1</Subscript> Population of Non-inbred Parents in Cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

A genetic linkage map of cassava has been generated with total of 355 molecular markers, 231 amplified fragment length polymorphisms (AFLPs), 41 simple sequence repeats (SSRs), 48 sequence-related amplified polymorphisms (SRAPs), and 35 expressed sequence tag (EST)-SSRs segregating from an F₁ population of an intraspecific cross between SC6, as female parent, and Mianbao, as male parent. The genetic map consisted of 18 linkage groups and spanned a 1,707.9 cM genetic distance, with the average marker interval being 4.81 cM. Thirty-five EST-SSR markers that were developed by our laboratory were mapped onto this map. The genetic map generated in this study will enrich the information of structural genomics in cassava, facilitating to identify quantitative trait loci (QTL) of interest and to serve as a useful complement for construction of an integrated map in the future.