甘蓝型油菜花瓣缺失基因的图谱定位

在无花瓣品系APT02和正常有花瓣品种中双4号构建的的F2分离群体中,运用AFLP和SRAP两种标记技术对甘蓝型油菜花瓣缺失基因进行分子标记和图谱定位。在两亲本间筛选20对AFLP引物和170对SRAP 引物,进一步通过BSA法筛选,获得了与甘蓝型油菜花瓣缺失基因WHB连锁的1个SRAP标记e8m3_4（600bp）和1个AFLP标记E3247_15（150bp）,标记与基因WHB之间的遗传距离分别为5 cM和13.5cM;构建了一个甘蓝型油菜(Brassica napus.L )的分子标记遗传连锁图谱,该图谱共包含213个AFLP标记、56个SRAP标记和1个形态标记,分布于17个主要连锁群、两个三联体和4个连锁对中,遗传图距总长2487.1cM,标记间平均距离为10.09 cM。通过图谱定位,控制花瓣缺失性状的基因WHB被定位到第4连锁群(LG4)上。     

利用向日葵重组自交系构建遗传图谱

以向日葵自选系K55为母本、K58为父本杂交组合,通过单粒传得到的187个F_5：6代重组自交系群体为作图材料,联合应用SSR和AFLP标记构建遗传连锁图谱。经过78对SSR引物和48对AFLP引物组合选择性扩增,分别得到341和1119条带,共1460条,分别获得多态性条带184条和393条,共577条多态性条带,占所有条带的39.52%。SSR和AFLP标记各有84个和108个多态性标记偏离孟德尔分离比例(P=0.05),共192个偏分离标记。采用JoinMap4.0软件进行连锁分析,构建了1张总长度为2759.4 cM、包含17个连锁群、连锁495个多态性标记的遗传图谱,其中偏分离标记170个,标记间的平均图距为5.57 cM。每个连锁群上分布有5~72个标记,长68.88~250.17 cM。本图谱为向日葵永久性图谱,为向日葵重要性状QTL定位和基因克隆奠定基础。     

An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.     

Anchoring of canine linkage groups with chromosome-specific markers

A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest. Received: 23 February 1999 / Accepted: 28 April 1999     

Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch

In temperate locations, terminal apices on evergrowing (also called evergreen) peach trees keep growing in winter until killed by low temperatures, while the lateral buds go into dormancy. A recessive allele of a single gene (evergrowing or evg) controls this trait in peach. The amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis were applied to construct a local genetic linkage map for the evg gene from the cross Empress op op dwarf x Evergrowing (P.I. 442380). This map, comprising nine AFLP markers and the evg locus, covers a total genetic distance of 79.3 cM. Four dominant AFLP markers (EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC) were linked to the evg locus at distances of 1, 5.3, 6.7, and 11.7 cM, respectively. EAT/MCAC and EAT/MCTA were converted into polymorphic sequence-tagged sites. Microsatellite markers in the evg region were developed from peach bacterial artificial chromosome (BAC) clones that hybridized to the AFLP marker fragments. Using three microsatellite anchor markers (pchgms12, pchgms17, and pchgms19), the local genetic linkage map was integrated into one minor linkage group of a previously constructed peach rootstock genetic linkage map. Three AFLP markers from the rootstock genetic linkage map were found linked to the evg locus.     

花椰菜遗传图谱的构建及NBS-LRR类抗性同源基因在图谱中的定位

利用AFLP和NBS profiling技术, 以花椰菜自交系“AD白花”与高代自交不亲和系“C-8”杂交得到的F1代自交产生的F2代分离群体为材料, 构建了第一个花椰菜遗传连锁图谱。该图谱由234个AFLP标记和21个NBS标记构成了9个连锁群, 总图距为668.4 cM, 标记间平均距离为2.9 cM。每个连锁群包含的位点数从12到47个, 相邻两标记之间的距离范围是0~14.9 cM。NBS标记分布在8个连锁群中, 这些标记大部分聚在一起。本研究为今后的基因定位及重要农艺性状的分析提供框架图。此外, 研究NBS profiling 方法在花椰菜中的稳定性和有效性以及NBS-LRR类RGA在花椰菜基因组中的分布和特点。     

An integrated genetic map of Populus deltoides based on amplified fragment length polymorphisms

Amplified fragment length polymorphism (AFLP) is an efficient molecular technique for generating a large number of DNA-based genetic markers in Populus. We have constructed an integrated genetic map for a Populus backcross population derived from two selected P. deltoides clones using AFLP markers. A traditional strategy for genetic mapping in outcrossing species, such as forest trees, is based on two-way pseudo-testcross configurations of the markers (testcross markers) heterozygous in one parent and null in the other. By using the markers segregating in both parents (intercross markers) as bridges, the two parent-specific genetic maps can be aligned. In this study, we detected a number of non-parental heteroduplex markers resulting from the PCR amplification of two DNA segments that have a high degree of homology to one another but differ in their nucleotide sequences. These heteroduplex markers detected have served as bridges to generate an integrated map which includes 19 major linkage groups equal to the Populus haploid chromosome number and 24 minor groups. The 19 major linkage groups cover a total of 2,927 cM, with an average spacing between two markers of 23. 3 cM. The map developed in this study provides a first step in producing a highly saturated linkage map of the Populus deltoides genome. Received: 10 September 1999 / Accepted: 3 November 1999     

Genetic Linkage Maps of <Emphasis Type="Italic">Betula platyphylla</Emphasis> Suk Based on ISSR and AFLP Markers

Two separate genetic linkage maps for Chinese silver birch based on inter-simple sequence repeat (ISSR) and amplified fragment-length polymorphism (AFLP) were constructed by a pseudo-testcross mapping strategy. Eighty F₁ progenies were obtained from the cross between two parental trees with desirable traits (the paternal one selected from ‘Qinghai’ and the maternal one from ‘Wangqing’). A total of 46 ISSR primers and 31 AFLP primers were employed to generate 102 ISSR and 355 AFLP polymorphic markers in the F₁ progenies. About 5.7% of all the markers displayed high segregation distortion with a P value below 0.01 and such markers were not used for map constructions. The paternal map consisted of 137 loci, spread over 13 groups and spanned 694.2 cM at an average distance of 5.1 cM between the markers, while in the maternal map, 147 loci were distributed in 14 groups covering a map distance about 949.62 cM at an average distance of 6.5 cM. These initial maps can serve as the basis for developing a more detailed genetic map.     

A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic kinkage map of Mycosphaerella graminicola,the septoria tritici leaf blotch pathogen of wheat

An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.     

AFLP-based genetic linkage map for the red flour beetle (Tribolium castaneum)

The red flour beetle (Tribolium castaneum) is a major pest of stored grain and grain products and a popular model species for a variety of ecological, evolutionary, and developmental biology studies. Development of a linkage map based on reproducible and highly polymorphic molecular markers would greatly facilitate research in these disciplines. We have developed a genetic linkage map using 269 amplified fragment length polymorphism (AFLP) markers. Ten previously known random amplified polymorphic DNA (RAPD) markers were used as anchor markers for linkage group assignment. The linkage map was constructed through genotyping two independent F(2) segregating populations with 48 AFLP primer combinations. Each primer combination generated an average of 4.6 AFLP markers eligible for linkage mapping. The length of the integrated map is 573 cM, giving an average marker resolution of 2.0 cM and an average physical distance per genetic distance of 350 kb/cM. A cluster of loci on linkage group 3 exhibited significant segregation distortion. We have also identified six X-linked and two Y-linked markers. Five mapped AFLP fragments were sequenced and converted to sequence-tagged site (STS) markers.     

A first linkage map of olive (Olea europaea L.) cultivars using RAPD,AFLP, RFLP and SSR markers

The first linkage map of the olive (Olea europaea L.) genome has been constructed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLP) as dominant markers and a few restriction fragment length polymorphisms (RFLP) and simple-sequence repeats (SSR) as codominant markers. Ninety-five individuals of a cross progeny derived from two highly heterozygous olive cultivars, Leccino and Dolce Agogia, were used by applying the pseudo test-cross strategy. From 61 RAPD primers 279 markers were obtained - 158 were scored for Leccino and 121 for Dolce Agogia. Twenty-one AFLP primer combinations gave 304 useful markers - 160 heterozygous in Leccino and 144 heterozygous in Dolce Agogia. In the Leccino map 249 markers (110 RAPD, 127 AFLP, 8 RFLP and 3 SSR) were linked. This resulted in 22 major linkage groups and 17 minor groups with fewer than four markers. In the Dolce Agogia map, 236 markers (93 RAPD, 133 AFLP, 6 RFLP and 4 SSR) were linked; 27 major linkage groups and three minor groups were obtained. Codominant RFLPs and SSRs, as well as few RAPDs in heteroduplex configuration, were used to establish homologies between linkage groups of both parents. The total distance covered was 2,765 cM and 2,445 cM in the Leccino and Dolce Agogia maps, respectively. The mean map distance between adjacent markers was 13.2 cM in Leccino and 11.9 cM in Dolce Agogia, respectively. Both AFLP and RAPD markers were homogeneously distributed in all of the linkage groups reported. The stearoyl-ACP desaturase gene was mapped on linkage group 4 of cv. Leccino.     

Construction of a genetic linkage map of black gram, Vigna mungo (L.) Hepper, based on molecular markers and comparative studies

A genetic linkage map of black gram, Vigna mungo (L.) Hepper, was constructed with 428 molecular markers using an F9 recombinant inbred population of 104 individuals. The population was derived from an inter-subspecific cross between a black gram cultivar, TU94-2, and a wild genotype, V. mungo var. silvestris. The linkage analysis at a LOD score of 5.0 distributed all 428 markers (254 AFLP, 47 SSR, 86 RAPD, and 41 ISSR) into 11 linkage groups. The map spanned a total distance of 865.1 cM with an average marker density of 2 cM. The largest linkage group spanned 115 cM and the smallest linkage group was of 44.9 cM. The number of markers per linkage group ranged from 11 to 86 and the average distance between markers varied from 1.1 to 5.6 cM. Comparison of the map with other published azuki bean and black gram maps showed high colinearity of markers, with some inversions. The current map is the most saturated map for black gram to date and will provide a useful tool for identification of QTLs and for marker-assisted selection of agronomically important characters in black gram.     

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.     

Evaluation of AFLP for genetic mapping in Pinus radiata D. Don

Efficient construction of reasonable density genetic linkage maps is an essential component of QTL detection programmes. The AFLP technique has been used to produce genetic linkage maps in a range of species. We have developed protocols to generate reproducible AFLP profiles in Pinus radiata and have evaluated the inheritance and informativeness of AFLP markers in this important timber species. The large genome size of P. radiata necessitated increased levels of selection at both the pre-amplification and selective amplification steps of the AFLP protocol to generate reproducible AFLP profiles. Once optimised ca. 41.3 scorable AFLP bands were resolvable through denaturing gels, of which 48.4% were polymorphic in a screen of eight unrelated trees. This level of polymorphism is ca. three times higher than with RAPD markers. The total number of bands and the number of polymorphismic bands per PCR were ca. halved when AFLPs were electrophoresed on non-denaturing gels and stained with ethidium bromide. Using the protocols developed, AFLP is an efficient method for generating the DNA markers required for genetic linkage map construction in P. radiata. This revised version was published online in June 2006 with corrections to the Cover Date.     

Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch

Genetic linkage maps have been increasingly developed for a wide variety of plants, using segregating populations such as F₂s or backcrosses between inbred lines. These pedigrees are rarely available in outbred species like forest trees which have long generation times. Thus genetic mapping studies have to use peculiar pedigrees and markers in appropriate configurations. We constructed single-tree genetic linkage maps of European larch (Larix decidua Mill.) and Japanese larch [Larix kaempferi (Lamb.) Carr.] using segregation data from 112 progeny individuals of an hybrid family. A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, i.e.heterozygous in one parent and null in the other parent, were grouped at LOD 4.0, θ=0.3. The maternal parent map (L. decidua)consisted of 117 markers partitioned within 17 linkage groups (1152 cM) and the paternal parent map (L. kaempferi) had 125 markers assembled into 21 linkage groups (1206 cM). The map distance covered by markers was determined by adding a 34.7-cM independence distance at the end of each group and unlinked marker. It reached 2537 cM and 2997 cM respectively for European larch and Japanese larch, and represented respectively a 79.6% and 80.8% coverage of the overall genome. A few 3:1 segregating markers were used to identify homologous linkage groups between the European larch and the Japanese larch genetic maps. The PCR-based molecular markers allowed the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies. Received: 15 March 1999 / Accepted: 29 March 1999     

A high-density molecular map for ryegrass (Lolium perenne) using AFLP markers

AFLP markers have been successfully employed for the development of a high-density linkage map of ryegrass (Lolium perenne L.) using a progeny set of 95 plants from a testcross involving a doubled-haploid tester. This genetic map covered 930 cM in seven linkage groups and was based on 463 amplified fragment length polymorphism (AFLP) markers using 17 primer pairs, three isozymes and five EST markers. The average density of markers was approximately 1 per 2.0 cM. However, strong clustering of AFLP markers was observed at putative centromeric regions. Around these regions, 272 markers covered about 137 cM whereas the remaining 199 markers covered approximately 793 cM. Most genetic distances between consecutive pairs of markers were smaller than 20 cM except for five gaps on groups A, C, D, F and G. A skeletal map with a uniform distribution of markers can be extracted from this high-density map, and can be applied to detect and map QTLs. We report here the application of AFLP markers to genome mapping, in Lolium as a prelude to quantitative trait locus (QTL) identification for diverse agronomic traits in ryegrass and for marker-assisted plant breeding. Received: 4 November 1998 / Accepted:15 March 1999     

A genetic linkage map of cowpea (Vigna unguiculata) developed from a cross between two inbred, domesticated lines

 We have constructed a genetic linkage map within the cultivated gene pool of cowpea (2n=2x=22) from an F₈ recombinant inbred population (94 individuals) derived from a cross between the inbreds IT84S-2049 and 524B. These breeding lines, developed in Nigeria and California, show contrasting reactions against several pests and diseases and differ in several morphological traits. Parental lines were screened with 332 random RAPD decamers, 74 RFLP probes (bean, cowpea and mung bean genomic DNA clones), and 17 AFLP primer combinations. RAPD primers were twice as efficient as AFLP primers and RFLP probes in detecting polymorphisms in this cross. The map consists of 181 loci, comprising 133 RAPDs, 19 RFLPs, 25 AFLPs, three morphological/classical markers, and a biochemical marker (dehydrin). These markers identified 12 linkage groups spanning 972 cM with an average distance of 6.4 cM between markers. Linkage groups ranged from 3 to 257 cM in length and included from 2 to 41 markers, respectively. A gene for earliness was mapped on linkage group 2. Seed weight showed a significant association with a RAPD marker on linkage group 5. This map should facilitate the identification of markers that “tag” genes for pest and disease resistance and other traits in the cultivated gene pool of cowpea. Received: 16 September 1996 / Accepted: 25 April 1997     

A combined RFLP and AFLP linkage map of upland rice (Oryza sativa L.) used to identify QTLs for root-penetration ability

Acombined RFLP and AFLP linkage map of an F₆ recombinant inbred population, which was derived from a previously mapped F₂ of a cross between the two drought resistant upland rice varieties Bala and Azucena, is presented. The map contains 101 RFLP and 34 AFLP markers on 17 linkage groups covering 1680 cM. Also presented is the approximate mapping position of a further four RFLP and 75 AFLP markers, which either could not be given a unique place on the map or for which the available data is not sufficient to allow confident positioning, and the result of quantitative trait locus (QTL) mapping of traits related to root-penetration ability. Root penetration was assessed by counting the number of root axes that penetrated a 3 mm-thick layer consisting of 80% wax and 20% white soft paraffin. Good root penetration would be expected to increase drought resistance where soil strength is high. Single-marker analysis revealed seven QTLs for the number of roots which penetrate the wax layer. In identical locations were seven QTLs for the ratio of penetrated to the total number of roots. Transgressive inheritance of positive alleles from Bala explained four of these QTLs. Comparison of the QTLs identified here with previous reports of QTLs for root morphology suggest that alleles which improve root penetration ability may also either make the roots longer or thicker. Received: 3 February 1999 / Accepted: 30 April 1999     

Inheritance and mapping of 11 avirulence genes in Phytophthora sojae

Two new crosses involving four races (races 7, 16, 17, and 25) of the soybean root and stem rot pathogen Phytophthora sojae were established (7/16 cross; 17/25 cross). An F2 population derived from each cross was used to determine the genetic basis of avirulence towards 11 different resistance genes in soybean. Avirulence was found to be dominant and determined by a single locus for Avr1b, 1d, 1k, 3b, 4, and 6, as expected for a simple gene-for-gene model. We also observed several cases of segregation, inconsistent with a single dominant gene being solely responsible for avirulence, which suggests that the genetic background of the different crosses can affect avirulence. Avr4 and 6 cosegregated in both the 7/16 and 17/25 crosses and, in the 7/16 cross, Avr1b and 1k were closely linked. Information from segregating RAPD, RFLP, and AFLP markers screened on F2 progeny from the two new crosses and two crosses described previously (a total of 212 F2 individuals, 53 from each cross) were used to construct an integrated genetic linkage map of P. sojae. This revised genetic linkage map consists of 386 markers comprising 35 RFLP, 236 RAPD, and 105 AFLP markers, as well as 10 avirulence genes. The map is composed of 21 major linkage groups and seven minor linkage groups covering a total map distance of 1640.4cM.     

AFLP-based genetic linkage map of the Colorado potato beetle Leptinotarsa decemlineata: sex chromosomes and a pyrethroid-resistance candidate gene

A genetic linkage map was constructed from an intraspecific cross of the Colorado potato beetle, Leptinotarsa decemlineata. This is an initial step toward mapping the loci that underlie important phenotypes associated with insect adaptation to an agroecosystem. The map was made with 172 AFLP and 10 anonymous codominant markers segregating among 74 backcross (BC(1)) individuals. Markers were mapped to 18 linkage groups and a subset of the markers with a mean intermarker distance of 11.1 cM is presented. A pyrethroid-resistance candidate gene, LdVssc1, was placed onto the map as well. The sex chromosome was identified by exploiting the XO nature of sex determination in this species using patterns of variation at LdVssc1 and the codominant markers.