Construction of a linkage map for watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai) using random amplified polymorphic DNA (RAPD)

Summary A linkage map for watermelon (Citrullus lanatus) was constructed on the basis of RADP, ribosomal DNA restriction fragment length polymorphism (RFLP), isozyme, and morphological markers using F₁BC1. A segregating population of 78 individuals was the result of a backcross of a cultivated inbred line (H-7; Citrullus lanatus; 2n=22) and a wild form (SA-1; C. lanatus; 2n=22), in which the latter was the recurrent (male) parent. A total of 69 RAPD, one RFLP, one isozyme, and three morphological markers was found to segregate in the BC1 population. Linkage analysis revealed that 62 loci could be mapped to 11 linkage groups that extended more than 524 centimorgans (cM), while 12 loci segregated independently of all other markers. The locus for exocarp color was linked to two RAPD markers within a region of 5 cM on linkage group 4. The locus for flesh color was linked to a RAPD marker within a region of 30 cM on linkage group 6. The isozyme marker GOT was located on the linkage group 1. Linkage group 2 contained a locus for ribosomal DNA within 5 cM of a RAPD marker. Half of the RAPD markers on the linkage group 7 displayed severely distorted segregation. The construction of linkage map using molecular markers is necessary for the breeding of watermelon to introduce useful gene of wild watermelon efficiently. However the linkage map that was constructed for the most part on the basis of RAPD markers could not cover significant parts of the genome, the linkage map provides breeders of watermelons the possibility of tagging useful agronomic traits, as well as the gene for exocarp color.Abbreviations RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - GOT glutamate oxaloacetate transaminase - MDH malate dehydrogenase - ACP acid phosphatase - 6PGH 6-phosphogluconate dehydrogenase     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

A genetic linkage map of rhodesgrass based on an F1 pseudo-testcross population

The linkage relationships between 164 polymorphic amplified fragment length polymorphism (AFLP) and 25 restriction fragment length polymorphism (RFLP) fragments assayed in a pseudo‐testcross population generated from the mating of single genotypes from two divergent cultivars were used to construct female, ‘Katambora’ (‘Kat’) and male, ‘Tochirakukei’ (‘Toch’) parental genetic maps for rhodesgrass. The ‘Kat’ genetic map consists of 84 marker loci (72 AFLP and 12 RFLP markers) distributed on 14 linkage groups and spans a total length of 488.3 cM, with an average distance of 7.8 cM between adjacent markers. The ‘Toch’ genetic map consists of 61 marker loci (52 AFLP and nine RFLP) mapped on 12 linkage groups spanning a total length of 443.3 cM, with an average spacing of 9.0 cM between adjacent markers. About 23% of the markers remained unassigned. The level of segregation distortion observed in this cross was 11.1%. In both maps, linked duplicated RFLP loci were found. These linkage maps will serve as a starting point for linkage studies in rhodesgrass with potential application for marker‐assisted selection in breeding programmes.     

A skeletal linkage map of Hordeum bulbosum L. and comparative mapping with barley (H. vulgare L.)

A restriction fragment length polymorphism (RFLP) based linkage map of a cross between two diploid Hordeum bulbosum (2n = 2x = 14) clones, PB1 and PB11, was constructed from 46 recombinant progeny clones. Since both parents are heterozygous, separate and combined parental maps were constructed. All of the RFLP markers screened had previously been mapped in barley (H. vulgare L.) so that comparative maps could be produced. The PB1 linkage map consists of 20 RFLP marker loci assigned to four linkage groups covering 94.3 cM. The PB11 linkage map consists of 27 RFLP marker loci assigned to six linkage groups covering 149.1 cM. Thirteen markers polymorphic in both parents were used as ‘anchors’ to create a combined linkage map consisting of 38 loci assigned to six linkage groups and covering a genetic distance of 198 cM. Marker order was highly conserved in a comparison with the linkage map of H. vulgare (Laurie etal., 1995). However, in contrast, the genetic distances for the same markers were very different being 649 cM and 198 cM respectively, a genetic distance ratio of 1: 3.3. Thus although the map was short, it can be presumed to cover half the genome of H. bulbosum. This study provides further confirmation of the close relationship between the two species and gives a basis for the development of marker mediated introgression through interspecific hybridisation between the two species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

Rhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants. The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA411₁₈₀₀ and AS7₁₁₀₀ at 9.5 and 18.5cM, respectively. Moreover, in our I₂ population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’-type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’-based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.     

Identification of resistance gene analogs as markers of disease resistance loci in oats, using near-isogenic lines

Genomic sequences with features of the major class of disease resistance genes and which bear nucleotide‐binding leucine‐rich repeat sequences (resistance gene analogs; RGA) were tested as potential markers of crown rust resistance loci in hexaploid oats. Two collections of paired near‐isogenic lines carrying resistance to different isolates of crown rust, Puccinia coronata were screened. Two out of the four RGAs assayed showed restriction fragment length polymorphism (RFLP) between one line of each collection and its recurrent parent. The paired lines X466 and D494 were polymorphic for RGA III2.2 and the pair of lines X470 and D504 were polymorphic for RGA III2.18. The III2.18 polymorphism was located in the hexaploid map Avena byzantina cv. ‘Kanota’ × A. sativa cv. ‘Ogle’ in linkage group KO17 in a region previously associated with crown rust resistance. In addition, 220 random primers were used for random amplified polymorphic DNA (RAPD) analysis to screen the two sets of NILs. Only one polymorphic band was obtained that differentiated the paired lines X470 and D504 from their parents. The RAPD band was used as a probe and the relevant RFLP that differentiated the NILs X470 and D504 was found at 1.7 cM from the III2.18 marker in KO17. RFLP analysis using probes previously mapped in KO17 confirmed differences for X470 and D504 in the region around the III2.18 marker. These results suggest that the resistance locus shared by this pair of NILs is probably linked to the markers revealed by RGA III2.18. The use of RGAs as RFLP probes in the screening of NILs with differences in crown rust resistance has proved to be more effective than RAPDs for finding polymorphic markers possibly linked to resistance loci.     

Inheritance and mapping of a light green mutant in cultivated diploid potatoes

A light green mutant was found in a population of adapted cultivated diploid potatoes. Genetic analysis indicates that this trait is controlled by a single nuclear gene. The gene symbol lg is proposed. The segregation ratios fit a pattern which strongly suggest that there is a close linkage between the Lg allele and a locus which confers lethality in its homozygous recessive state. Some crossing over between the lg locus and the lethal was found to occur but LgLg genotypes were not observed in progenies from sib-matings. The lg locus mapped to the potato linkage group VI between the restriction fragment length polymorphism (RFLP) loci CP18 and GP24. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mapping a new source of resistance to powdery mildew in mungbean

Both restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) analyses were employed to map a new source of resistance to powdery mildew in mungbean. Disease scores of an F₂ population derived from the cross between a moderately resistant breeding line VC1210A and a susceptible wild relative (Vigna radiata var. sublobata, accession TC1966) showed a continuous distribution and was treated as a quantitative trait. Although no significant quantitative trait loci (QTL) that can explain the variation was detected by QTL analysis based on the reconstructed RFLP linkage map, new marker loci associated with resistance were discovered by AFLP analysis. The RFLP loci detected by two of the cloned AFLP bands are associated with resistance and constitute a new linkage group. A major resistance quantitative trait locus was found on this linkage group that accounted for 64.9% of the variation in resistance to powdery mildew. One of the probes developed in this study has the potential to assist in breeding for powdery mildew resistance in mungbean.     

An Extended Linkage Map of Sugar Beet (Beta vulgaris L.) Including Nine Putative Lethal Genes and the Restorer Gene X

An extended genetic map of sugar beet (Beta vulgaris L.) is presented encompassing 177 segregating markers (2 morphological traits, 7 isozymes, and 168 RFLP markers) on 9 linkage groups. The linkage map comprises 1057.3 cM equivalent to an average genetic spacing of 6.0 cM/marker. The length of individual linkage groups varies between 80.7 (group VIII) and 167.4 cM (group VIII). The number of markers per linkage group ranges between 13 and 24. No indication of duplicate regions was found, confirming the true diploid nature of B. vulgaris. Twenty-six markers (15 %) deviated significantly (a = 0.01) from the expected segregation ratio. This distorted segregation was probably caused by linkage with lethal genes. Four such genes (designated Let Ib, Let 5b, Let 6b, Let 8) could be located at discrete positions due to their absolute linkage to skewed RFLP markers. The restorer gene X has been located terminally on linkage group ÜI, 9.6 cM distant from RFLP marker pKP1238.     

RFLP-based mapping of the Sec-2 and Sec-5 loci encoding 75K γ-secalins of rye

For mapping the Sec2 and Sec5 loci of rye which determine expression of 75K γ-secalins, a partial genetic map of chromosome 2R spanning 64 cM was constructed. The map was developed using an F₂ population of 103 plants from a cross between two inbred lines. Both loci were mapped distally on the short arm of chromosome 2R and clearly tagged in relation to 12 restriction fragment length polymorphism (RFLP) markers. The Sec2 locus was localized between the Xiag57 and Xpsr109a loci in an 11 cM interval. The Sec5 locus co-segregated to Xiag57 and was tightly linked to the Sec2 locus at a map distance of 0.5cM.     

Construction of a linkage map for quantitative trait loci associated with economically important traits in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is the most widely cultivated and high-value turfgrass species. Genetic linkage maps of creeping bentgrass were constructed for quantitative trait loci (QTL) analysis of gray snow mold (Typhula incarnata) resistance, recovery and leaf width. A segregating population of 188 pseudo-F₂ progeny was developed by two-way pseudo-testcross mapping strategy. Amplified fragment length polymorphism, new developed Agrostis specific expressed sequence tag-single sequence repeat (SSR), random amplified polymorphic DNA and genomic SSR markers corresponding to DNA polymorphisms heterozygous in one parent and null in the other, were scored and placed on two separate genetic linkage maps, representing each parent. In the male parent map, 100 markers were mapped to 14 linkage groups covering a total length of 793?cM with an average interval of 8.2?cM. In the female parent map, 146 markers were clustered in another 14 linkage groups spanning 805?cM with an average distance of 5.9?cM between adjacent markers. We identified three putative QTL for leaf width and one QTL for snow mold disease resistance. The construction of a linkage map and QTL analysis are expected to facilitate the development of disease resistant creeping bentgrass cultivars by using molecular marker-assisted selection.     

Development of PCR-based markers for a major locus conferring powdery mildew resistance in mungbean (Vigna radiata)

Powdery mildew (PM) can cause significant yield loss in mungbean and several loci conferring resistance to this disease have been identified. A restriction fragment length polymorphism (RFLP) marker (VrCS65) linked closely to one of these loci was used to screen a mungbean bacterial artificial chromosome (BAC) library and positive BAC clones identified were used to develop simple sequence repeat (SSR or microsatellite) and sequence tagged site (STS) markers. Four of the new PCR markers (including two SSRs and two STSs) co-segregated with the original RFLP marker VrCS65, and another SSR marker (VrCS SSR2) was located 0.5 cM away from it. These PCR-based and locus-specific markers could be useful in breeding cultivars with enhanced resistance to PM and in the further characterization of the locus including the isolation of gene(s) responsible for the resistance.     

大豆重组自交系群体NJRIKY遗传图谱的加密及其应用效果

作物基因组研究,包括基因或数量性状位点(QTL)定位、图位克隆以及物理图谱构建等,首先必须建立具有丰富标记信息的高密度遗传连锁图谱。由科丰1号和南农1138-2杂交组合衍生的重组自交系群体NJRIKY已经构建了4张大豆遗传连锁图谱,但由于遗传信息和标记数目不够充分,在基因和QTL作图时仍然存在精确度和准确度问题。为增加NJRIKY图谱密度,本研究在967对SSR引物中获得了401个多态性SSR标记。结合其他分子数据,使用作图软件Mapmaker/Exp3.0b,获得一张含有553个遗传标记,25个连锁群,总长2071.6cM,平均图距3.70cM的新遗传连锁图谱,其中SSR标记316个,RFLP标记197个,EST标记39个,形态标记1个。连锁群上大于20cM的标记间隔由原来42个减少到2个。原图谱的3个SMV抗性基因定位于D1b连锁群末端的开放区间上且仅与一个RFLP标记连锁,利用加密图谱对Rsc-3、Rsc-7、Rsc-9、Rsc-13、Rsa、Rn1和Rn3等7个SMV抗性基因重定位,全部位于D1b连锁群,与相邻分子标记距离均小于6cM,其中Rsc-9、Rn1、Rsa的距离小于1cM,Rsc-13与EST标记GMKF168a共分离。对本群体农艺性状进行QTL重定位,获得8个性状相关的42个主效QTL,其中20个QTL遗传贡献率大于10%,与原图谱比较,新定位的各QTL的标记区间明显缩短,与相邻标记的连锁更加紧密。     

Evaluation of F2 and F3 plants introgressed with QTLs for clubroot resistance in cabbage developed by using SCAR markers

Clubroot disease caused by Plasmodiophora brassicae is one of the major diseases of Brassica crops, often devastating to the cultivation of cruciferous crops in temperate regions. In a previous study (Moriguchi et al. 1999) identified three major quantitative trait loci (QTLs) for clubroot resistance, each in a separate linkage group, in a population derived from a cross between a clubroot‐susceptible inbred cabbage line, Y2A and a resistant inbred kale line, K269. In this study, the original random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were converted into sequence‐characterized amplified region (SCAR) markers to facilitate large‐scale marker‐assisted screening of clubroot resistance in cabbage breeding. Of 15 RAPD markers closely linked to the three QTLs, nine SCARs were developed as dominant markers after cloning and sequencing. In addition, two RAPD markers were converted into co‐dominant cleaved amplified polymorphic sequence (CAPS) markers, and one RFLP marker out of three tested was converted to a dominant SCAR marker. The effect of selection for resistance by the improved markers was evaluated in progeny plants in the F2 and F3. A total of 138 F2 plants were genotyped with nine SCARs and 121 well‐distributed makers consisting of 98 RAPD, 19 RFLP, two isozymes, and two morphological markers in order to estimate the level of resistance and the proportion of undesirable alleles from the kale in non‐target areas in each of the F2 populations. An F2 plant, YK118, had kale alleles at QTL1, QTL3 and QTL9. Three F2 plants, namely, YK107, YK25 and YK51 had kale alleles at only QTL1, QTL3 and QTL9, respectively. These F2 plants were selected for their low proportion of alleles derived from kale in non‐target regions. YK118, like the resistant kale parent, expressed very high resistance to three field isolates of Plasmodiophora brassicae, whereas the mean disease index in the F2 and F3 plants carrying only single QTLs was intermediate. The QTLs showed no differential response to the isolates. These plants with improved resistance will be useful as parental inbred lines for F1 hybrids.     

RFLP/AFLP mapping of a brown planthopper (Nilaparvata lugens Stål) resistance gene Bph1 in rice

We have constructed a linkage map of the rice brown planthopper (BPH)resistance gene, Bph1. RFLP and AFLP markers were selected by thebulked segregant analysis and used in the mapping study of 262 F₂sthat were derived from a cross of `Tsukushibare', a susceptible japonica cultivar, and `Norin-PL3', an authentic japonicaBph1-introgression line. Twenty markers were mapped within a 28.9-cMregion containing the Bph1 locus on the long arm of rice chromosome12. Combining the result of segregation analysis of BPH resistance by themass seedling test and that of the markers, the Bph1 locus wasmapped within a 5.8-cM region between two flanking markers. The closestAFLP markers, em5814N and em2802N, was at 2.7 cM proximal to theBph1 locus. Together with the previously constructed high-resolutionmap of bph2 locating the locus at ca. 10 cM proximal to the Bph1 locus, this improved version of the linkage map would facilitatepyramiding these two important BPH resistance genes.     

Mapping genes for double podding and other morphological traits in chickpea

Seed traits are important considerations for improving yield and product quality of chickpea (Cicer arietinum L.). The purpose of this study was to construct an intraspecific genetic linkage map and determine map positions of genes that confer double podding and seed traits using a population of 76 F₁₀ derived recombinant inbred lines (RILs) from the cross of ‘ICCV-2’ (large seeds and single pods) × ‘JG-62’ (small seeds and double podded). We used 55 sequence-tagged microsatellite sites (STMS), 20 random amplified polymorphic DNAs (RAPDs), 3inter-simple sequence repeats (ISSR) and 2 phenotypic markers to develop a genetic map that comprised 14 linkage groups covering297.5 cM. The gene for double podding (s) was mapped to linkage group 6 and linked to Tr44 and Tr35 at a distance of7.8 cM and 11.5 cM, respectively. The major gene for pigmentation, C, was mapped to linkage group 8 and was loosely linked to Tr33 at a distance of 13.5 cM. Four QTLs for 100 seed weight (located on LG4 and LG9), seed number plant^-1 (LG4), days to 50% flower (LG3) were identified. This intraspecific map of cultivated chickpea is the first that includes genes for important morphological traits. Synteny relationships among STMS markers appeared to be conserved on six linkage groups when our map was compared to the interspecific map presented by Winter et al. (2000). This revised version was published online in August 2006 with corrections to the Cover Date.     

A genetic linkage map of Vigna vexillata

Vigna vexillata is a wild cross‐incompatible relative of cowpea. It is highly resistant to several diseases and pests plaguing cowpea. A linkage map was developed for V. vexillata comprising 120 markers, including 70 random amplified polymorphic DNAs, 47 amplified fragment length polymorphisms, one simple sequence repeat and two morphological traits namely, the cowpea mottle carmovirus resistance locus (CPMo V) and leaf shape (La), utilizing an F₂ generation of the intra‐specific cross Tvnu 1443’× Tvnu 73′. The genetic map comprised 14 linkage groups spanning 1564.1 cM of the genome. Thirty‐nine quantitative trait loci (QTLs) associated with nine traits were detected on the linkage map, explaining between 15.62 and 66.58% of their phenotypic variation. Seven chromosomal intervals contained QTLs with effects on multiple traits.     

SSR作为锚定标记构建白菜×芜菁分子遗传图谱

为了将已有的大白菜分子遗传图谱和已发表的A基因组参考图谱对应起来,本研究利用国际上发表的大白菜和甘蓝型油菜A基因组特异SSR标记作为锚定标记,重新构建了白菜×芜菁分子遗传图谱。利用双亲和F1对326个SSR标记进行了筛选,共获得86个多态性分子标记。在此基础上整合了已有的400个标记,最终构建了一张由10个连锁群组成,包含了347个标记的分子连锁图谱,图谱总长度为1008.7cM,标记间的平均图距为2.91cM。此图谱上包含了已在A基因组参考图谱上定位的56个SSR标记,分布于10个连锁群上,从而将各个连锁群与参考图谱的连锁群对应起来。每个连锁群上的标记数在25～58个之间,连锁群长度在60.6～177.0cM范围内,平均图距在1.33～4.92cM之间。该图谱为白菜重要性状的遗传定位奠定了良好基础。     

DAF marker tightly linked to a major locus for Ascochyta blight resistance in chickpea (Cicer arietinum L.)

Resistance of chickpea against the disease caused by the ascomycete Ascochyta rabiei is encoded by two or three quantitative trait loci, QTL1, QTL2 and QTL3. A total of 94 recombinant inbred lines developed from a wide cross between a resistant chickpea line and a susceptible accession of Cicer reticulatum, a close relative of cultivated chickpea, was used to identify markers closely linked to QTL1 by DNA amplification fingerprinting in combination with bulked segregant analysis. Of 312 random 10mer oligonucleotides, 3 produced five polymorphic bands between the parents and bulks. Two of them were transferred to the population on which the recent genetic map of chickpea is based, and mapped to linkage group 4. These markers, OPS06-1 and OPS03-1, were linked at LOD-scores above 5 to markers UBC733B and UBC181A flanking the major ascochyta resistance locus. OPS06-1 mapped at the peak of the QTL between markers UBC733B (distance 4.1 cM) and UBC181A (distance 9.6 cM), while OPS03-1 mapped 25.1 cM away from marker UBC733B on the other flank of the resistance locus. STMS markers localised on this linkage group were transferred to the population segregating for ascochyta resistance. Three of these markers were closely linked to QTL1. Twelve of 14 STMS markers could be used in both populations. The order of STMS markers was essentially similar in both populations, with differences in map distances between them. The availability of flanking STMS markers for the major resistance locus QTL1 will help to elucidate the complex resistance against different Ascochyta pathotypes in future. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mapping of EST-derived CAPS markers in Italian ryegrass (Lolium multiflorum Lam.)

New molecular markers derived from expressed sequence tag (EST) sequences were mapped on linkage maps of Italian ryegrass by a two-way pseudo-testcross strategy. cDNA sequences were obtained from various tissues of Italian ryegrass ( Lolium multiflorum ) and converted into cleaved amplified polymorphic sequence (CAPS) markers. Of 260 EST primer pairs that amplified a single band, 74 generated bands that showed clear polymorphisms among individuals of an F₁ mapping family. Of the 74 polymorphic marker loci, 69 were mapped on an Italian ryegrass linkage map previously constructed using amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) markers. The newly-developed EST-CAPS markers would be useful as an efficient tool to identify genetic markers and to identify candidate genes for quantitative trait loci (QTLs) associated with important traits in Italian ryegrass.