Construction of a genetic map of melon with molecular markers and horticultural traits, and localization of genes associated with ZYMV resistance

Abstract: A partial linkage map of melon was constructed from a cross between PI414723 and Dulce. Twenty-two SSR, 46RAPD, 2 ISSR markers and four horticultural markers [female flower form (a), Fusarium resistance, striped epicarp (st), and fruit flesh pH (pH)] were analyzed in an F₂/F₃ population to produce a map spanning 14 linkage groups. We report for the first time map positions for the st, a, and pH genes. One SSR marker was tightly linked to pH. Mapping the a gene for the female flower form to molecular linkage group 4 enabled the merging of the map of horticultural traits with the of molecular markers in this region. Using the 22 SSR markers of this map, two of the three postulated ZYMV resistance genes were located using a BC₁ population (PI414723 recurrent parent). One SSR marker was tightly linked to a ZYMV resistance gene, designated Zym-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of AFLP and derived CAPS markers for root-knot nematode resistance in cotton

Resistance to root-knot nematode (Meloidogyne incognita) is determined by a single major gene rkn1 in Gossypium hirsutum Acala NemX cotton. Bulked segregant analysis (BSA) combined with amplified fragment length polymorphism (AFLP) was used to identify molecular markers linked to rkn1. DNA pools from homozygous susceptible (S) and resistant (R) bulks of an F_2:3 originating from the intraspecific cross NemX × SJ-2 were screened with 128 EcoR1/Mse1 primer combinations. Putative AFLP markers were then screened with 60 F_2:7 RIL plants and four AFLP markers were found linked to rkn1. The linkage of AFLP markers to rkn1 was also confirmed in a F₂ population. The closest AFLP marker was converted to a cleaved amplified polymorphic sequence (CAPS) marker (designated GHACC1) by aligning the sequences from both susceptible and resistant parents. GHACC1 linkage to rkn1 was confirmed in the F₂ (1R:3S), F_2:7 RIL (1R:1S) and the backcross population SJ-2 × F₁ (NemX × SJ-2) (1 heterozygous: 1 homozygous). The four AFLP markers, GHACC1 plus two SSR markers (CIR316 and BNL1231) linked to rkn1 from previous work were mapped to intervals of 2.6–14.2 cM from the rkn1 locus, and the genomic region around rkn1 was spanned to about 28.2 cM in the F_2:7 population. The PCR-based GHACC1 and CIR316 markers were tested on 21 nematode resistant and susceptible cotton breeding lines and cultivars. GHACC1 was suitable for nematode resistance screening within G.␣hirsutum, but not G. barbadense, whereas CIR316 was useful in both species, indicating their␣potential for utilization in marker-assisted selection.     

Mapping Co, a gene controlling the columnar phenotype of apple, with molecular markers

The columnar phenotype is a very valuable genetic resource for apple breeding because of its compact growth form determined by the dominant gene Co. Using bulked segregant analysis combined with several DNA molecular marker techniques to screen the F₁ progeny of Spur Fuji × Telamon (heterozygous for Co), 9 new DNA markers (6 RAPD, 1 AFLP and 2 SSRs) linked to the Co gene were identified. A total of 500 10-mer random primers, 56 pairs of selective AFLP primers and 8 SSR primer pairs were screened. One RAPD marker S1142₆₈₂, and the AFLP marker, E-ACT/M-CTA₃₄₆, were converted into SCAR markers designated SCAR₆₈₂ and SCAR₂₁₆, respectively. These markers will enable early selection in progenies where Co is difficult to identify. The Co gene was located between the SSR markers CH03d11 and COL on linkage group 10 of the apple genetic linkage map. Finally, a local genetic map of the region around the Co gene was constructed by linkage analysis of the nine new markers and three markers developed earlier.     

Mapping and validation of QTLs for resistance to an Indian isolate of Ascochyta blight pathogen in chickpea

Ascochyta blight (AB) caused by Ascochyta rabiei, is globally the most important foliar disease that limits the productivity of chickpea (Cicer arietinum L.). An intraspecific linkage map of cultivated chickpea was constructed using an F₂ population derived from a cross between an AB susceptible parent ICC 4991 (Pb 7) and an AB resistant parent ICCV 04516. The resultant map consisted of 82 simple sequence repeat (SSR) markers and 2 expressed sequence tag (EST) markers covering 10 linkage groups, spanning a distance of 724.4 cM with an average marker density of 1 marker per 8.6 cM. Three quantitative trait loci (QTLs) were identified that contributed to resistance to an Indian isolate of AB, based on the seedling and adult plant reaction. QTL1 was mapped to LG3 linked to marker TR58 and explained 18.6% of the phenotypic variance (R ²) for AB resistance at the adult plant stage. QTL2 and QTL3 were both mapped to LG4 close to four SSR markers and accounted for 7.7% and 9.3%, respectively, of the total phenotypic variance for AB resistance at seedling stage. The SSR markers which flanked the AB QTLs were validated in a half-sib population derived from the same resistant parent ICCV 04516. Markers TA146 and TR20, linked to QTL2 were shown to be significantly associated with AB resistance at the seedling stage in this half-sib population. The markers linked to these QTLs can be utilized in marker-assisted breeding for AB resistance in chickpea.     

Detection of quantitative trait locus for leaffolder (Cnaphalocrocis medinalis (Guenée)) resistance in rice on linkage group 1 based on damage score and flag leaf width

Rice leaffolder (RLF) (Cnaphalocrocis medinalis (Guenée) is a destructive and widespread insect pest throughout the rice growing regions in Asia. The genetics of resistance to RLF in rice is very complex and not thoroughly explored. The present study was conducted to detect the quantitative trait loci (QTL) associated with RLF resistance involving 176 recombinant inbred lines (RILs) of F₈ generation derived from a cross between IR36, a leaffolder susceptible variety and TNAULFR831311, a moderately resistant indica rice culture. Simple sequence repeat (SSR) markers were used to construct specific linkage groups of rice. All the RILs were screened to assess their level of resistance to RLF by measuring the leaf area damaged. Besides this, the length and width of the flag leaf of each RIL were measured since these two parameters were considered as correlated traits to the RLF resistance in rice. All the above parameters observed across the RILs showed quantitative variation. Correlation analysis revealed that damage score based on greenhouse screening was positively correlated with length and width of the flag leaf. Out of 364 SSR markers analysed, 90 were polymorphic between the parents. Multi-point analysis carried out on segregating 69 SSR marker loci linkage group wise resulted in construction of linkage map with eleven groups of 42 SSR markers. Through single marker analysis, 19 SSR markers were found to have putative association with the three phenotypic traits studied. Of these markers, RM472 was identified as a locus having major effect on RLF resistance trait based on length of the flag leaf. Interval mapping detected two QTLs on linkage group 1. Among these QTLs, the QTL flanked by RM576–RM3412 were found to be associated with width of the flag leaf and RLF resistance. The putative SSR markers associated with leaffolder resistance identified in the present study may be one of the loci contributing resistance to RLF in rice.     

SSR marker tightly linked to the Ti locus in Soybean [Glycine max (L.) Merr.]

Soybean is a major source of protein meal in the world. Soybean kunitz trypsin inhibitor (SKTI) protein is a responsible for the inferior nutritional quality of unheated or incompletely heated soybean meal. The primary objective of this research was to identify DNA markers linked to the Ti locus controlling presence and absence of kunitz trypsin inhibitor protein. Two mapping populations were developed. Population 1 was derived from a cross between cultivar Jinpumkong2 (TiTi) and C242 (titi). Population 2 was made from a mating between cultivar Clark (TiTi) and C242. The F₁ plants were grown in the greenhouse to produce F₂ seeds. Each F₂ seed from F₁ plants was analyzed electrophoretically to determine the presence of the SKTI protein band. One-thousand RAPD primers, 342 AFLP primer sets, and 35 SSR primers were used to map Ti locus in population 1 and 2. The presence of SKTI protein was dominant to the lack of a SKTI protein and kunitz trypsin inhibit protein band was controlled by a single locus. Twelve DNA markers (4 RAPD, 4 AFLP, and 3 SSR) and Ti locus were found to be genetically linked in population 1 consisted with 94 F₂ individual plants. Three SSR markers (Satt409, Satt228, and Satt429) were linked with Ti locus within 10 cM. Satt228 marker was tightly linked with Ti locus. Satt228 marker was tightly linked within 0–3.7 cM of the Ti locus and may be useful in a marker assisted selection program.     

Genetic mapping of quantitative trait loci for fiber quality and yield trait by RIL approach in Upland cotton

The improvement of cotton fiber quality has become more important because of changes in spinning technology. Stable quantitative trait loci (QTLs) for fiber quality will enable molecular marker-assisted selection to improve fiber quality of future cotton cultivars. A simple sequence repeat (SSR) genetic linkage map consisting of 156 loci covering 1,024.4 cM was constructed using a series of recombinant inbred lines (RIL) developed from an F₂ population of an Upland cotton (Gossypium hirsutum L.) cross 7235 × TM-1. Phenotypic data were collected at Nanjing and Guanyun County in 2002 and 2003 for 5 fiber quality and 6 yield traits. We found 25 major QTLs (LOD ≥ 3.0) and 28 putative QTLs (2.0 < LOD < 3.0) for fiber quality and yield components in two or four environments independently. Among the 25 QTLs with LOD ≥ 3, we found 4 QTLs with large effects on fiber quality and 7 QTLs with large effects on yield components. The most important chromosome D8 in the present study was densely populated with markers and QTLs, in which 36 SSR loci within a chromosomal region of 72.7 cM and 9 QTLs for 8 traits were detected.     

Genetic mapping of AFLP markers in Japanese bunching onion (<Emphasis Type="Italic">Allium fistulosum</Emphasis>)

Summary The first genetic linkage map of Japanese bunching onion (Allium fistulosum) based primarily on AFLP markers was constructed using reciprocally backcrossed progenies. They were 120 plants each of (P₁)BC₁ and (P₂)BC₁ populations derived from a cross between single plants of two inbred lines: D1s-15s-22 (P₁) and J1s-14s-20 (P₂). Based on the (P₂)BC₁ population, a linkage map of P₁ was constructed. It comprises 164 markers – 149 amplified fragment length polymorphisms (AFLPs), 2 cleaved amplified polymorphic sequences (CAPSs), and 12 simple sequence repeats (SSRs) from Japanese bunching onion, and 1 SSR from bulb onion (A. cepa) – on 15 linkage groups covering 947 centiMorgans (cM). The linkage map of P₂ was constructed with the (P₁)BC₁ population and composed of 120 loci – 105 AFLPs, 1 CAPS, and 13 SSRs developed from Japanese bunching onion and 1 SSR from bulb onion – on 14 linkage groups covering 775 cM. Both maps were not saturated but were considered to cover the majority of the genome. Nine linkage groups in P₂ map were connected with their counterparts in P₁ map using co-dominant anchor markers, 13 SSRs and 1 CAPS.     

An SSR marker in the nitrate reductase gene of common bean is tightly linked to a major gene conferring resistance to common bacterial blight

A simple sequence repeat (SSR) marker composed of a tetra nucleotide repeat is tightly linked to a major gene of common bean (Phaseolus vulgaris L.) conferring resistance to common bacterial blight (CBB) incited by Xanthomonas axonopodis pv. phasoli (Xap). This SSR is located in the third intron region of the common bean nitrate reductase (NR) gene, which is mapped to linkage group (LG) H7, corresponding to LG B7 of the bean Core map. Co-segregation analysis between the SSR marker and CBB resistance in a recombinant inbred line (RIL) population demonstrated a tight linkage between the NR gene-specific marker and the major gene for CBB resistance. In total, the marker explained approximately 70% of the phenotypic variation in the population. Because it is co-dominant, this SSR marker should be more efficient for marker-assisted selection (MAS) than dominant/recessive random amplified polymorphic DNA (RAPD) or sequence characterized amplified region (SCAR) markers that have been developed, especially for early generation selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Construction of a linkage map and identification of DNA markers linked to Fom-1, a gene conferring resistance to Fusarium oxysporum f.sp. melonis race 2 in melon

Resistance to Fusarium oxysporum f.sp. melonis race 2 is conferred by a single dominant gene, Fom-1 in melon. Here, we identified DNA markers tightly linked to Fom-1 that could be used for marker assisted selection in breeding programs. First, we developed 125 F₂ plants derived from the cross between melon lines P11 (fom-1fom-1) and MR-1 (Fom-1Fom-1). Using the F₂ population, we constructed a linkage map including 14 SSR markers which had not been mapped previously. Fom-1 was confirmed to be allocated to linkage group 7. Then, we identified four AFLP markers using bulked segregant analysis. The AFLP marker TAG/GCC-470 was completely linked to Fom-1 and other three markers were mapped near Fom-1. TAG/GCC-470 and TCG/GGT-400 were respectively converted to STS and CAPS markers. Usefulness of DNA markers was confirmed in the analysis with several melon cultivars and lines.     

The first genetic maps of cashew (Anacardium occidentale L.)

Cashew (Anacardium occidentale) is a widespread tropical tree crop that is grown primarily for its nuts and has a global production of over 2 million Mt. In spite of its economic importance to many countries, however, no linkage map containing STS anchor sites has yet been produced for this species. This is largely attributable to a prolonged juvenile phase of the tree (limiting mapping to F₁ progenies) and difficulty in effecting sufficient hand-pollinations to create mapping populations of effective size. Here, we produce an F₁ mapping population of 85 individuals from a cross between CP 1001 (dwarf commercial clone) and CP 96 (giant genotype), and use it to generate two linkage genetic maps comprising of 205 genetic markers (194 AFLP and 11 SSR markers). The female map (CP 1001) contains 122 markers over 19 linkage groups and the male map (CP 96) comprises 120 markers assembled over 23 linkage groups. The total map distance of the female map is 1050.7 cM representing around 68% genome coverage, whereas the male map spans 944.7 cM (64% coverage). The average map distance between markers is 8.6 cM in the female map and 7.9 cM in the male map. Homology between the two maps was established between 13 linkage groups of the female map and 14 of the male map using 46 bridging markers that include 11 SSR markers. These maps represent a platform from which to identify loci controlling economically important traits in this crop.     

Simple sequence repeat markers linked to a major QTL controlling pod shattering in soybean

Shattering of soybean pods prior to harvest leads to a reduction in yield. In order to identify simple sequence repeat (SSR) markers linked to quantitative trait loci (QTLs) conditioning pod shattering, QTL analysis was conducted using an recombinant inbred line (RIL) population segregating for this trait. The degrees of pod‐shattering resistance were evaluated by heat treatment applied to pods harvested from plants in the field and in a growth chamber. Composite interval mapping identified one major QTL between SSR markers Sat_093 and Sat_366 on linkage group J for both environments. The position and the effect of this QTL were confirmed in an F₂ population derived from a cross between the pod shattering‐susceptible parental cultivar and a pod shattering‐resistant RIL. The SSR markers linked to the major QTL will be useful for marker‐assisted selection in soybean‐breeding programmes.     

利用3个F_2群体整合高密度栽培种花生遗传连锁图

遗传图谱的构建及整合是开展花生分子育种研究的基础,利用多个作图群体整合遗传图谱是解决图谱标记密度低的有效途径。本研究采用基于锚定SSR标记的作图策略,构建3个F_2群体3张遗传连锁图,利用Join Map 3.0软件整合图谱,获得一张包含20个连锁群、792个位点、总遗传距离为2079.50 c M,标记间平均距离为2.63 c M的整合图谱,各连锁群标记数在20~66个之间,遗传距离在59.10~175.80 c M之间。将3个分离群体中检测到的与荚果及种子大小相关的QTL区段与整合连锁图的标记比较发现,各群体中检测到的位于各染色体上的QTL在整合图谱中都能出现,有些QTL标记区间在整合图谱中存在更多的标记,为今后利用这些标记进行精细定位奠定了基础。     

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

Apple 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 R_gls, 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 R_gls locus. Thus, a total of 11 SSR markers were in linkage with R_gls, and mapped at distances ranging from 0.5 to 33.8 cM. The closest marker to the R_gls locus was S0405127, which showed a genetic distance of approximately 0.5 cM. The first mapping of the gene R_gls 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.     

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     

An SSR-based molecular genetic map of cassava

Summary Microsatellites or simple sequence repeats (SSR) are the markers of choice for molecular genetic mapping and marker-assisted selection in many crop species. A microsatellite-based linkage map of cassava was drawn using SSR markers and a F₂ population consisting of 268 individuals. The F₂ population was derived from selfing the genotype K150, an early yielding genotype from an F₁ progeny from a cross between two non-inbred elite cassava varieties, TMS 30572 and CM 2177-2 from IITA and CIAT respectively. A set of 472 SSR markers, previously developed from cassava genomic and cDNA libraries, were screened for polymorphism in K150 and its parents TMS 30572 and CM 2177-2. One hundred and twenty two polymorphic SSR markers were identified and utilized for linkage analysis. The map has 100 markers spanning 1236.7 cM, distributed on 22 linkage groups with an average marker distance of 17.92 cM. Marker density across the genome was uniform. This is the first SSR based linkage map of cassava and represents an important step towards quantitative trait loci mapping and genetic analysis of complex traits in M. esculenta species in national research program and other institutes with minimal laboratory facilities. SSR markers reduce the time and cost of mapping quantitative trait loci (QTL) controlling traits of agronomic interest, and are of potential use for marker-assisted selection (MAS).     

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in 'Peking'

Amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSp_eking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F₂ population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F₂ population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to Rcs_Peking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to Res_Peking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the Res_Peking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.     

Two microsatellite markers flanking a dominant gene for resistance to soybean cust nematode race 3

The purpose of this work was to identifymicrosatellite markers linked to a gene forresistance to Heterodera glycinesIchinohe (Soybean Cyst Nematode – SCN) insoybean cultivar Hartwig. ABC₁F₂ mapping population derivedfrom a cross between Hartwig (resistant)and the Brazilian soybean line Y23(susceptible) was used. About 200microsatellite or simple sequence repeat(SSR) primer pairs were tested in a bulkedsegregant analysis (BSA). Those thatshowed clear polymorphisms were amplifiedin the BC₁F₂ population, whichhad been previously inoculated andevaluated for resistance/susceptibility toSCN Race 3. Three SSR markers linked toSCN resistance were detected in thepopulation. Two of them, Satt 038 and Satt163, flanking a dominant resistant gene(d/a = –0.90), explained 37% of thephenotypic variance. This gene was mappedat the edge of molecular linkage group G. Broad and narrow sense heritabilities wereestimated to be 50.54% and 37.73%,respectively. A selection efficiency of91.18% was obtained with the simultaneoususe of the two markers. The identified SSRmarkers will be useful tools for assistingthe selection of homozygous genotypes andfor expediting the introgression of the SCNresistance locus from cv. Hartwig tosoybean elite cultivars.     

Molecular mapping of a major QTL conferring resistance to SCMV based on immortal RIL population in maize

Sugarcane mosaic virus (SCMV) is one of devastating pathogens in maize (Zea mays L.), and causes serious yield loss in susceptible cultivars. An effective solution to control the virus is utilizing resistant genes to improve the resistance of susceptible materials, whereas the basic work is to analyze the genetic basis of resistance. In this study, maize inbred lines Huangzao4 (resistant) and Mo17 (susceptible) were used to establish an F₉ immortal recombinant inbred line (RIL) population containing 239 RILs. Based on this segregation population, a genetic map was constructed with 100 simple sequence repeat (SSR) markers selected from 370 markers, and it covers 1421.5 cM of genetic distance on ten chromosomes, with an average interval length of 14.2 cM. Analysis of the genetic map and resistance by mapping software indicated that a major quantitative trait locus (QTL) was between bin6.00 and bin6.01 on chromosome 6, linked with marker Bnlg1600 (0.1 cM of interval). This QTL could account for 50.0% of phenotypic variation, and could decrease 27.9% of disease index.