Construction of AFLP-based genetic linkage maps for the Chinese shrimp Fenneropaeneus chinensis

Fenneropaeneus chinensis is an important species in marine fishery resources and aquaculture in China. A genetic linkage map is essential for improving the efficiency of its breeding by marker-assisted selection and identifying commercially important genes. Linkage maps of F. chinensis were constructed with an F2 mapping population （110 progenies） using amplified fragment length polymorphic （AFLP） marker in this study. Fifty-five AFLP primer combinations produced 532 AFLP markers fitting for map strategy in mapping family. The markers with 3：1 segregating ratios were analyzed using F2 intercross model for the common linkage map, while the markers with 1：1 ratio were analyzed using the pseudo-testcross strategy. The maps of male, female and common were constructed. The female map included 103 markers that formed 28 linkage groups, covering a total length of 1090 cM. All markers were linked with the linkage groups. Segregation distortion was observed for 6 of 103 markers in the female map. The average distance between markers was 14.53 cM and ranged from 4.4 to 24.8 cM. The male map included 144 markers that formed 35 linkage groups. Ten markers remained unlinked in male map. Segregation distortion was observed for 7 of 144 markers in the male map. The total distance of male map covered 1617 cM. The average distance between markers was 16.36 cM. The male map was 32.6% longer than the female map, which may reflect sex-specific recombination rates in Chinese shrimp. The common map was composed of 216 markers, including in 44 linkage groups covering a total distance of 1772.1 cM. Two markers remained unlinked. No distorted markers of 216 markers were shown in the common map. The distance between markers was 10.42 cM. An average estimated genome size for the Chinese shrimp was 2420 cM, which was consistent with the relative size of the Penaeid genome. The distribution of AFLP markers was relatively even in chromosomes of Chinese shrimp maps. The linkage analysis presented in this work provided some insight     

Construction of AFLP-based genetic linkage maps for the Chinese shrimp Fenneropaeneus chinensis

Fenneropaeneus chinensis is an important species in marine fishery resources and aquaculture in China. A genetic linkage map is essential for improving the efficiency of its breeding by marker-as- sisted selection and identifying commercially important genes. Linkage maps of F. chinensis were constructed with an F2 mapping population (110 progenies) using amplified fragment length polymor- phic (AFLP) marker in this study. Fifty-five AFLP primer combinations produced 532 AFLP markers fitting for map strategy in mapping family. The markers with 3:1 segregating ratios were analyzed using F2 intercross model for the common linkage map, while the markers with 1:1 ratio were analyzed using the pseudo-testcross strategy. The maps of male, female and common were constructed. The female map included 103 markers that formed 28 linkage groups, covering a total length of 1090 cM. All mark- ers were linked with the linkage groups. Segregation distortion was observed for 6 of 103 markers in the female map. The average distance between markers was 14.53 cM and ranged from 4.4 to 24.8 cM. The male map included 144 markers that formed 35 linkage groups. Ten markers remained unlinked in male map. Segregation distortion was observed for 7 of 144 markers in the male map. The total dis- tance of male map covered 1617 cM. The average distance between markers was 16.36 cM. The male map was 32.6% longer than the female map, which may reflect sex-specific recombination rates in Chinese shrimp. The common map was composed of 216 markers, including in 44 linkage groups covering a total distance of 1772.1 cM. Two markers remained unlinked. No distorted markers of 216 markers were shown in the common map. The distance between markers was 10.42 cM. An average estimated genome size for the Chinese shrimp was 2420 cM, which was consistent with the relative size of the Penaeid genome. The distribution of AFLP markers was relatively even in chromosomes of Chi- nese shrimp maps. The linkage analysis presented in this work provided some insight into the level of polymorphism and genetic variation of Chinese shrimp.     

Localization of genes for lateral branch and female sex expression and construction of a molecular linkage map in cucumber (Cucumis sativus L. ) with RAPD markers

A cucumber ( Cucumis sativus L. ) molecular linkage map, including 79 random-amplified polymorphic DNAs (RAPD)and two genes , lb for lateral branch and f for female sex expression, is constructed from a cross between a line, S52, with weak lateral growing ability and staminate from Dabieshan Mountains area in China and another line, S06, with strong lateral growing ability and gynoecious from Europe. The map contains nine linkage groups and spans 1110.0 cM with an average distance of 13.7 cM between loci. The lb locus is located in a longer linkage group LG-2 and flanked by two markers, OP-Q5-1 and OP-M-2-2, at 9.3 cM and 15.9 cM, respectively. In the meantime, the RAPD loci, OP-Q5-2 and BC151, in a short linkage group were found to flank f at 13.7 cM and 13.4 cM,respectively. The construction of RAPD map has paved a way for further study of the genes for lateral branch, female sex expression and other agronomic traits in cucumber.     

Genetic linkage map of Brassica campestris L.using AFLP and RAPD markers

A genetic linkage map comprised of 131 loci was constructed with an F2 population derived from an inter-subspecific cross between Brassica campestris L.ssp.chinensis cv.aijiaohang” and ssp.rapifera cv.,”‘isihai”.The genetic map included 93 RAPD loci,36 AFLP loci and 2 morphological loci organized into 10 main linkage groups(LGs) and 2 small groups,covering 1810.9cM with average distance between adjacent markers being approximately 13.8cM.The map is suitable for identification of molecular markers linked to important agronomic traits.QTL analysis,and even for marker-assisted selection in breeding programs of Chinese cabbage and turnip.     

Construction of a BAC library from cucumber （Cucumis sativus L.） and identification of linkage group specific clones

A bacterial artificial chromosome （BAC） library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber （Cucumis sativus L.） inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions （SCAR） and seven simple sequence repeats （SSR） markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction （PCR）. Fifteen markers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

Fine mapping of an incomplete recessive gene for leaf rolling in rice （Oryza sativa L.）

Genetic analysis and fine mapping of genes controlling leaf rolling were conducted using two backcrossed generations （BC4F2, BC4F3） derived from a cross between QMX, a non-rolled leaf cultivar as a recurrent parent, and JZB, a rolled leaf NIL of ZB as a donor parent. Results indicated that leaf rolling was mainly controlled by an incompletely recessive major gene, namely rl（t）, and at the same time, affected by quantitative trait loci （QTLs） and/or the environment. A genetic linkage map was constructed using MAPMAKER/EXP3.0 with eight polymorphic markers on chromosome 2, which were screened by BAS method from 500 SSR markers and 15 newly developed insertion/deletion （InDel） markers. The position of rl（t） was estimated with composite interval mapping （CIM） method using WinQTLcart2.5. Gene rl（t） was mapped between markers InDel 112 and RM3763, and 1.0 cM away from InDel 112 using 241 plants in BC4F2 population. To fine map r（t）, one BC4F3 line with 855 plants was generated from one semi-rolled leaf plant in BC4F2. Four new polymorphic InDel markers were developed, including InDel 112.6 and InDel 113 located between markers InDe1112 and RM3763. Based on the information of recombination offered by 191 rolled leaf plants and 185 non-rolled leaf plants from the BC4F3 line ,we mapped r（t） to a 137-kb region between markers InDel 112.6 and InDel 113. Homologous gene analysis suggested that r（t）was probably related to the process of leaf development regulated by microRNA.     

Reconstruction of linkage maps in the distorted segregation populations of backcross, doubled haploid and recombinant inbred lines

Non-Mendelian segregation of markers, known as distorted segregation, is a common biological phenomenon. Although segregation distortion affects the estimation of map distances and the results of quantitative trait loci (QTL) mapping, the effects of distorted markers are often ignored in the construction of linkage maps and in QTL mapping. Recently, we have developed a multipoint method via a Hidden Markov chain method to reconstruct linkage maps in an F2 population that corrects for bias of map distances between distorted markers. In this article, the method is extended to cover backcross, doubled haploid and recombinant inbred line (RIL) populations. The results from simulated experiments show that: (1) the degree that two linked segregation distortion loci (SDL) affect the estimation of map distances increases as SDL heritability and interval length between adjacent markers increase, whereas sample size has little effect on the bias; (2) two linked SDL result in the underesti- mation of linkage distances for most cases, overestimation for an additive model with opposite additive effects, and unbiased estimation for an epistatic model with negative additive-by-additive effects; (3) the proposed method can obtain the unbiased estimation of linkage distance. This new method was applied to a rice RIL population with severely distorted segregation to reconstruct the linkage maps, and a bootstrap method was used to obtain 95% confidence intervals of map distances. The results from real data analysis further demonstrate the utility of our method, which provides a foundation for the inheritance analysis of quantitative and viability traits.     

Fine mapping of the dominant glandless Gene Gl2^e in Sea-island cotton （Gossypium barbadense L.）

Gle2 is a mutant gene that controls glandless trait in cotton plants and seeds. It is an important gene resource to gossypol-free cottonseed breeding. The objective of this research was to develop SSR markers tightly linked with Gle2 by using the F2 segregating population containing 1599 plants derived from the cross of G. hirsutum genetic standard line TM-1 and G. barbadense glandless mutant line Hai-1. Genetic analysis suggested that the Gle2 was an incomplete dominant gene. Based on the backbone of genetic linkage map from G. hirsutum × G. barbadense BC1 published by our laboratory,Gle2 was lo-cated between CIR362 and NAU2251b,NAU3860b,STV033,with a genetic distance 9.27 and 0.96 cM,respectively. This result is useful for cloning Gle2 gene by map-based cloning method.     

Construction of a genetic linkage map for cotton based on SRAP

DNA markers have been widely used in construction of molecular genetic linkage maps in plants. The first genetic linkage map of cotton was constructed by Reinish in 1994 using RFLP (restriction fragment length polymorphism)[1], which included 705 polymorphic loci on 41 linkage groups with a total length of 4675 cM. Afterwards, several genetic linkage maps were constructed[2—7], but no map is comparable to this one in marker density. A high-density genetic linkage map could be applied effec…     

Construction of a genetic map and localization of QTLs for yield traits in tomato by SSR markers

Using an F2 population derived from the hybrid of Lycopersicon esculentum Mill. ‘XF 98-7’× Lycopersicon pimpinellifolium LA2184, a SSR genetic linkage map of tomato is constructed. The map contains 112 markers and spans 808.4 cM with an average distance of 7.22 cM between loci. Two quantitative trait loci (QTLs) for first flower node on chromosomes 5 and 11, two QTLs for number of flowers per truss on chromosomes 2 and 5, and five QTLs for fruit weight on chromosomes 1, 2, 3, 9 and 12 are identified.     

Genetic analysis and mapping of rice （Oryza sativa L.） male-sterile （OsMS-L） mutant

A rice male-sterile mutant OsMS-L of japonica cultivar 9522 background, was obtained in M4 population treated with ^60Co γ-Ray. Genetic analysis indicated that the male.sterile phenotype was controlled by a single recessive gene. Results of tissue section showed that at microspore stage, OsMS-L tapetum was retarded. Then tapetal calls expanded and microspores degenerated. No matured pollens were observed in OsMS-L anther locus. To map OsMS-L locus, an F2 population was constructed from the cross between the OsMS-L (japonica) and LongTeFu B(indica). Firstly, the OsMS-L locus was roughly mapped between two SSR markers, RM109 and RM7562 on chromosome 2. And then eleven polymorphic markers were developed for further fine fine-mapping. At last the OsMS-L locus was mapped between the two lnDel markers, Lhsl0 and Lhs6 with genetic distance of 0.4 cM, respectively. The region was delimited to 133 kb. All these results were useful for further cloning and functional analysis of OsMS-L.     

Characterization and mapping of a white panicle mutant gene in rice

A spontaneous white panicle mutant was found from the F6 progenies of an indicajaponica cross.The mutant exhibits white stripes on its basal leaves while the panicles,rachis and pedicel are milky white colored at flowering stage.Genetic analysis in an F2 population from the cross of Zhi7/white panicle mutant indicates that the white panicle phenotype is controlled by a single recessive nuclear gene,tentatively termed as wp(t).Using microsatellite markers,the wp(t) gene was anchored between the markers of SSR101 and SSR63.9 with a map distance of 2.3 and 0.8cM,respectively,and co-segregated with the marker of SSR17 on rice chromosome 1.     

Fine mapping of a semidwarf gene sd-g in indica rice（Oryza sativa L.）

The semidwarf gene sd-g which has been usedin indiea rice breeding in southern China is a new one, non-allelic to sd-1. To map sd-g, an F2 population derived fromthe cross between Xinguiaishuangai and 02428 was con-structed. The sd-g was roughly mapped between two mi-crosatellite markers RM440 and RM163, with genetic dis-tances of 0.5 and 2.5 cM, respectively. Then nine new poly-morphic microsatellite markers were developed in this region.The sd-g was further mapped between two microsatellitemarkers SSR5-1 and SSR5-51, with genetic distances of 0.1and 0.3 cM, respectively, while cosegregated with SSR418. ABAC contig was found to span the sd-g locus, the region be-ing delimited to 85 kb. This result was very useful for cloningof the sd-g gene.     

Simultaneous estimation of genetic linkage and preferential pairing factor for a triploid population with unphased markers

Triploids,recognized to occur more frequently in natural and experimental populations of many species than previously appreciated,display important economic and biological values.Despite this,however,linkage analysis for triploids has not been well explored.We develop a statistical model for estimating and testing the linkage between molecular markers in a triploid population derived from a tetraploid and diploid parent.The model incorporates one important meiotic feature of tetraploids by which more homologous chromosomes pair with a greater likelihood than less homologous chromosomes.By implementing the EM algorithm within the maximum likelihood framework,the model provides a procedure for simultaneous estimation of the linkage and preferential pairing factor.The model accommodates the segregating patterns of pseudotest markers and intercross markers with different amounts of informativeness.The utility of the model was validated through a real data analysis and simulation studies.The model provides a statistical tool for linkage analysis in a triploid population by taking into account meiotic behavior of tetraploids.Results from the model will help to shed light on the genetic diversity and origin of a polyploid population.     

Fine mapping of the <Emphasis Type="Italic">Ht2 (Helminthosporium turcicum</Emphasis> resistance 2) gene in maize

Fine mapping of Helminthosporium turcicum resistance gene Ht2 is extremely valuable for map-based cloning of the Ht2 gene,gaining a better knowledge of the distribution of resistance genes in maize genome and marker-assisted selection in maize breeding.An F2 mapping population was developed from a cross between a resistant inbred line 77Ht2 and a susceptible inbred line Huobai.With the aid of RFLP marker analyses,the Ht2 gene was mapped between the RFLP markers UMC89 and BNL2.369on chromosome 8,with a genetic distance of 0.9cM to BNL2.369.There was a linkage between SSR markers UMC1202,BNLG1152,UMC1149 and the Ht2 gene by SSR assay,Among the SSR markers,the genetic distance between UMC1149 and the Ht2 gene was 7.2cM,By bulked segregant analysis 7 RAPD-amplified products which were probably linked to the Ht2 gene were selected after screening 450 RAPD primers and converted the single-copy ones into SCAR markers.Linkage analysis showed that the genetic distance between the SCAR marker SD-06633 and the Ht2 gene was 0.4cM.From these results,a part of linkage map around the Ht2 gene was constructed.     

Genetic analysis and fine mapping of the pubescence gene GL6 in rice (Oryza sativa L.)

The pubescence of the leaf blade surface is an important agronomic characteristic for rice morphology and significantly influences rice growth as well as physiological characteristics. This characteristic was analyzed in F1 and F2 plants derived by crossing cultivar 75-1-127 with the indica cultivar Minghui 63, as well as the glabrous cultivar Lemont and indica cultivar 9311. Results indicated that the pubescence of the leaf blade surface was a dominant trait and controlled by a single gene. The GL6 gene was primarily mapped on rice chromosome 6 with recessive F2 population derived from 75-1-127/Minghui 63 by combining bulked segregation analysis and recessive class analysis using the Mapmaker3.0/MapDraw software. The genetic distances between the simple sequence repeat markers RM20491 and RM20547 were 7.2 and 2.2 cM, respectively. The GL6 gene was fine mapped in the interval between InDel-106 and InDel-115 at genetic distances of 0.3 and 0.1 cM, respectively. The large, recessive F2 population was derived from 75-1-127/Minghui 63. A high-resolution genetic and physical map of GL6 was constructed. Derived from the map-based sequences published by the International Rice Genome Sequencing Project, the GL6 gene was localized at an interval of 79 (japonica) and 116.82 kb (9311) bracketed by InDel-106 and InDel-115 within the BAC accession numbers AP008403 and AP005760. Seven annotated genes (japonica) and eight annotated genes (9311) were present. The basis was further set for GL6 cloning and function analysis.     

Genetic mapping of telomere-associated sequences in soybean ( Glycine max )

Two telomere-associated sequences (TAS), named STAS8 and STAS10, were cloned from soybean genomic DNA using polymerase chain reaction (PCR) amplification. Southern analysis showed that they were sequences with moderate copy number in soybean genome. Sequence analysis demonstrated that STAS10 had tandemly arrayed con sensus sequences of TTTAGGG and TIAGGG . The mapping of these two TAS was performed with a population of F8 re combinant inbred line using restriction fragment length polymorphisms(RFLP). Seven out of nine polymorphic fragments were mapped to the most distal position of five linkage groups, Dla, F, G2, H and Q of soybean, and the other two loci were closely linked and mapped to two interstitial positions within linkage group D1a. The mapping of TAS in soybean is essential for completeness of a molecular genetic map of soybean.     

Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

Construction of a linkage map and QTL mapping for fiber quality traits in upland cotton (Gossypium hirsutum L.)

With the development in spinning technology, the improvement of cotton fiber quality is becoming more and more important. The main objective of this research was to construct a high-density genetic linkage map to facilitate marker assisted selection for fiber quality traits in upland cotton (Gossypium hirsutum L.). A genetic linkage map comprising 421 loci and covering 3814.3 cM, accounting for approximately 73.35% of the cotton genome, was constructed using an F2 population derived from cross GX1135 (P 1 )×GX100-2 (P 2 ). Forty-four of 49 linkage groups were assigned to the 26 chromosomes. Fiber quality traits were investigated in F2 population sampled from individuals, and in F2:3 , and F2:4 generations sampled by lines from two sites and one respectively, and each followed a randomized complete block design with two replications. Thirty-nine quantitative trait loci were detected for five fiber quality traits with data from single environments (separate analysis each): 12 for fiber length, five for fiber uniformity, nine for fiber strength, seven for fiber elongation, and six for fiber micronaire, whereas 15 QTLs were found in combined analysis (data from means of different environments in F2:3 generation). Among these QTLs, qFL-chr5-2 and qFL-chr14-2 for fiber length were detected simultaneously in three generations (four environments) and verified further by combined analysis, and these QTLs should be useful for marker assisted selection to improve fiber quality in upland cotton.     

Mapping the Nuclear Fertility Restorer Gene in Rice with Simple Sequence Length Polymorphism （SSLP）

Bulked segregant analysis (BSA) of a F2 population derived from D62A/Ruby B was used to map the nuclear fertility restorer gene for wild abortive (WA) cytoplasmic male sterility. Three hundred and ninety-seven microsatellite primer pairs which distributed on 12 chromosomes were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants. One microsatellite marker RM182 located on chromosome 7 produced polymorphic products. The nuclear fertility restorer gene for WA cytoplasmic male sterility was mapped on chromosome 7, 7.4cM from RM182.