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.     

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     

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.     

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.     

Construction of a genetic map with SRAP markers and localization of the gene responsible for the first-flower-node trait in cucumber (Cucumis sativus L.)

With an F2 population from the cross of two cucumber inbred lines, S06 and S52, sequence-related amplified polymorphism (SRAP) was used to construct a genetic linkage map in cucumber (Cucumis sativus L.). Sixty-four SRAP primer combinations generated 108 polymorphic bands in the F2 population analysis. The average of polymorphic bands produced by one primer pair was 1.5, and the maximum was 5. Using Mapmaker 3.0, a linkage map was constructed, which consisted of 77 SRAP markers distributed in nine linkage groups (LOD≥3.0) and spanned 1114.2 cM with an average interval of 14.5 cM between markers. The gene for the first-flower-node trait, termed ffn, was mapped to linkage group Ⅸ, flanked by DC1EM5 and ME7EM2A at 10.3 cM and 12.1 cM distance, respectively.     

Estimation of genome length

The genome length is a fundamental feature of a species. This note outlined the general concept and estimation method of the physical and genetic length. Some formulae for estimating the genetic length were derived in detail. As examples, the genome genetic length of Pinus pinaster Ait. and the genetic length of chromosome Ⅵ of Oryza sativa L. were estimated from partial linkage data.     

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.     

Positive correlation between recombination rate and nucleotide diversity is shown under domestication selection in the chicken genome

Positive correlation between recombination rate and nucleoUde diversity has been observed in a wide variety of eukaryotes on megabase scale. On the basis of genome-wide chicken genetic variation map generated by comparing three domestic breeds with wild ancestor and the positions of markers on the genetic linkage map, we found that SNPs rates were similar for all chromosomes while the recombination rates increased in micro chromosomes. In other words no correlation exists in chromosome size. Nevertheless, when we scanned the genome by calculating the values of each characteristic within non-overlapping windows, instead of single value for each chromosomes, the nucleoUde diversity was found to be significantly correlated with the recombination rate （r=0.27, P〈0.0005）. Furthermore, the significant association not only existed between these two features, but also existed between all 6 pairwise combinations of nucleoUde diversity, recombination rate, GC content and average gene length. This co-variation is very meaningful for the studies of sequence evolution.     

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.     

Effects of cutoff thresholds for minor allele frequencies on HapMap resolution: A real dataset-based evaluation of the Chinese Han and Tibetan populations

Genomic variation is the genetic basis of phenotypic diversity among individuals, including variation in disease susceptibility and drug response. The greatest promise of the International HapMap is to provide roadmaps for identifying genetic variants predisposing to complex diseases. Single nucleotide polymorphism （SNP） is the fundamental element of the HapMap. Allele frequency of SNPs is one of the major factors affecting the resulting HapMap, being the factor upon which linkage disequilibrium （LD） is calculated, haplotypes are constructed, and tagging SNPs （tagSNPs） are selected. The cutoff thresholds for the frequency of minor alleles used in the making of the map therefore have profound effects on the resolution of that map. To date most researchers have adopted their own cutoff thresh- olds, and there has been little real dataset-based evaluation of the effects of different cutoff thresholds on HapMap resolution. In an attempt to assess the implications of different cutoff values, we analyzed our own data for the centromeric genes on Chromosome 15 in Chinese Han and Tibetan populations, with respect to minor allele frequency cutoff values of 〉0.01 （0.01 group）, 〉0.05 （0.05 group）, and 〉0.10 （0.10 group）, and constructed HapMaps from each of the datasets. The resolution, study power and cost-effectiveness for each of the maps were compared. Our results show that the 0.01 threshold provides the greatest power （P= 0.019 in Han and P= 0.029 in Tibetan for 0.01 vs. 0.05 threshold） and de- tects most population-specific haploypes （P= 0.012 for 0.01 vs. 0.05 threshold）. However, in the regions studied, the 0.05 cutoff threshold did not significantly increase power above the 0.10 threshold （P = 0.191 in Han; 1.000 in Tibetans）, and did not improve resolution over the 0.10 value for population- specific haplotypes （P= 0.592） neither. Furthermore the 0.05 and 0.10 values produced the same figures for tagging efficiency, LD block number, LD length, study power and cost-savings in the Tibetan population. These results suggest that a lower cutoff value is more appropriate for studies in which population-specific haplotypes are crucial, and that the most appropriate cutoff value may differ between populations. Due to the limited genes studied in this project more studies should be conducted to further address this important issue.     

Construction of a genetic map with SRAP markers and localization of the gene responsible for the first-flower-node trait in cucumber (Cucumis sativus L.)

Cucumber (Cucumis sativus L.) is an annualclimber originated from the tropic rain forest area inthe south of Himalayas, which belongs to the Cucur bitaceae family. Cucumber is one of the importantvegetables in the world, and its planting area is sec ond only to that of tomato[1]. However genetics re search on cucumber obviously drops behind that of thelatter. The classic genetic map of cucumber, with sixlinkage groups, is composed of more than 100 genesfor color, morphology, and dise…     

春小麦重组自交系及多种SSR标记构建遗传图谱

 以春小麦重组自交系(RIL)宁春4 号×宁春27 号为作图群体,利用SSR 标记构建小麦遗传连锁图谱。结果表明,用1001对SSR 引物选出亲本间表现多态性的引物307 对,多态性频率为30.7%。利用307 对多态性引物对RIL 群体进行分析,共检测到266 个多态性标记位点。通过χ²检测(P<0.05),有147 个SSR 标记表现为偏分离,偏分离率为55.3%,129 个偏向母本宁春4号,其偏分离位点主要分布在B 和D 基因组上。用Mapmaker 3.0 和Mapdraw 2.1 软件将266 个SSR 位点绘制在小麦遗传连锁图上,该图谱覆盖小麦基因组全长2187.79 cM,标记间的平均遗传距离为8.22 cM。     

Mining,characterization, and exploitation of EST-derived microsatellites in <Emphasis Type="Italic">Gossypium barbadense</Emphasis>

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of ex-pressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were de-termined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barba-dense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Further-more, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.     

棉花种质资源多样性的ISSR聚类分析及主成分分析

利用ISSR分子标记对48份棉花种质资源的亲缘关系及遗传多样性进行分析,结果显示,筛选出的10个ISSR引物扩增出的112条带中,多态性条带占101条,为总条带的90.18%.每个引物均可扩增4～21个多态性位点,平均10.10个,引物平均多样性信息指数(PIC)和多态性谱带百分率(PPB)分别为0.76和87.88%.利用NT-SYSpc 2.1统计分析软件中的UPMGA法对48份棉花种质资源构建亲缘关系树状图,在遗传相似性系数(GS)为0.66水平上可将所有参试材料聚成两大类,且与主成分分析(PCA)结果基本吻合.ISSR标记揭示出这48份棉花种质资源遗传多样性较小,遗传基础比较狭窄.     

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.     

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.     

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.     

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.