Construction of a high-density SSR genetic linkage map and identification of QTL for storage-root yield and dry-matter content in sweetpotato

Sweetpotato(Ipomoea batatas(L.)Lam.)is a widely grown food crop especially in developing countries.Increasing storage-root yield and dry-matter content has been the main breeding objective of the crop,and DNA marker-assisted breeding is needed for this purpose.In this study,using a mapping population of 500 F1 individuals from a cross between Xushu 18(female)and Xu 781(male),we constructed a highdensity genetic linkage map of sweetpotato using 601 simple-sequence repeat(SSR)primer pairs.The Xushu 18 map contained 90 linkage groups with 5547 SSR markers and spanned 18,263.5 cM,and the Xu 781 map contained 90 linkage groups with 4599 SSR markers and spanned 18,043.7 cM,representing the highest genome coverage yet reported for sweetpotato.We identified 33 QTL for storage-root yield and 16 QTL for dry-matter content,explaining respectively 6.5%–47.5%and 3.2%–18.9%of variation.These results provide a foundation for fine-mapping and cloning of QTL and for marker-assisted breeding in sweetpotato.

Genetic linkage mapping and QTL identification for salinity tolerance in Indian mustard(Brassica juncea L.Czern and Coss.)using SSR markers

Soil salinity is one of the major environmental constraints that limits crop yield and nearly 7%of the total area worldwide is affected by salinity.Salinity-induced oxidative stress causes membrane damage during germination and seedling growth.Indian mustard is a major oilseed crop in India and its production and productivity are severely affected by salt stress.Breeding Brassica cultivars for salinity tolerance by conventional means is very difficult and time-consuming.Therefore,understanding the molecular components associated with salt tolerance is needed to facilitate breeding for salt tolerance in Brassica.In this investigation,quantitative trait loci(QTLs)associated with salt tolerance were identified using F_(2:3)mapping population developed from a cross between CS52(salinity tolerant)and RH30(salinity sensitive).Parents and F_(2:3)were evaluated under controlled and salinity stress conditions for 14 morpho-physiological traits for two consecutive generations(F2 and F_(2:3)),explaining proportion of the phenotypic variance under control condition.Simple sequence repeat(SSR)markers were used for mapping studies.A genetic linkage map based on 42 simple sequence repeats(SSRs)markers was constructed covering 2298.5 cM(Haldane)to identify the loci associated with salt tolerance in Brassica juncea.Forty-one SSRs showing polymorphism in the parents(CS52 and RH30)were mapped on 8 linkage groups(C1–C8).One marker(nga 129)did not map to any of the linkage group and was excluded from mapping.Linkage group 5(C5;317.9 cM)was longest and linkage group 1(C1,255.0 cM)was shortest.Further,we identified 15 QTLs controlling 8 traits using F_(2:3)population.These QTLs explained 12.44–60.63%of the phenotypic variation with a LOD score range of 3.62–5.97.Out of these QTLs,QMI4.1 related to membrane injury showed 51.28%phenotypic variance with a LOD score of 3.34.QTL QBYP8.1 related to biological yield per plant showed 60.63%phenotypic variance at a LOD score of 3.62.The highest LOD score of 5.97 was recorded for QTL related to seed yield per plant(QSYP4.1).Major QTLs were QTL for biological yield per plant(QBYP8.1),QTL for siliquae per plant(QSP4.1),QTL for primary branches(QPB4.1),QTLs for seed per siliqua(QSS4.1,QSS4.2),QTL for seed yield per plant(QSYP4.1),and QTL for membrane injury(QMI8.1)which showed more than 50%phenotypic variance.These QTLs identified in our study need to be confirmed in other populations as well so that these can be used in marker-assisted selection and breeding to enhance salt tolerance in Brassica juncea.

Construction of Genetic Linkage Map Based on SSR Markers in Peanut(Arachis hypogaea L.)

Molecular genetic maps of crop species can be used in a variety of ways in breeding and genomic research such as identification and mapping of genes and quantitative trait loci （QTLs） for morphological, physiological and economic traits of crop species. However, a comprehensive genetic linkage map for cultivated peanut has not yet been developed due to the extremely low frequency of DNA polymorphism in cultivated peanut. In this study, 142 recombinant inbred lines （RILs） derived from a cross between Yueyou 13 and Zhenzhuhei were used as mapping population in peanut （Arachis hypogaea L.）. A total 652 pairs of genomic-SSR primer and 392 pairs of EST-SSR primer were used to detect the polymorphisms between the two parents. 141 SSR primer pairs, 127 genomic-SSR and 14 EST-SSR ones, which can be used to detect polymorphisms between the two parents, were selected to analyze the RILs population. Thus, a linkage genetic map which consists of 131 SSR loci in 20 linkage groups, with a coverage of 679 cM and an average of 6.12 cM of inter-maker distance was constructed. The putative functions of 12 EST-SSR markers located on the map were analyzed. Eleven showed homology to gene sequences deposited in GenBank. This is the first report of construction of a comprehensive genetic map with SSR markers in peanut （Arachis hypogaea L.）. The map presented here will provide a genetic framework for mapping the qualitative and quantitative trait in peanut.

Development of a high-density SSR genetic linkage map in sweet potato

Simple sequence repeat(SSR) markers have been proved to be a very powerful tool for quantitative trait locus(QTL) mapping, marker-assisted selection and comparative genomics research in many crop species. However, a high-density SSR genetic linkage map is still lacking because there are only a few SSR markers available in sweet potato. In this study, a total of 2545 simple sequence repeat(SSR) primer pairs, including 1215 genomic SSR(gSSR) primer pairs and 1330 BES-SSR(bSSR) primer pairs designed from the genome sequence and BAC-end sequence of sweet potato, respectively, were screened with sweet potato cultivars Luoxushu 8 and Zhengshu 20 and their randomly sampled two F1 individuals and 571 of them generated polymorphic bands. The selected 571 polymorphic SSR primer pairs and 35 EST-based SSR(eSSR) primer pairs developed at our laboratory were used to genotype 240 F1 individuals derived from a cross between Luoxushu 8 and Zhengshu 20. A double pseudo-test-cross strategy was applied for linkage analysis. The Luoxushu 8 map included 90 linkage groups with 5057 SSR markers and covered 13,299.9 cM with a marker density of 2.6 cM, and the Zhengshu 20 map contained 90 linkage groups with 3009 SSR markers and covered 11,122.9 cM with a marker density of 3.7 cM. Fifteen homologous groups were identified in both parent maps. These are the first SSR linkage maps consisting of the complete 90 linkage groups and 15 homologous groups, which are consistent with the autohexaploid nature of sweetpotato, and are also the linkage maps with the highest SSR marker density reported to date.These results provide a basis for QTL mapping, marker-assisted breeding and comparative genomics research of sweet potato.

A SSR Linkage Map of Maizex Teosinte F_2 Population and Analysis of Segregation Distortion

In this study,a linkage genetic map was constructed using a F2 population derived from a cross between a elite maize inbred,B73,and its progenitor,Teosinte（Z.mays ssp.mexicana）,through 205 simple sequence repeat（SSR） markers and one morphological marker.By Mapmaker 3.0,polymorphic markers were clustered into 10 groups,covering 10 chromosomes of maizexteosinte,with a total length of 2 002.4 cM and an average interval of 9.7 cM.Genotyping errors were detected using R/QTL（LOD=2.0） in 109 markers referring to 176 individuals,distributed across all 10 chromosomes with a ratio 1.2%.Projected error loci were re-run and 304 out of the 460 were confirmed as errors and replaced.A new linkage map was constructed,in which markers maintained the same order but the total map length decreased to 1 947.8 cM,with an average interval of 9.4 cM between markers.In total,25.2%（P0.05） markers were identified to have segregation distortion,in which 34.6% deviated towards the pollination parent（B73）,30.8% deviated towards Teosinte,32.7% deviated towards heterozygote and 1.9% deviated towards both parents.This map was also compared with published maizexteosinte and maize IBM map.

A Genetic Linkage Map of Brassica campestris L.ssp. pekinensis (syn. B. rapa L. ssp. pekinensis)

A molecular genetic map of Chinese cabbage was constructed with a 102 recombinant inbred (RI) population from a cross of two cultivated Chinese cabbage lines 177 and 276, using AFLP and RAPD markers. 352 markers including 265 AFLP markers and 87 RAPD markers were integrated into 17 linkage groups. It covered a total of 2 665. 7 cM with an average interval of 7. 6 cM. AFLP marker is efficient for map construction while it easily forms clusters to cause big gaps in map. A total of 13.92 % abnormal segregation markers distributed in the map. The molecular genetic map is fundamental for gene localization, comparative genomics, and QTL mapping of important agronomic traits.

A genetic linkage map with 178 SSR and 1901 SNP markers constructed using a RIL population in wheat (Triticum aestivum L.)

The construction of high density genetic linkage map provides a powerful tool to detect and map quantitative trait loci(QTLs) controlling agronomically important traits. In this study, simple sequence repeat(SSR) markers and Illumina 9K i Select single nucleotide polymorphism(SNP) genechip were employed to construct one genetic linkage map of common wheat(Triticum aestivum L.) using 191 recombinant inbred lines(RILs) derived from cross Yu 8679×Jing 411. This map included 1 901 SNP loci and 178 SSR loci, covering 1 659.9 c M and 1 000 marker bins, with an average interval distance of 1.66 c M. A, B and D genomes covered 719.1, 703.5 and 237.3 c M, with an average interval distance of 1.66, 1.45 and 2.9 c M, respectively. Notably, the genetic linkage map covered 20 chromosomes, with the exception of chromosome 5D. Bioinformatics analysis revealed that 1 754(92.27%) of 1 901 mapped SNP loci could be aligned to 1 215 distinct wheat unigenes, among which 1 184(97.4%) were located on o ne single chromosome, and the rest 31(2.6%) were located on 2 to 3 chromosomes. By performing in silico comparison, 214 chromosome deletion bin-mapped expressed sequence tags(ESTs), 1 043 Brachypodium genes and 1 033 rice genes were further added onto the genetic linkage map. This map not only integrated genetic and physical maps, SSR and SNP loci, respectively, but also provided the information of Brachypodium and rice genes corresponding to 1 754 SNP loci. Therefore, it will be a useful tool for comparative genomics analysis, fine mapping of QTL/gene controlling agronomically important traits and marker-assisted selection breeding in wheat.

Genome-wide association and linkage mapping strategies reveal genetic loci and candidate genes of phosphorus utilization in soybean

Insufficient available phosphorus in soil has become an important limiting factor for the improvement of yield and quality in soybean. The mining of QTLs and candidate genes controlling soybean phosphorus utilization related traits is a necessary strategy to solve this problem. In this study, 11 phosphorus utilization related traits of a natural population of 281 typical soybean germplasms and a recombinant inbred line(RIL) population of 270 lines were evaluated under different phosphorus conditions at two critical stages: the four-leaf stage as the seedling critical stage was designated as the Tstage, and the six-leaf stage as the flowering critical stage was designated as the Tstage. In total, 200 single nucleotide polymorphism(SNP) loci associated with phosphorus utilization related traits were identified in the natural population, including 91 detected at the Tstage, and 109 detected at the Tstage. Among these SNP loci, one SNP cluster(s715611375, ss715611377, ss715611379 and ss715611380) on Gm12 was shown to be significantly associated with plant height under the low phosphorus condition at the Tstage, and the elite haplotype showed significantly greater plant height than the others. Meanwhile, one pleiotropic SNP cluster(ss715606501, ss715606506 and ss715606543) on Gm10 was found to be significantly associated with the ratio of root/shoot, root and total dry weights under the low phosphorus condition at the Tstage, and the elite haplotype also presented significantly higher values for related characteristics under the phosphorus starvation condition. Furthermore, four co-associated SNP loci(ss715597964, ss715607012, ss715622173 and ss715602331) were identified under the low phosphorus condition at both the Tand Tstages, and 12 QTLs were found to be consistent with these genetic loci in the RIL population. More importantly, 14 candidate genes, including MYB transcription factor, purple acid phosphatase, sugar transporter and HSP20-like chaperones superfamily genes, etc., showed differential expression levels after low phosphorus treatment, and three of them were further verified by q RT-PCR. Thus, these genetic loci and candidate genes could be applied in markerassisted selection or map-based gene cloning for the genetic improvement of soybean phosphorus utilization.

Constructing the wolfberry(Lycium spp.) genetic linkage map using AFLP and SSR markers

Genetic linkage maps are important for quantitative trait locus(QTL) and marker-assisted selection breeding. The wolfberry(Lycium spp.) is an important food and traditional medicine in China. However, few construction genetic linkage maps have been reported because of the lack of genomic and genetic resources. In this study, a population of 89 F1 seedings was derived from a cross between two heterozygous parents, L. chinense var. potaninii ‘BF-01’(female)and L. barbarum var. auranticarpum ‘NH-01’(male), in order to construct a genetic linkage map using simple sequence repeat(SSR) and amplified fragment length polymorphism(AFLP) markers based on the double pseudo-test cross mapping strategy. The resulting genetic map consisted of 165 markers(74 AFLPs and 91 SSRs) distributed across 12 linkage groups and spanned a total length of 557.6 cM with an average distance of 3.38 cM between adjacent markers.The 12 linkage groups contained 3 to 21 markers and ranged in length from 8.6 to 58.3 cM. Twenty-nine segregated markers distributed in the map were mainly located on LG4 and LG9 linkage groups at P<0.05. This is the first linkage map of Lycium species using SSR and AFLP markers, which can serve as basis for improving genes and selective breeding of the genome assembly.

A Genetic Linkage Map of Brassica rapa Based on AFLP Markers

A F2 mapping population was developed by crossing a Chinese cabbage-pe-tsai variety CC156 and an oil type Rapid cycling RC144 which were different from each other in morphology, maturity, self-compatibility, plant height, etc. Using 244 AFLP markers a map was constructed containing 10 main linkage groups covering a total distance of 857 cM, corresponding to 3.5 cM per marker. Length of linkage groups varied from 43 to 125 cM and the number of AFLP markers linkage to each group ranged from 7 to 41.

Polymorphism analysis of microsatellites and construction of linkage map in part regions of four chromosomes in chicken

Based on chicken＇ consensus map issued in 2000, 17 microsatellites near 4 candidate genes such as IGF2, OBR, GDF8 and APOA 1 in 4 chromosomes （chromosome 5, 7, 8 and 24） were chosen for polymorphism analysis and construction of linkage map. Combining the technique of PCR and the fluorescent semi-automated detection, genome scanning was performed for 440 chickens, which was derived from China Agricultural University chicken resource families within three generations. The individuals of this resource families were genotyped. The results showed that the number of alleles ranged from 4 to 14; heterozygosity （H） of markers was between 0.3116 and 0.9148. Polymorphic information content （PIC） varied from 0.2672 to 0.8679. Microsatellites along with above-mentioned 4 candidate genes doing as general markers were used to construct linkage map. The spans of 4 linkage maps constructed in the part region of chromosome 5, 7, 8 and 24 were 263.5, 79.9, 206.2 and 104.2 cM, respectively. The order of markers was consistent with that of counterpart of reported consensus map. However, The spans of linkage map were larger than that of consensus map. The constructed linkage maps laid the foundation for mapping quantitative trait loci （QTL） responsible for economically important traits in chicken.

Rapid Construction of a High-Density Rice Linkage Map by High Efficiency Genome Scanning (HEGS) System

High-density linkage maps are essential tools for genome analysis of various biological traits. Our developed compact multi-gel system, HEGS （high efficiency genome scanning） is a high-throughput and high-cost-performance electrophoresis apparatus. Using this system, a high-density （average interval 2.3 cM） map with 1 065 AFLP and 63 SSR markers was constructed from recombinant inbred lines of a japonica and indica hybrid in just two months of electrophoreses by a single person. More than 50% of the mapped AFLP markers were commonly polymorphic for several combinations between japonica and indica rice and 15% were applicable for genetically closer crosses between upland and lowland types of japonica rice. This system can be used for rapid analyses of all kinds of markers.

QTL Mapping for Fiber Quality Traits Based on a Dense Genetic Linkage Map with SSR,TRAP,SRAP and AFLP Markers in Cultivated Tetraploid Cotton

Cotton is one of the most important economic crops in the world,and it provides natural fiber for the textile industry.With the advancement of the textile technology and increased consumption demands on cotton fiber,both cotton yield and quality should be enhanced.However,cotton yield

Intron-Targeted Intron-Exon Splice Conjunction (IT-ISJ) Marker and Its Application in Construction of Upland Cotton Linkage Map

To develop a new DNA maker, which could be used in genetic diversity analysis and genetic map construction in plants, IT-ISJ （intron targeted intron-exon splice junction） primer combinations, which were designed according to the intronexon splice junction conserved sequences, were used to construct cotton genetic linkage map in the present study. 49 out of 704 IT-ISJ primer combinations showed polymorphism between upland cotton high quality cultivar Yumian 1 and multiple dominant gene line T586, and the polymorphic primer combinations accounted for 7.0% of total primer combinations. 49 IT-ISJ primer combinations were used to genotype 270 F2：7 recombinant inbred lines developed from （Yumian 1 × T586） F2, and 58 IT-ISJ loci were obtained. 58 IT-ISJ, together with 150 SSR and 8 morphological loci, were used to conduct linkage analysis, and a linkage map including 22 linkage groups and 113 loci （49 IT-ISJ, 62 SSR, and 2 morphological loci） was constructed. The linkage map covered 714.5 cM with an average interval of 6.3 cM between two markers, accounting for 16.1% of cotton genome. The present study demonstrated that the polymorphism of IT-ISJ marker is high, and it could be effectively applied in plant genetic map construction.

Constructing Genetic Linkage Maps Using the Whole Genome Sequence of Pacific Bluefin Tuna (Thunnus orientalis) and a Comparison of Chromosome Structure among Teleost Species

Pacific bluefin tuna (Thunnus orientalis) is one of the most economically important species in the Percomorpha group of teleost fishes. Their migrations are extensive and depend upon continuous swimming at a high rate of speed throughout their life. The draft genome sequence of this species has been reported but remains highly fragmented. We constructed a Pacific bluefin tuna genetic linkage map using microsatellite markers developed on each of the scaffolds from the draft genome sequence to link these genome fragments and understand the genomic structure of species in Percomorpha. Of the 606 polymerase chain reaction microsatellite primer pairs tested, 473 were polymorphic in the mapping populations for the linkage analysis. We constructed sex-specific maps for 24 linkage groups consisting of 470 markers, which allowed us to place scaffolds that cumulatively represented 20.8% (153.8 Mb) of the sequenced genome onto the linkage groups. The distribution of orthologous genes on the chromosomes of tuna and four other teleost fish species suggested that the constitution of tuna chromosomes is closest to that of medaka. Both species have the 24 chromosomes of the ancestral teleost, including several chromosomal inversions. The integrated map developed in this study will be useful to construct a complete physical map to conduct comparative teleost genomics and genetic studies on economically useful traits in Pacific bluefin tuna.

Construction of high-density genetic linkage map of Pyropia yezoensis(Bangiales,Rhodophyta)and identifi cation of red color trait QTLs in the thalli

Pyropia yezoensis is an important macroalga because of its extensive global distribution and economic importance.Color is an important trait in the thalli of P.yezoensis,it is also an effective marker to identify the hybridization in genetic breeding.In this study,a high-density genetic linkage map was constructed based on high-throughput single nucleotide polymorphism(SNP)markers,and used for analyzing the quantitative trait loci(QTLs)of red color trait in the thalli of P.yezoensis.The conchospore undergoes meiosis to develop into an ordered tetrad,and each cell has a haploid phenotype and can grow into a single individual.Based on this theory,F1 haploid population was used as the mapping population.The map included 531 SNP markers,394.57 cM long on average distance of 0.74 cM.Collinear analysis of the genetic linkage map and the physical map indicated that the coverage between the two maps was 79.42%.Furthermore,QTL mapping identified six QTLs for the chromosomal regions associated with the red color trait of the thalli.The value of phenotypic variance explained(PVE)by an individual QTL ranged from 4.71%-63.11%.And QTL qRed-1-1,with a PVE of 63.11%,was considered the major QTL.Thus,these data may provide a platform for gene and QTL fine mapping,and marker-assisted breeding in P.yezoensis in the future.

Constructing Molecular Marker Linkage Maps of Chromosome 14Sh and 22Sh and QTL Mapping for Major Traits by Use of Substitution Lines of Gossypium hirsutum L.

CSB14Sh,which is isogenic for its recurrent parent TM-1 except for chromosome 14 short arm,was crossed with TM-1,and the F2 population was produced.A total of 3800 SSR primer pairs covering the whole genome were used to screen polymorphism among two parents,TM-1 and CSB14Sh,

Genetic Linkage Map and Comparative Genome Analysis for the Atlantic Killifish (Fundulus heteroclitus)

Genetic linkage maps are valuable tools in evolutionary biology;however, their availability for wild populations is extremely limited. Fundulus heteroclitus (Atlantic killifish) is a non-migratory estuarine fish that exhibits high allelic and phenotypic diversity partitioned among subpopulations that reside in disparate environmental conditions. An ideal candidate model organism for studying gene-environment interactions, the molecular toolbox for F. heteroclitus is limited. We identified hundreds of novel microsatellites which, when combined with existing microsatellites and single nucleotide polymorphisms (SNPs), were used to construct the first genetic linkage map for this species. By integrating independent linkage maps from three genetic crosses, we developed a consensus map containing 24 linkage groups, consistent with the number of chromosomes reported for this species. These linkage groups span 2300 centimorgans (cM) of recombinant genomic space, intermediate in size relative to the current linkage maps for the teleosts, medaka and zebrafish. Comparisons between fish genomes support a high degree of synteny between the consensus F. heteroclitus linkage map and the medaka and (to a lesser extent) zebrafish physical genome assemblies.

Identification and fine mapping of qSW2 for leaf slow wilting in soybean

Drought is one of the abiotic stresses limiting the production of soybean(Glycine max).Elucidation of the genetic and molecular basis of the slow-wilting(SW)trait of this crop offers the prospect of its genetic improvement.A panel of 188 accessions and a set of recombinant inbred lines produced from a cross between cultivars Liaodou 14 and Liaodou 21 were used to identify quantitative-trait loci(QTL)associated with SW.Plants were genotyped by Specific-locus amplified fragment sequencing and seedling leaf wilting was assessed under three water-stress treatments.A genome-wide association study identified 26 SW-associated single-nucleotide polymorphisms(SNPs),including three located in a 248-kb linkage-disequilibrium(LD)block on chromosome 2.Linkage mapping revealed a major-effect QTL,qSW2,associated with all three treatments and adjacent to the LD block.Fine mapping in a BC_(2)F_(3) population derived from a backcross between Liaodou 21 and R26 confined qSW2 to a 60-kb interval.Gene expression and sequence variation analysis identified the gene Glyma.02 g218100,encoding an auxin transcription factor,as a candidate gene for qSW2.Our results will contribute significantly to improving drought-resistant soybean cultivars by providing genetic information and resources.

The linkage maps of Dendrobium species based on RAPD and SRAP markers

Dendrobium plants are used commonly as tonic herbs and health food in many Asian countries, especially in China. Here we report the genetic map construction of two Dendrobium species with a double pseudo-testcross strategy using random amplified polymorphic DNA （RAPD） and sequence-related amplified polymorphism （SRAP） markers. A F1 mapping population of 90 individuals was developed from a cross between D. officinale and D. hercoglossum. A total of 307 markers, including 209 RAPD and 98 SRAP, were identified and used for genetic linkage group （LG） analysis. The D. officinale linkage map consisted of 11 major linkage groups and 3 doublets, which covered 629.4 cM by a total of 62 markers with an average locus distance of 11.2 cM between two adjacent markers. The D. hercoglos- sum linkage map contained 112 markers mapped on 15 major and 4 minor linkage groups, spanning a total length of 1,304.6 cM with an average distance of I 1.6 cM between two adjacent markers. The maps constructed in this study covered 92.7% and 82.7% of the D. hercoglossum and D. officinale genomes respectively, providing an important basis for the mapping of horticultural and medicinal traits and for the application of marker-assisted selection in Dendrobium breeding program.