Rapid Constructing a Genetic Linkage Map by AFLP Technique and Mapping a New Gene tms5

In this study, we reported the repaid construction of a molecular marker linkage map of rice (Oryza sativa L.). An F-2 population from the cross between Annong S-1 and Nanjing 11 was used to construct a genetic linkage map of rice. Total of 142 newly screened AFLP markers and 30 anchor markers (25 SSR markers and 5 RFLP markers) were mapped on the 12 chromosomes covering 1537.4 cM of rice genome. The average interval between these markers was 9.0 cM. The total work which usually was finished in more than one year was finished within only 3 months by one person. This is the first plant AFLP map developed in China. A new thermosensitive genic male sterile gene in rice, tms5, was Egged and mapped onto chromosome 2 during the development of the linkage map.

Construction of Molecular Genetic Linkage Map Based on an RIL Population of Rice and Detection of QTLs for Tiller Angle

In this study, an RIL （recombinant inbred line） population containing 240 lines was developed by single seed descent method （SSD） based on a parent com- bination of small-grain indica cultivar Kasalath and large-grain japanica cultivar TD70 with significant differences in plant type traits, to construct the molecular genetic linkage map. Totally 838 SSR （Simple Sequence Repeat） markers were used for polymorphism screening between parents, 302 SSR markers with polymorphism were detected, with a frequency of 36.04%; 141 SSR markers with clear amplified bands and uniform distribution in the genome were finally used for genotype analysis of the RIL population. According to the experimental results, the frequency of male and female genotype in this RIL population was respectively 53% and 47%, suggesting good balance in population structure. A molecular genetic linkage map of rice was constructed by 141 markers based on a RIL population of 240 lines, with a total genetic distance of about 1 832.47 cM covering all 12 chromosomes, an average genetic distance between markers of 12.70 cM and a range of genetic distance be- tween markers of 0.43-36.11 cM, which is consistent with basic requirements of quantitative trait locus （QTL） mapping. Except for few markers on chromosomes 1 and 8, the order and location of markers is similar to the published sequences of Nipponbare. QTL analysis for the tiller angle was conducted with this RIL population of 240 lines, and results showed that three QTLs controlling tiller angle were detected on chromosome 8, 9 and 11, which were named qTA8, qTA9 and qTA11, with a contribution rate of 4.10%, 26.08% and 4.35%, respectively. To be specific, qTA9 contained Tiller Angle Controlling （TAC1） gene. The construction of this molecular genetic linkage map laid the foundation for genetic analysis and QTL mapping of various traits in the progeny of indica and japonica.

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.

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

A genetic linkage map comprised of 131 loci was constructed with an F 2 population derived from an inter subspecific cross between Brassica campestris L. ssp. chinensis cv. 'aijiaohuang' and ssp. rapifera cv. 'qisihai'. 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 Genetic Linkage Map of Mango Based on SRAP, AFLP and ISSR Markers

A hybrid Ft population was obtained by crossing between mango cultivars ＇ Jinhuang＇ and ＇ Guifei＇. Among the hybrid population, 98 F1 plants were selected as the mapping population. The molecular genetic map of mango was constructed by linkage analysis by SRAP, AFLP and ISSR markers using Joinmap4. 0 software. The genetic linkage map consisted of 33 linkage groups, with a total genetic distance of 1 561.1 cM. Moreover, the genetic linkage map involved 245 polymorphic markers, including 149 SRAP markers, 90 AFLP markers and 6 ISSR markers, with an average genetic distance of 6.37 cM. This study laid a founda- tion for further investigation of the important agronomic traits of mango.

A Genetic Linkage Map of Kenaf (Hibiscus cannabinus L.) Based on SRAP, ISSR and RAPD Markers

Kenaf （Hibiscus cannabinus L.） is one of the most economically important crops for non-wood fiber production. The objective of this study was to establish a genetic linkage map of kenaf with higher density of molecular markers. A semi- wild variety Ga42 and a cultivar Alain kenaf were used as parents to construct an F2 population consisting of 155 plants. The genetic linkage map comprising 134 marker loci was constructed, including 65 sequence-related amplified polymorphism （SRAP）, 56 inter-simple sequence repeat （ISSR）, and 13 randomly amplified polymorphic DNA （RAPD） markers. This map spans 2 108.9 cM and contains 20 linkage groups with an average marker density of 15.7 cM between the adjacent markers.

High density genetic map and quantitative trait loci(QTLs)associated with petal number and flower diameter identified in tetraploid rose

Rose is one of the most important ornamental and economic plants in the world.Modern rose cultivars are primarily tetraploid,and during meiosis,they may exhibit double reduction or preferential chromosome pairing.Therefore,the construction of a high density genetic map of tetraploid rose is both challenging and instructive.In this study,a tetraploid rose population was used to conduct a genetic analysis using genome sequencing.A total of 17382 single nucleotide polymorphism(SNP)markers were selected from 2308042 detected SNPs.Combined with 440 previously developed simple sequence repeats(SSR)and amplified fragment length polymorphism(AFLP)markers,a marker dosage of 6885 high quality markers was successfully assigned by GATK software in the tetraploid model.These markers were used in the construction of a high density genetic map,containing the expected seven linkage groups with 6842 markers,a total map length of 1158.9 c M,and an average inter-marker distance of 0.18 c M.Quantitative trait locus(QTL)analysis was subsequently performed to characterize the genetic architecture of petal number and flower diameter.One major QTL(qpnum-3-1)was detected for petal number in three consecutive years,which explained 20.18–22.11%of the variation in petal number.Four QTLs were detected for flower diameter;the main locus,qfdia-2-2,was identified in two consecutive years.Our results will benefit the molecular marker-assisted breeding of modern rose cultivars.In addition,this study provides a guide for the genetic and QTL analysis of autotetraploid plants using sequencing-based genotyping methods.

QTL mapping of general combining abilities of four traits in maize using a high-density genetic map

General combining abilities （GCAs） are very important in utilization of heterosis in maize breeding. However, its genetic basis is unclear. In the present study, a set of 118 doubled haploid （DH） lines were induced from F1 generations produced from the cross between the inbred line Zheng 58 and the inbred line W499 belonging to the Reid subgroup. Using the MaizeSNP50 BeadChip, a high-density genetic map was constructed based on the DH population which included 1 147 bin markers with an average interval length of 2.00 cM. Meanwhile, the DH population was crossed with three testers including W16-5, HD568, and W556, which belong to the Sipingtou subgroup. The GCAs of the ear height （EH）, the kernel moisture content （KMC）, the kernel ratio （KR）, and the yield per plant （YPP） were estimated using these hybrids in three environments. Combining the high-density genetic map and the GCAs, a total of 14 QTLs were detected for the GCAs of the four traits. Especially, one pleiotropic QTL was identified on chromosome 1 between the SNP SYN16067 and the SNP PZE-101169244 which was simultaneously associated with the GCAs of the EH, the KR, and the YPP. These QTLs pave the way for further dissecting the genetic architecture underlying GCAs of the traits, and they may be used to enhance GCAs of inbred lines under the fixed heterotic pattern ReidxSipingtou in China through a marker-assisted selection approach.

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.

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.

An Integrated Map of Soybean Physical Map and Genetic Map

Soybean is a major crop in the world, and it is a main source of plant proteins and oil. A lot of soybean genetic maps and physical maps have been constructed, but there are no integrated map between soybean physical map and genetic map. In this study, soybean genome sequence data, released by JGI （US Department of Energy＇s Joint Genome Institute）, had been downloaded. With the software Blast 2.2.16, a total of 161 super sequences were mapped on the soybean public genetic map to construct an integrated map. The length of these super sequences accounted for 73.08% of all the genome sequence. This integrated map could be used for gene cloning, gene mining, and comparative genome of legume.

Construction of high-density SNP genetic maps and QTL mapping for dwarf-related traits in Litchi chinensis Sonn

Litchi chinensis Sonn is widely cultivated in subtropical regions and has an important economic value.A high-density genetic map is a valuable tool for mapping quantitative trait loci(QTL)and marker-assisted breeding programs.In this study,a single nucleotide polymorphism(SNP)-based high-density linkage map was constructed by a genotyping-by-sequencing(GBS)protocol using an F1 population of 178 progenies between two commercial litchi cultivars,‘Ziniangxi’(dwarf)and‘Feizixiao’(vigorous).The genetic map consisted of 3027 SNP markers with a total length of 1711.97 cM in 15 linkage groups(LGs)and an average marker distance of 0.57 cM.Based on this high-density linkage map and three years of phenotyping,a total of 37 QTLs were detected for eight dwarf-related traits,including length of new branch(LNB),diameter of new branch(DNB),length of common petiole(LCP),diameter of common petiole(DCP),length of internode(LI),length of single leaf(LSL),width of single leaf(WSL),and plant height(PH).These QTLs could explain 8.0 to 14.7%(mean=9.7%)of the phenotypic variation.Among them,several QTL clusters were observed,particularly on LG04 and LG11,which might show enrichment for genes regulating the dwarf-related traits in litchi.There were 126 candidate genes identified within the QTL regions,55 of which are differentially expressed genes by RNA-seq analysis between‘Ziniangxi’and‘Feizixiao’.These DEGs were found to participate in the regulation of cell development,material transportation,signal transduction,and plant morphogenesis,so they might play important roles in regulating plant dwarf-related traits.The high-density genetic map and QTLs identification related to dwarf traits can provide a valuable genetic resource and a basis for marker-assisted selection and genomic studies of litchi.

QTL mapping for berry shape based on a high-density genetic map constructed by whole-genome resequencing in grape

Grape berry shape is an important agricultural trait.Clarifying its genetic basis is significant for cultivating grape varieties that meet market demands.However,the current study by forward genetics has not achieved in-depth results.Here,a high-density map was constructed to identify quantitative trait loci(QTLs)for berry shape.A total of 358709 polymorphic SNPs were obtained using whole-genome resequencing(WGS)based on 208 F2 individuals derived from round grape‘E42-6’and oblong grape‘Rizamat’.The 1635.65 cM high-density map was divided into 19 linkage groups with an average distance of 0.37 cM.Using this map,three significant QTLs for fruit shape index(ShI:ratio of berry length to berry width)identified over three years were mapped onto LG4 and LG5,including one stable QTL on Chr5 with the genomic region of 0.47–1.94 Mb.Combining with gene annotation and expression patterns based on RNA-seq data from two contrasting F2 individuals with round and oblong berry(their average ShI was 1.89 and 1.10,respectively)at four developmental stages,four candidate genes were selected from the above QTLs.They were mainly involved in DNA replication,cell wall modification,and phytohormone biosynthesis.Further analysis of RNA-seq data revealed that several important phytohormone synthesis and metabolic pathways were enriched based on differentially expressed genes(DEGs),which was consistent with the results of QTL mapping for genes related to plant hormone biosynthesis in the F2 population.Furthermore,a comparison of plant hormone content showed that there were significant differences in IAA and tZ content between the two contrasting F2 individuals at different developmental stages.Our findings provide molecular insights into the genetic variation in grape berry shape.Stable QTLs and their tightly linked markers offer the possibility of marker-assisted selection to accelerate berry shape breeding.

High-density genetic mapping identified a major locus for environmental sex expression in pumpkin(Cucurbita moschata Duch.)

Long-day length and high temperature inhibit sex expression in pumpkin(Cucurbita moschata Duch.),and therefore directly impact the production potential.In this study,female flowering patterns in photoperiod-insensitive(PPIS)and photoperiod-sensitive(PPS)germplasms differed significantly in a moderately long day and high temperature environment.However,both germplasms exhibited a similar response in short day with either low temperature or high temperature environment.Photoperiod sensitivity led to this difference in sex expression between the germplasms.For the traits of 1st female flowering node(FFFN)and number of female flowers(NFF),high-density linkage map construction and quantitative trait locus(QTL)mapping were performed using SLAF-seq technology and 162 F_(2) individuals generated from PPIS and PPS.In total,4655 SLAFs were selected and mapped on 20 linkage groups(LGs).The total map length was 2502.01 cM with an average interval distance of 0.75 cM.Major QTLs for both FFFN and NFF were detected on LG6 with intervals of 7.89 and 17.67 cM and PVE values of 30.5%and 22.9%,respectively.Further analyses of the major locus for FFFN revealed 73 protein-coding genes.Among them,4 were related to sex expression,photoperiod flowering,and hormone response.An InDel(insertion and deletion)marker partially correlated with FFFN of the F_(2) population was also developed.Our study identified the QTL for the sex expression response to environmental factors using the high-density linkage map.The identified candidate genes and markers will provide useful information about the molecular interaction between the environment and sex expression and for marker-assisted selection of pumpkin environment-insensitive resources.

Construction of a high-density adzuki bean genetic map and evaluation of its utility based on a QTL analysis of seed size

Adzuki bean(Vigna angularis(Willd.)Ohwi&Ohashi)is an annual cultivated leguminous crop commonly grown in Asia and consumed worldwide.However,there has been limited research regarding adzuki bean genetics,which has prevented the efficient application of genes during breeding.In the present study,we constructed a high-density genetic map based on whole genome re-sequencing technology and validated its utility by mining QTLs related to seed size.Moreover,we analyzed the sequences flanking insertions/deletions(In Dels)to develop a set of PCR-based markers useful for characterizing adzuki bean genetics.A total of 2904 markers were mapped to 11 linkage groups(LGs).The total length of the map was 1365.0 cM,with an average distance between markers of 0.47 cM.Among the LGs,the number of markers ranged from 208(LG7)to 397(LG1)and the total distance ranged from 97.4 cM(LG9)to 155.6 cM(LG1).Twelve QTLs related to seed size were identified using the constructed map.The two major QTLs in LG2 and LG9 explained 22.1 and 18.8%of the total phenotypic variation,respectively.Ten minor QTLs in LG4,LG5 and LG6 explained 3.0–10.4%of the total phenotypic variation.A total of 9718 primer pairs were designed based on the sequences flanking In Dels.Among the 200 selected primer pairs,75 revealed polymorphisms in 24 adzuki bean germplasms.The genetic map constructed in this study will be useful for screening genes related to other traits.Furthermore,the QTL analysis of seed size and the novel markers described herein may be relevant for future molecular investigations of adzuki bean and will be useful for exploiting the mechanisms underlying legume seed development.

Construction of the first high-density genetic map for growth related QTL analysis in Ancherythroculter nigrocauda

Ancherythroculter nigrocauda is a fish endemic to the upper areas of the Changjiang(Yangtze)River in China.Quantitative trait locus(QTL)mapping is a powerful tool to identify potential genes affecting traits of economic importance in domestic animals.In this study,a high-density genetic map was constructed with 5901 single nucleotide polymorphism(SNP)makers by sequencing 92 individual fish from a F1 family using the specific-locus amplified fragment sequencing approach.Initially,48 QTLs for total length,body length,body height,and body weight were identified according to the high density of the genetic map with 24 LGs,a total length of 3839.4 cM,and marker spacing of about 0.82 cM.These QTLs explained 27.1%-49.9%of phenotypic variance.The results of this study suggest that major QTLs are responsible for the growth of A.nigrocauda,and these are potentially useful in comparative genomics research,genome assembly,and marker-assisted breeding programs for this species.

Construction of Soybean Genetic Map with RIL Population by Charleston ×Dongnong 594

F2：10 RIL population with 154 lines, crossed by Charleston as female parent and Dongnong 594 as male parent were used. 164 SSR primers were screened with the two parents and amplified on the 154 lines. A new soybean molecular genetics map, named NEAUSRI-GMS, was constructed by Mapmaker. The total length of the soybean genetic map is 1 913.5 cM, and the average distance among markers is 11.89 cM. The length of linkage group varied from 0.4 to 309.5 cM, and the markers on the linkage group varied from 2 to 28. The distribution of SSR markers on every linkage group is not even. High density region of markers existed on linkage group A1, C2, and Dla. Compared with 5 soybean genetic maps constructed at home and abroad, NEAUSRI-GMS has high homologous with the public genetic map abroad.

Rice molecular markers and genetic mapping:Current status and prospects

Dramatic changes in climatic conditions that supplement the biotic and abiotic stresses pose severe threat to the sustainable rice production and have made it a difficult task for rice molecular breeders to enhance production and productivity under these stress factors. The main focus of rice molecular breeders is to understand the fundamentals of molecular pathways involved in complex agronomic traits to increase the yield. The availability of complete rice genome sequence and recent improvements in rice genomics research has made it possible to detect and map accurately a large number of genes by using linkage to DNA markers. Linkage mapping is an effective approach to identify the genetic markers which are co-segregating with target traits within the family. The ideas of genetic diversity, quantitative trait locus（QTL） mapping, and marker-assisted selection（MAS） are evolving into more efficient concepts of linkage disequilibrium（LD） also called association mapping and genomic selection（GS）, respectively. The use of cost-effective DNA markers derived from the fine mapped position of the genes for important agronomic traits will provide opportunities for breeders to develop high-yielding, stress-resistant, and better quality rice cultivars. Here we focus on the progress of molecular marker technologies, their application in genetic mapping and evolution of association mapping techniques in rice.

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.

Advances and prospects of genetic mapping of Verticillium wilt resistance in cotton

Verticillium wilt is one of the most important diseases affecting cotton production in China.The fungus,Verticillium dahliae,has a wide host range and a high degree of genetic variability.No resistance resources have been found in the available planting resources,thus presenting difficulties and challenges for our study.The long-term production practice shows that selection of disease-resistant varieties is the most economical and effective measure to control Verticillium wilt of cotton to reduce the yield loss and quality decline of cotton.In this paper,we summarized the genetic mapping population,the analysis method of genetic localization,the discovery,mining and cloning of disease-resistant quantitative trait loci/markers,and the analysis of their genetic functions,so as to provide information for the molecular breeding approach of disease-resistant cotton.