Construction Of Genetic Linkage Map Research Articles

Novel microsatellite markers in Pistacia vera L. and their transferability across the genus Pistacia

This study was undertaken primarily to develop new simple sequence repeat (SSR) markers for pistachio (Pistacia vera L.) and related Pistacia species using genomic libraries enriched for dinucleotide (GA)n and trinucleotide (AAG)n microsatellite motifs. A total of 192 clones were sequenced from each library, 135 primer pairs were designed, and 110 generated PCR products. Forty-six loci from the GA library and 18 loci from the AAG library produced polymorphic fragments following testing of 12 pistachio cultivars. A total of 264 alleles were generated from the polymorphic loci with an average of 4.13 alleles per locus. Polymorphism information content (PIC) was 0.51, whereas the observed (Ho) and expected heterozygosity (He) values were 0.52 and 0.56, respectively. Transferability of 110 SSR primer pairs were tested in ten wild Pistacia species, and 100 were transferable to at least one of the Pistacia species. Pistacia eurycarpa Yalt. had the highest number of transferable loci, whereas Pistacia texana Swingle and Pistacia lentiscus L. had the lowest one. This is the first study to report candidate SSR markers for P. eurycarpa, Pistacia palaestina and P. texana. It is apparent that SSR markers developed in this study could aid germplasm characterization, construction of genetic linkage maps and comparative mapping in the genus Pistacia.

Current Status of Knowledge and Research Perspectives in Korean Pear Genomics

The pear (Pyrus spp.) is most important fruit crop in the world. The genus Pyrus belongs to the subfamily Maloideae in the Rosaceae family and contains at least 22 primary species; however, only a few species, including P. pyrifolia, P. ussuriensis, P. bretschneideri, and P. communis have been utilized for fruit production. In Korea, awareness of the importance of the fruit industry and fruit tree breeding is low, and there is little support for genetic and genomic studies of fruit trees. In foreign countries, studies have focused on obtaining genomic information of fruit crops and the development of important agronomic trait-related molecular markers, providing a genomic framework for fruit tree breeding. Although Korea does not actively participate in research on the genomics of fruit trees, it is not far behind other countries in terms of technology and is therefore still competitive in research and development. The resequencing of 'Whangkeumbae' and 'Minibae' pears has been performed using the Illumina Hiseq 2000 platform as a part of the Biogreen 21 project, offering novel, rapid methods for identification of molecular marker, such as single nucleotide polymorphisms, insertion-deletions, and simple sequence repeats, through next-generation sequencing (NGS) technology. These NGS-based molecular markers are useful for genetic studies of Asian pears, e.g., for construction of genetic linkage maps, mapping of quantitative trait loci, and marker-assisted selection.

Construction of the High-Density Genetic Linkage Map and Chromosome Map of Large Yellow Croaker (Larimichthys crocea).

High-density genetic maps are essential for genome assembly, comparative genomic analysis and fine mapping of complex traits. In this study, 31,191 single nucleotide polymorphisms (SNPs) evenly distributed across the large yellow croaker (Larimichthys crocea) genome were identified using restriction-site associated DNA sequencing (RAD-seq). Among them, 10,150 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 5451.3 cM with an average distance of 0.54 cM between loci. This represents the densest genetic map currently reported for large yellow croaker. Using 2889 SNPs to target specific scaffolds, we assigned 533 scaffolds, comprising 421.44 Mb (62.04%) of the large yellow croaker assembled sequence, to the 24 linkage groups. The mapped assembly scaffolds in large yellow croaker were used for genome synteny analyses against the stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes). Greater synteny was observed between large yellow croaker and stickleback. This supports the hypothesis that large yellow croaker is more closely related to stickleback than to medaka. Moreover, 1274 immunity-related genes and 195 hypoxia-related genes were mapped to the 24 chromosomes of large yellow croaker. The integration of the high-resolution genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits in large yellow croaker.

A genetic linkage map with 178 SSR and 1 901 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 iSelect 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 cM and 1 000 marker bins, with an average interval distance of 1.66 cM. A, B and D genomes covered 719.1, 703.5 and 237.3 cM, with an average interval distance of 1.66, 1.45 and 2.9 cM, 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 one 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.

The First High-Density Genetic Map Construction in Tree Peony (Paeonia Sect. Moutan) using Genotyping by Specific-Locus Amplified Fragment Sequencing.

Genetic linkage maps, permitting the elucidation of genome structure, are one of most powerful genomic tools to accelerate marker-assisted breeding. However, due to a lack of sufficient user-friendly molecular markers, no genetic linkage map has been developed for tree peonies (Paeonia Sect. Moutan), a group of important horticultural plants worldwide. Specific-locus amplified fragment sequencing (SLAF-seq) is a recent molecular marker development technology that enable the large-scale discovery and genotyping of sequence-based marker in genome-wide. In this study, we performed SLAF sequencing of an F1 population, derived from the cross P. ostti ‘FenDanBai’ × P. × suffruticosa ‘HongQiao’, to identify sufficient high-quality markers for the construction of high-density genetic linkage map in tree peonies. After SLAF sequencing, a total of 78 Gb sequencing data and 285,403,225 pair-end reads were generated. We detected 309,198 high-quality SLAFs from these data, of which 85,124 (27.5%) were polymorphic. Subsequently, 3518 of the polymorphic markers, which were successfully encoded in to Mendelian segregation types, and were in conformity with the criteria of high-quality markers, were defined as effective markers and used for genetic linkage mapping. Finally, we constructed an integrated genetic map, which comprised 1189 markers on the five linkage groups, and spanned 920.699 centiMorgans (cM) with an average inter-marker distance of 0.774 cM. There were 1115 ‘SNP-only’ markers, 18 ‘InDel-only’ markers, and 56 ‘SNP&InDel’ markers on the map. Among these markers, 450 (37.85%) showed significant segregation distortion (P < 0.05). In conclusion, this investigation reported the first large-scale marker development and high-density linkage map construction for tree peony. The results of this study will serve as a solid foundation not only for marker-assisted breeding, but also for genome sequence assembly for tree peony.

Transcriptome sequencing of mung bean (Vigna radiate L.) genes and the identification of EST-SSR markers.

Mung bean (Vigna radiate (L.) Wilczek) is an important traditional food legume crop, with high economic and nutritional value. It is widely grown in China and other Asian countries. Despite its importance, genomic information is currently unavailable for this crop plant species or some of its close relatives in the Vigna genus. In this study, more than 103 million high quality cDNA sequence reads were obtained from mung bean using Illumina paired-end sequencing technology. The processed reads were assembled into 48,693 unigenes with an average length of 874 bp. Of these unigenes, 25,820 (53.0%) and 23,235 (47.7%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases, respectively. Furthermore, 19,242 (39.5%) could be classified into gene ontology categories, 18,316 (37.6%) into Swiss-Prot categories and 10,918 (22.4%) into KOG database categories (E-value < 1.0E-5). A total of 6,585 (8.3%) were mapped onto 244 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. Among the unigenes, 10,053 sequences contained a unique simple sequence repeat (SSR), and 2,303 sequences contained more than one SSR together in the same expressed sequence tag (EST). A total of 13,134 EST-SSRs were identified as potential molecular markers, with mono-nucleotide A/T repeats being the most abundant motif class and G/C repeats being rare. In this SSR analysis, we found five main repeat motifs: AG/CT (30.8%), GAA/TTC (12.6%), AAAT/ATTT (6.8%), AAAAT/ATTTT (6.2%) and AAAAAT/ATTTTT (1.9%). A total of 200 SSR loci were randomly selected for validation by PCR amplification as EST-SSR markers. Of these, 66 marker primer pairs produced reproducible amplicons that were polymorphic among 31 mung bean accessions selected from diverse geographical locations. The large number of SSR-containing sequences found in this study will be valuable for the construction of a high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.

QTL IciMapping: Integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations

QTL IciMapping is freely available public software capable of building high-density linkage maps and mapping quantitative trait loci (QTL) in biparental populations. Eight functionalities are integrated in this software package: (1) BIN: binning of redundant markers; (2) MAP: construction of linkage maps in biparental populations; (3) CMP: consensus map construction from multiple linkage maps sharing common markers; (4) SDL: mapping of segregation distortion loci; (5) BIP: mapping of additive, dominant, and digenic epistasis genes; (6) MET: QTL-by-environment interaction analysis; (7) CSL: mapping of additive and digenic epistasis genes with chromosome segment substitution lines; and (8) NAM: QTL mapping in NAM populations. Input files can be arranged in plain text, MS Excel 2003, or MS Excel 2007 formats. Output files have the same prefix name as the input but with different extensions. As examples, there are two output files in BIN, one for summarizing the identified bin groups and deleted markers in each bin, and the other for using the MAP functionality. Eight output files are generated by MAP, including summary of the completed linkage maps, Mendelian ratio test of individual markers, estimates of recombination frequencies, LOD scores, and genetic distances, and the input files for using the BIP, SDL, and MET functionalities. More than 30 output files are generated by BIP, including results at all scanning positions, identified QTL, permutation tests, and detection powers for up to six mapping methods. Three supplementary tools have also been developed to display completed genetic linkage maps, to estimate recombination frequency between two loci, and to perform analysis of variance for multi-environmental trials.

High-density genetic linkage map construction and QTL mapping of grain shape and size in the wheat population Yanda1817 × Beinong6.

High-density genetic linkage maps are necessary for precisely mapping quantitative trait loci (QTLs) controlling grain shape and size in wheat. By applying the Infinium iSelect 9K SNP assay, we have constructed a high-density genetic linkage map with 269 F 8 recombinant inbred lines (RILs) developed between a Chinese cornerstone wheat breeding parental line Yanda1817 and a high-yielding line Beinong6. The map contains 2431 SNPs and 128 SSR & EST-SSR markers in a total coverage of 3213.2 cM with an average interval of 1.26 cM per marker. Eighty-eight QTLs for thousand-grain weight (TGW), grain length (GL), grain width (GW) and grain thickness (GT) were detected in nine ecological environments (Beijing, Shijiazhuang and Kaifeng) during five years between 2010–2014 by inclusive composite interval mapping (ICIM) (LOD≥2.5). Among which, 17 QTLs for TGW were mapped on chromosomes 1A, 1B, 2A, 2B, 3A, 3B, 3D, 4A, 4D, 5A, 5B and 6B with phenotypic variations ranging from 2.62% to 12.08%. Four stable QTLs for TGW could be detected in five and seven environments, respectively. Thirty-two QTLs for GL were mapped on chromosomes 1B, 1D, 2A, 2B, 2D, 3B, 3D, 4A, 4B, 4D, 5A, 5B, 6B, 7A and 7B, with phenotypic variations ranging from 2.62% to 44.39%. QGl.cau-2A.2 can be detected in all the environments with the largest phenotypic variations, indicating that it is a major and stable QTL. For GW, 12 QTLs were identified with phenotypic variations range from 3.69% to 12.30%. We found 27 QTLs for GT with phenotypic variations ranged from 2.55% to 36.42%. In particular, QTL QGt.cau-5A.1 with phenotypic variations of 6.82–23.59% was detected in all the nine environments. Moreover, pleiotropic effects were detected for several QTL loci responsible for grain shape and size that could serve as target regions for fine mapping and marker assisted selection in wheat breeding programs.

PAV markers in Sorghum bicolour: genome pattern, affected genes and pathways, and genetic linkage map construction.

Key message 5,511 genic small-size PAVs in sorghum were identified and examined, including the pattern and the function enrichment of PAV genes. 325 PAV markers were developed to construct a genetic map. Presence/absence variants (PAVs) correlate closely to the phenotypic variation, by impacting plant genome sizes and the adaption to the environment. To shed more light on their genome-wide patterns, functions and the possibility of using them as molecular markers, we generated next generation genome sequencing data for four sorghum inbred lines and used associated bioinformatic pipelines to identify small-size PAVs (40–10 kb). Five thousand five hundreds and eleven genic PAVs (40–10 kb) were identified and found to affect 3,238 genes. These PAVs were mainly distributed on the sub-telomeric regions, but the highest proportions occurred in the vicinity of the centromeric regions. One of the prominent features of the PAVs is the high occurrence of long terminal repeats retrotransposons and DNA transposons. PAVs caused various alterations to gene structure, primarily including the coding sequence variants, intron variants, transcript ablation, and initiator codon changes. The genes affected by PAVs were significantly enriched in those involved in stress responses and protein modification. We used 325 PAVs polymorphic between two sorghum inbred lines Ji2731 and E-Tian, together with 49 SSR markers, and constructed a genetic map, which consisted of 10 linkage groups corresponding to the 10 chromosomes of sorghum and spanned 1,430.3 cM in length covering 97 % of the physical genome. The resources reported here should be useful for genetic study and breeding of sorghum and related species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-015-2458-4) contains supplementary material, which is available to authorized users.

Development of novel and polymorphic microsatellite DNA loci from Haliotis ovina.

Twelve microsatellite loci were developed from Haliotis ovina by the magnetic bead hybridization method. Genetic variability was assessed using 30 individuals from 3 wild populations. The number of alleles per locus ranged from 2 to 5, and the polymorphism information content ranged from 0.1228 to 0.6542. Observed and expected heterozygosities ranged from 0.0000 to 0.7778 and 0.1288 to 0.6310, respectively. These loci should provide useful information for genetic studies such as genetic diversity, pedigree analysis, construction of genetic linkage maps, and marker-assisted selection breeding in H. ovina.

Screening the first set of polymorphic microsatellite loci in Lunella coronata granulata (Turbinidae).

Lunella coronata granulata, from the family Turbini-dae, is an economically important species. The first set of 10 poly-morphic microsatellite loci was screened from L. coronata granulata, and 30 individuals were used to analyze the degree of polymorphism in these loci. The level of observed and expected heterozygosity was 0.0667-0.7333 and 0.0644-0.6628, respectively. The polymorphism information content varied from 0.305 to 0.559. Eight loci were in Hardy-Weinberg equilibrium (HWE) (P > 0.05), while two loci devi-ated significantly from the HWE after Bonferroni's correction (P < 0.005). The isolated microsatellite loci can be utilized in studies of population genetic analysis and they provide important genetic mark-ers for construction of genetic linkage maps and genetic breeding of L. coronata granulata resources.

菲律宾蛤仔EST_SSR标记与生长性状的相关分析

PDF HTML阅读 XML下载 导出引用 引用提醒 菲律宾蛤仔EST_SSR标记与生长性状的相关分析 DOI: 10.5846/stxb201305161083 作者: 作者单位: 大连海洋大学,大连海洋大学,大连海洋大学,大连海洋大学,大连海洋大学 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金(31302183);国家高技术研究发展计划"863"项目(2012AA10A400);现代农业产业技术体系建设专项(CARS-48) Identification of EST_SSR markers associated with growth-related traits in the Manila clam Ruditapes philippinarum Author: Affiliation: Dalian Ocean University,Engineering and Technology Research Center of Shellfish Breeding of Liaoning Province,Key Laboratory of Mariculture Stock Enhancement in North China’s Sea,Ministry of Agriculture,Dalian Ocean University,Engineering and Technology Research Center of Shellfish Breeding of Liaoning Province,Key Laboratory of Mariculture Stock Enhancement in North China’s Sea,Ministry of Agriculture,Dalian Ocean University,Engineering and Technology Research Center of Shellfish Breeding of Liaoning Province,Key Laboratory of Mariculture Stock Enhancement in North China’s Sea,Ministry of Agriculture,Dalian Ocean University,Engineering and Technology Research Center of Shellfish Breeding of Liaoning Province,Key Laboratory of Mariculture Stock Enhancement in North China’s Sea,Ministry of Agriculture,Dalian Ocean University,Engineering and Technology Research Center of Shellfish Breeding of Liaoning Province,Key Laboratory of Mariculture Stock Enhancement in North China’s Sea,Ministry of Agriculture Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:研究利用20 个微卫星标记对菲律宾蛤仔斑马蛤F2代家系107 个个体进行遗传多样性分析,并对标记位点与生长相关性状进行分析。在20 个微卫星位点共检测到41 个等位基因,各位点等位基因数为2-3 个,等位基因片段大小为109-430 bp,平均等位基因数为2.05 个。平均有效等位基因数为1.71个,观测杂合度平均值为0.504,期望杂合度的平均值为0.431,平均多态信息含量为0.324。经卡方检验,3 个位点SSR11,SSR164和SSR213的基因型分布显著偏离了孟德尔定律(P < 0.01)。运用SPSS 20.0对20 个微卫星位点与菲律宾蛤仔斑马蛤家系生长性状的相关性(壳长、壳宽、壳高和体重)进行连锁显著性检验。结果表明,SSR9位点与壳高存在显著的相关关系(P < 0.05),SSR135和SSR164位点与壳宽呈显著相关(P < 0.05),SSR142位点与体重呈显著性相关(P < 0.05)。研究结果可为菲律宾蛤仔的分子标记辅助选育提供参考。 Abstract:The Manila clam, Ruditapes philippinarum, which is widely distributed along the coasts of China, is an economically important marine bivalve species in China's aquaculture industry. The world production of this species was 3.6 million metric tons in 2010. China is the first largest country in the world in terms of production of the Manila clam, producing about 3.0 million metric tons annually, which accounts for about 90% of global production. This species has several pedigrees including White, Zebra, Liangdao Red and Marine Red distributing in the coastal areas in North China. Microsatellites or simple sequence repeats (SSRs) are tandemly repeated motifs of 1-6 genetic base pairs. Microsatellite markers are powerful molecular markers due to their high polymorphism, stability, and co-dominance, and they are used widely in studies of genetic diversity, parentage assignment, genetic linkage map construction, and trait-related marker screening. In this study, 20 microsatellite DNA markers were used to analyze the genetic diversity of 107 individuals of a Zebra F2 pedigree of R. philippinarum. Forty-one alleles were detected, and the number of alleles (Na) was 2-3 at each locus (average, 2.05). The effective number of alleles (Ne) was 1.71, and the DNA fragment length was 109-430 base pairs. The mean values of observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were 0.504, 0.431, and 0.324, respectively. The probability value of the chi-square test showed that three loci significantly deviated from Mendelian segregation (P < 0.01), which suggested that these loci might link with the adaptive gene, and two loci (SSR11 and SSR164) may link with recessive homozygous lethal genes. The general linear model procedure in SPSS20.0 was used to analyze the correlation between the 20 microsatellites and growth-related traits of R. philippinarum (i.e., shell length, shell width, shell height, and body weight). Four loci were significantly related to the growth traits (P < 0.05): SSR135 and SSR164 were significantly related to shell width (P < 0.05), SSR9 was significantly related to shell height (P < 0.05), and SSR142 was significantly related to total weight (P < 0.05). Favorable genotypes for each growth trait were identified by a multiple comparison among the loci. Allele A for SSR9 had a significant impact on shell height (P < 0.05) and had the highest phenotype value; thus, it can be used as a molecular marker for selective breeding. SSR135 and SSR164 had a significant impact on shell width and total weight (P < 0.05), respectively, and allele B for SSR135 and allele A for SSR142 had a positive effect on growth-related traits. The following four genotypes of these loci had a favorable effect on growth-related traits: AA for SSR9, BB for SSR135, AA for SSR142, and AB for SSR164. The trait-related microsatellite loci identified in this study will be valuable for marker-assisted breeding of R. philippinarum. 参考文献 相似文献 引证文献

Construction of dense genetic linkage maps of apple cultivars Kaşel-41and Williams’ Pride by simple sequence repeat markers

Abstract: Almost all commercially grown apple cultivars in Turkey are exotic, with local varieties serving as important genetic resources for breeding and marker development purposes. Among the latter is Amasya, which has a pleasing taste and flavor, but is unsuitable for commercial use because of its small fruit size and alternate-bearing characteristics. Genetic maps of apple cultivars are important resources to facilitate marker-assisted selection. Because of its good eating quality, Amasya is a good genetic resource for marker development to enhance fruit quality, especially flavor. In this study, we constructed a simple sequence repeat (SSR) marker-based genetic map of the Amasya apple cultivar using 96 F1 progenies generated from a cross between Kasel-41 (a selection from Amasya) and Williams' Pride cultivars. Of 187 bacterial artificial chromosome SSRs, 150 expressed sequence tag SSRs, and of 362 genomic SSR primer pairs, 400 markers were mapped, with parental and consensus maps constructed independently. A reference map was also constructed using 207 common SSR markers, thereby producing the most dense SSR-based reference map currently reported for apple. The reference genetic map was 1351 cM long, with a mean marker density of 6.5 cM. The consensus map obtained by mapping 396 markers had a length of 1476.4 cM and a 3.82-cM mean marker density. We also identified the positions of 57 SSR loci previously unmapped in apple. As a result, once additional markers have been incorporated into the maps to increase saturation, the evaluation of phenotypic traits in the mapping population should facilitate linkage between molecular markers and fruit quality traits. Additionally, the constructed apple reference genetic map, with 207 SSR markers, should also assist the construction of new genetic maps and aid integration of various apple linkage maps.

Distribution and analysis of SSR in mung bean (Vigna radiata L.) genome based on an SSR-enriched library

Simple sequence repeats (SSR) are widely distributed in plant genomes, have been popular genetic markers and can be involved in gene function. We report an SSR analysis of mung bean (Vigna radiata L.), based on an SSR-enriched library. A total of 308,509 SSRs (56.9 % simple and 43.1 % compound) were discovered from 167,628 sequences. Both di- and tri-nucleotide were the most prevalent repeat types (each accounts for 48.6 %). The most frequent motifs were AAC/GTT, accounting for 45.14 % of all, followed by AC/GT at 41.80 %. After filtering the SSRs, 70,104 flanking sequences were used as BLAST queries and corresponded to 574 non-redundant gene ontology terms against the protein database from Arabidopsis thaliana. The three main categories were biological processes (23.8 %), cellular components (44.4 %) and molecular functions (31.8 %). A total of 6,100 non-repeated primer pairs were designed and validated by PCR analysis. The results showed that 60 % of primers were effective in mung bean and 35.2, 34.0 and 25.9 % could be transferred to rice bean, adzuki bean and cowpea, respectively. 9.1 % of the 6,100 displayed polymorphism between a wild and cultivated mung bean genotype, and 367 were mapped onto chromosomes using a RIL population derived from a wild × a cultivated cross. However, only 49 of 1,700 effective primer pairs showed polymorphism among 32 Chinese cultivated mung bean accessions. A total of 46,565 loci on mung bean chromosomes from the draft genome were hit by the 70,104 flanking sequences using BLASTn. The present study, especially the newly published 387 markers that have been validated and mapped, will significantly enhance genetic linkage map construction, QTL mapping and marker-assisted selection in mung bean and breeding in closely related crop species.

Construction of a high-density, high-resolution genetic map and its integration with BAC-based physical map in channel catfish.

Construction of genetic linkage map is essential for genetic and genomic studies. Recent advances in sequencing and genotyping technologies made it possible to generate high-density and high-resolution genetic linkage maps, especially for the organisms lacking extensive genomic resources. In the present work, we constructed a high-density and high-resolution genetic map for channel catfish with three large resource families genotyped using the catfish 250K single-nucleotide polymorphism (SNP) array. A total of 54,342 SNPs were placed on the linkage map, which to our knowledge had the highest marker density among aquaculture species. The estimated genetic size was 3,505.4 cM with a resolution of 0.22 cM for sex-averaged genetic map. The sex-specific linkage maps spanned a total of 4,495.1 cM in females and 2,593.7 cM in males, presenting a ratio of 1.7 : 1 between female and male in recombination fraction. After integration with the previously established physical map, over 87% of physical map contigs were anchored to the linkage groups that covered a physical length of 867 Mb, accounting for ∼90% of the catfish genome. The integrated map provides a valuable tool for validating and improving the catfish whole-genome assembly and facilitates fine-scale QTL mapping and positional cloning of genes responsible for economically important traits.

Construction of genetic linkage map and graphical genotyping of pseudo-backcrossed F2 (BC’2) progeny to introduce a CTV resistance from Poncirus trifoliata (L.) Raf. into Citrus by introgression breeding

Most citrus tristeza virus (CTV) strains infect almost all Citrus species and have caused serious damage on citrus production in many countries. Poncirus trifoliata (trifoliate orange) has a single dominant resistance gene effective against a broad range of CTV strains. P. trifoliata is sexually compatible with Citrus species, and introgression breeding programs have been initiated to introduce the CTV resistance into Citrus. “Kankitsu Chukanbohon Nou 8 Gou” (Nou-8) was developed by crossing Citrus sp. “Kiyomi” with an F1 hybrid derived from Citrus hassaku × P. trifoliata, but it still had undesirable eating traits. In aim to progress marker-assisted introgression breeding for CTV resistance efficiently, we selected 189 DNA markers which could be applied to genotyping for Citrus and P. trifoliata and constructed a genetic linkage map of Nou-8. The map included 189 DNA markers on 10 linkage groups and extended about 722.8 cM in total length. CTV resistance locus was located at 2.4 cM position in linkage group 02_(1), with the same location found when progeny were scored for resistance using an ELISA assay and for DNA marker for CTV resistant (CTV103). Graphical genotypes of Nou-8 and its progeny suggested that (1) 36 % of the chromosome in the BC’2 population were inherited from Nou-8 without any recombination sites, (2) Nou-8 had been already completely substituted by Citrus genotype in two linkage groups, and (3) four of 93 progeny of Nou-8 had recombination sites in the flanking region of the CTV resistance gene locus.

Development of 107 SSR markers from whole genome shotgun sequences of Chinese bayberry (Myrica rubra) and their application in seedling identification.

Chinese bayberry (Myrica rubra Sieb. et Zucc.) is one of the important subtropical fruit crops native to the South of China and Asian countries. In this study, 107 novel simple sequence repeat (SSR) molecular markers, a powerful tool for genetic diversity studies, cultivar identification, and linkage map construction, were developed and characterized from whole genome shotgun sequences. M13 tailing for forward primers was applied as a simple method in different situations. In total, 828 alleles across 45 accessions were detected, with an average of 8 alleles per locus. The number of effective alleles ranged from 1.22 to 10.41 with an average of 4.08. The polymorphic information content (PIC) varied from 0.13 to 0.89, with an average of 0.63. Moreover, these markers could also be amplified in their related species Myrica cerifera (syn. Morella cerifera) and Myrica adenophora. Seventy-eight SSR markers can be used to produce a genetic map of a cross between 'Biqi' and 'Dongkui'. A neighbor-joining (NJ) tree was constructed to assess the genetic relationships among accessions, and the elite accessions 'Y2010-70', 'Y2012-140', and 'Y2012-145', were characterized as potential new genotypes for cultivation.

The genetic linkage maps of Anthurium species based on RAPD, ISSR and SRAP markers

We report the construction of genetic linkage maps of two species Anthurium ornatum (A. ornatum) and Anthurium andreanum (A. andreanum) based on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and sequence related amplified polymorphism (SRAP) markers using a double-pseudo test cross mapping strategy. Mapping population of 43 F1 individuals was developed using a natural mutant IIHR selection A1 (A. ornatum or Anthurium nymphaefolium) and a variety called Eternity (A. andreanum). A total of 228 markers, including 99 RAPDs, 21 ISSRs and 108 SRAP combinations were used for the linkage group analysis. The linkage map of A. ornatum consisted of 10 linkage groups and a doublet covering 1233.5 cM, with a total of 85 markers, whereas the linkage map of A. andreanum consisted of 12 linkage groups covering a distance of 1023.5cM with 78 markers. The constructed linkage map covered a total genome length of 77% and 73% of A. ornatum and A. Andreanum, respectively. This study provides an important basis for identification and mapping of horticultural traits, which would be exploited in Marker—Assisted Selection in Anthurium breeding programs.

Development and Application of Genic Simple Sequence Repeat Markers from the Transcriptome of Loquat

Deep transcriptome sequencing allows for the acquisition of large-scale microsatellite information, and it is especially useful for genetic diversity analysis and mapping in plants without reference genome sequences. In this study, a total of 14,004 simple sequence repeats (SSRs) were mined from 10,511 unigenes screening of 63,608 nonredundant transcriptome unigenes in loquat (Eriobotrya japonica) with a frequency of 22 SSR loci distributed over 100 unigenes. Dinucleotide and trinucleotide repeat SSRs were dominant, accounting for 20.62%, and 42.1% of the total, respectively. Seventy primer pairs were designed from partial SSRs and used for polymerase chain reaction (PCR) amplification. Of these primer pairs, 54 exhibited amplification and 33 were polymorphic. The number of alleles at these loci ranged from two to 17, and the polymorphism information content values ranged from 0.24 to 0.89. We tested the transferability of 33 SSR polymorphic primer pairs in apple and pear, and the transferability rates in these two species were 90.9% and 87.9%, respectively. A high level of marker polymorphism was observed in apple [Malus ×domestica (66.7%)], whereas a low level was observed in pear [Pyrus sp. (51.5%)]. In addition, the PCR products from seven SSR primer pairs were selected for sequence analysis, and 89.2% of the fragments were found to contain SSRs. SSR motifs were conserved among loquat, apple, and pear. According to our sequencing results for real SSR loci, ≈12,490 SSR loci were present in these loquat unigenes. The cluster dendrogram showed a distinct separation into different groups for these three species, indicating that these SSR markers were useful in the evaluation of genetic relationships and diversity between and within the species of Maloideae in the Rosaceae. The results of our identified SSRs should be useful for genetic linkage map construction, quantitative trait locus mapping, and molecular marker-assisted breeding of loquat and related species.

High-density genetic linkage map construction and identification of fruit-related QTLs in pear using SNP and SSR markers.

Pear (Pyrus spp) is an important fruit crop, grown in all temperate regions of the world, with global production ranked after grape and apples among deciduous tree crops. A high-density linkage map is a valuable tool for fine mapping quantitative trait loci (QTL) and map-based gene cloning. In this study, we firstly constructed a high-density linkage map of pear using SNPs integrated with SSRs, developed by the rapid and robust technology of restriction-associated DNA sequencing (RADseq). The linkage map consists of 3143 SNP markers and 98 SSRs, 3241 markers in total, spanning 2243.4 cM, with an average marker distance of 0.70 cM. Anchoring SSRs were able to anchor seventeen linkage groups to their corresponding chromosomes. Based on this high-density integrated pear linkage map and two years of fruit phenotyping, a total of 32 potential QTLs for 11 traits, including length of pedicel (LFP), single fruit weight (SFW), soluble solid content (SSC), transverse diameter (TD), vertical diameter (VD), calyx status (CS), flesh colour (FC), juice content (JC), number of seeds (NS), skin colour (SC), and skin smooth (SS), were identified and positioned on the genetic map. Among them, some important fruit-related traits have for the first time been identified, such as calyx status, length of pedicel, and flesh colour, and reliable localization of QTLs were verified repeatable. This high-density linkage map of pear is a worthy reference for mapping important fruit traits, QTL identification, and comparison and combination of different genetic maps.