Optimization of linkage mapping strategy and construction of a high-density American lotus linkage map.

Lotus is a diploid plant with agricultural, medicinal, and ecological significance. Genetic linkage maps are fundamental resources for genome and genetic study, and also provide molecular markers for breeding in agriculturally important species. Genotyping by sequencing revolutionized genetic mapping, the restriction-site associated DNA sequencing (RADseq) allowed rapid discovery of thousands of SNPs markers, and a crucial aspect of the sequence based mapping strategy is the reference sequences used for marker identification. We assessed the effectiveness of linkage mapping using three types of references for scoring markers: the unmasked genome, repeat masked genome, and gene models. Overall, the repeat masked genome produced the optimal genetic maps. A high-density genetic map of American lotus was constructed using an F1 population derived from a cross between Nelumbo nucifera 'China Antique' and N. lutea 'AL1'. A total of 4,098 RADseq markers were used to construct the American lotus 'AL1' genetic map, and 147 markers were used to construct the Chinese lotus 'China Antique' genetic map. The American lotus map has 9 linkage groups, and spans 494.3 cM, with an average distance of 0.7 cM between adjacent markers. The American lotus map was used to anchor scaffold sequences in the N. nucifera 'China Antique' draft genome. 3,603 RADseq markers anchored 234 individual scaffold sequences into 9 megascaffolds spanning 67% of the 804 Mb draft genome. Among the unmasked genome, repeat masked genome and gene models, the optimal reference sequences to call RADseq markers for map construction is repeat masked genome. This high density genetic map is a valuable resource for genomic research and crop improvement in lotus.

Zhang Q ，Li L ，VanBuren R ，Liu Y ，Yang M ，Xu L ，Bowers JE ，Zhong C ，Han Y ，Li S ，Ming R ... -  《BMC GENOMICS》

Genetic linkage maps for Asian and American lotus constructed using novel SSR markers derived from the genome of sequenced cultivar.

The genus Nelumbo Adans. comprises two living species, N. nucifera Gaertan. (Asian lotus) and N. lutea Pers. (American lotus). A genetic linkage map is an essential resource for plant genetic studies and crop improvement but has not been generated for Nelumbo. We aimed to develop genomic simple sequence repeat (SSR) markers from the genome sequence and construct two genetic maps for Nelumbo to assist genome assembly and integration of a genetic map with the genome sequence. A total of 86,089 SSR motifs were identified from the genome sequences. Di- and tri-nucleotide repeat motifs were the most abundant, and accounted for 60.73% and 31.66% of all SSRs, respectively. AG/GA repeats constituted 51.17% of dinucleotide repeat motifs, followed by AT/TA (44.29%). Of 500 SSR primers tested, 386 (77.20%) produced scorable alleles with an average of 2.59 per primer, and 185 (37.00%) showed polymorphism among two parental genotypes, N. nucifera 'Chinese Antique' and N. lutea 'AL1', and six progenies of their F1 population. The normally segregating markers, which comprised 268 newly developed SSRs, 37 previously published SSRs and 53 sequence-related amplified polymorphism markers, were used for genetic map construction. The map for Asian lotus was 365.67 cM with 47 markers distributed in seven linkage groups. The map for American lotus was 524.51 cM, and contained 177 markers distributed in 11 genetic linkage groups. The number of markers per linkage group ranged from three to 34 with an average genetic distance of 3.97 cM between adjacent markers. Moreover, 171 SSR markers contained in linkage groups were anchored to 97 genomic DNA sequence contigs of 'Chinese Antique'. The 97 contigs were merged into 60 scaffolds. Genetic mapping of SSR markers derived from sequenced contigs in Nelumbo enabled the associated contigs to be anchored in the linkage map and facilitated assembly of the genome sequences of 'Chinese Antique'. The present study reports the first construction of genetic linkage maps for Nelumbo, which can serve as reference linkage maps to accelerate characterization germplasm, genetic mapping for traits of economic interest, and molecular breeding with marker-assisted selection.

Yang M ，Han Y ，VanBuren R ，Ming R ，Xu L ，Han Y ，Liu Y ... -  《BMC GENOMICS》

Construction of a high-density, high-quality genetic map of cultivated lotus (Nelumbo nucifera) using next-generation sequencing.

The sacred lotus (Nelumbo nucifera) is widely cultivated in China for its edible rhizomes and seeds. Traditional plant breeding methods have been used to breed cultivars with increased yields and quality of rhizomes and seeds with limited success. Currently, the available genetic maps and molecular markers in lotus are too limited to be useful for molecular genetics based breeding programs. However, the development of next-generation sequencing (NGS) technologies has enabled large-scale identification of single-nucleotide polymorphisms (SNPs) for genetic map construction. In this study, we constructed an SNP-based high-density genetic map for cultivated lotus using double digest restriction site-associated DNA sequencing (ddRADseq). An F2 population of 96 individuals was derived from a cross between the rhizome lotus cultivar 'Juwuba' (male parent) and the seed lotus cultivar 'Mantianxing' (female parent). Genomic DNAs from this population were digested with the restriction enzymes EcoRI and MspI and then sequenced. In total, 133.65 Gb of raw data containing 1,088,935,610 pair-end reads were obtained. The coverage of reads on a reference genome was 7.2 % for the female parent, 6.56 % for the male parent, and 1.46 % for F2 individuals. From these reads, 10,753 valid SNP markers were used for genetic map construction. Finally, 791 bin markers (so-segregated adjacent SNPs treated as a bin marker), consisting of 8,971 SNP markers, were sorted into 8 linkage groups (LGs) that spanned 581.3 cM, with an average marker interval of 0.74 cM. A total of 809 genome sequence scaffolds, covering about 565.9 cM of the wild sacred lotus genome, were anchored on the genetic map, accounting for 70.6 % of the genome assembly. This study reports the large-scale discovery of SNPs between cultivars of rhizome and seed lotus using a ddRADseq library combined with NGS. These SNPs have been used to construct the first high-density genetic map for cultivated lotus that can serve as a genomic reference and will facilitate genetic mapping of important traits in the parental cultivars.

Liu Z ，Zhu H ，Liu Y ，Kuang J ，Zhou K ，Liang F ，Liu Z ，Wang D ，Ke W ... -  《BMC GENOMICS》

Improving Nelumbo nucifera genome assemblies using high-resolution genetic maps and BioNano genome mapping reveals ancient chromosome rearrangements.

Genetic and physical maps are powerful tools to anchor fragmented draft genome assemblies generated from next-generation sequencing. Currently, two draft assemblies of Nelumbo nucifera, the genomes of 'China Antique' and 'Chinese Tai-zi', have been released. However, there is presently no information on how the sequences are assembled into chromosomes in N. nucifera. The lack of physical maps and inadequate resolution of available genetic maps hindered the assembly of N. nucifera chromosomes. Here, a linkage map of N. nucifera containing 2371 bin markers [217 577 single nucleotide polymorphisms (SNPs)] was constructed using restriction-site associated DNA sequencing data of 181 F individuals and validated by adding 197 simple sequence repeat (SSR) markers. Additionally, a BioNano optical map covering 86.20% of the 'Chinese Tai-zi' genome was constructed. The draft assembly of 'Chinese Tai-zi' was improved based on the BioNano optical map, showing an increase of the scaffold N50 from 0.989 to 1.48 Mb. Using a combination of multiple maps, 97.9% of the scaffolds in the 'Chinese Tai-zi' draft assembly and 97.6% of the scaffolds in the 'China Antique' draft assembly were anchored into pseudo-chromosomes, and the centromere regions along the pseudo-chromosomes were identified. An evolutionary scenario was proposed to reach the modern N. nucifera karyotype from the seven ancestral eudicot chromosomes. The present study provides the highest-resolution linkage map, the optical map and chromosome level genome assemblies for N. nucifera, which are valuable for the breeding and cultivation of N. nucifera and future studies of comparative and evolutionary genomics in angiosperms.

Gui S ，Peng J ，Wang X ，Wu Z ，Cao R ，Salse J ，Zhang H ，Zhu Z ，Xia Q ，Quan Z ，Shu L ，Ke W ，Ding Y ... -  《-》

A SSR-based composite genetic linkage map for the cultivated peanut (Arachis hypogaea L.) genome.

The construction of genetic linkage maps for cultivated peanut (Arachis hypogaea L.) has and continues to be an important research goal to facilitate quantitative trait locus (QTL) analysis and gene tagging for use in a marker-assisted selection in breeding. Even though a few maps have been developed, they were constructed using diploid or interspecific tetraploid populations. The most recently published intra-specific map was constructed from the cross of cultivated peanuts, in which only 135 simple sequence repeat (SSR) markers were sparsely populated in 22 linkage groups. The more detailed linkage map with sufficient markers is necessary to be feasible for QTL identification and marker-assisted selection. The objective of this study was to construct a genetic linkage map of cultivated peanut using simple sequence repeat (SSR) markers derived primarily from peanut genomic sequences, expressed sequence tags (ESTs), and by "data mining" sequences released in GenBank. Three recombinant inbred lines (RILs) populations were constructed from three crosses with one common female parental line Yueyou 13, a high yielding Spanish market type. The four parents were screened with 1044 primer pairs designed to amplify SSRs and 901 primer pairs produced clear PCR products. Of the 901 primer pairs, 146, 124 and 64 primer pairs (markers) were polymorphic in these populations, respectively, and used in genotyping these RIL populations. Individual linkage maps were constructed from each of the three populations and a composite map based on 93 common loci were created using JoinMap. The composite linkage maps consist of 22 composite linkage groups (LG) with 175 SSR markers (including 47 SSRs on the published AA genome maps), representing the 20 chromosomes of A. hypogaea. The total composite map length is 885.4 cM, with an average marker density of 5.8 cM. Segregation distortion in the 3 populations was 23.0%, 13.5% and 7.8% of the markers, respectively. These distorted loci tended to cluster on LG1, LG3, LG4 and LG5. There were only 15 EST-SSR markers mapped due to low polymorphism. By comparison, there were potential synteny, collinear order of some markers and conservation of collinear linkage groups among the maps and with the AA genome but not fully conservative. A composite linkage map was constructed from three individual mapping populations with 175 SSR markers in 22 composite linkage groups. This composite genetic linkage map is among the first "true" tetraploid peanut maps produced. This map also consists of 47 SSRs that have been used in the published AA genome maps, and could be used in comparative mapping studies. The primers described in this study are PCR-based markers, which are easy to share for genetic mapping in peanuts. All 1044 primer pairs are provided as additional files and the three RIL populations will be made available to public upon request for quantitative trait loci (QTL) analysis and linkage map improvement.

Hong Y ，Chen X ，Liang X ，Liu H ，Zhou G ，Li S ，Wen S ，Holbrook CC ，Guo B ... -  《BMC PLANT BIOLOGY》