Developing abundant and reliable molecular marker is of great significance for genetic map construction. And SNP marker has been used for genetic map construction in this research. Comparing to the common method, SLAF-seq technology used for large-scale marker development provided a higher density, better consistency, better effectiveness and saved the cost[19]. SLAF technology has been used in a large amount of plant molecular marker development and genetic mapping for laurel[20], mango[18], duck[21], soybeans[17] and other important crops. The smooth development of this study proved that SLAF sequencing technology is an effective method for large-scale development of molecular markers and high-density genetic mapping.
In this study, SLAF reduced-representation genome sequencing method has been used for large-scale marker development of Job’s tear. The high-throughput sequencing of Job’s tears SLAF library has been achieved under Illumina sequencing platform leading to a raw data of 325.49Gb for total of 1,628,398,591 double-ended sequencing fragments. Total of 302,295 SLAF markers were obtained by comparing and clustering analysis, and 3,605 effective markers with high polymorphism were received after filtering and eliminating the low-quality markers which was used for Job’s tears genetic linkage map construction. In this study, large number of molecular markers of Job’s tears were constructed to supply an important foundation for genetic map construction. Abundant of genome information provided an important reference for further molecular biology investigation on Job’s tears.
An important advantage of SLAF sequencing technology is the development of large-scale molecular markers in a single experiment. However, the sequence data obtained by high-throughput sequencing inevitably included numerical missing and errors. Therefore, multi-selection must be used for eliminating the errors which can affect genetic map construction[19]. 79,364 polymorphic SLAF markers has been obtained from the raw sequence data in this study but only 3,605 effective SALF markers has been final received after eliminating the markers possibly due to parent-genotype missing, low integrity, Mendelian error and significant segregation. In addition, sequencing result indicated that the accuracy of SLAF can be effectively improved by increasing the sequencing depth which is consistent with previous studies[19, 22]. Therefore, it is necessary to improve the depth of sequencing in future experiments for improving the efficiency of marker developing.
In this study the GC content of Job’s tears SLAF library was 46.96% which is slightly lower than transcription group[23]. This might be due to the different DNA sequence origins (i.e. genomic DNA, cDNA or EST). The types of SNP site in SLAF markers showed that most of them belong to conversion types (71.76%) which was similar to mango, peonies and sesame seeds[16]. The polymorphism rate of SLAF markers among parents of Job’s tears in target group was 26.25% which was lower than EST-SSR polymorphism rate of 31.1%[23]. However, this polymorphic rate of SLAFs is higher than many other varieties[11, 12], indicating a high genetic diversity has been constructed among Job’s tears varieties through SLAF which is consistent to previous investigation[2, 24].
By comparison with the other annual herb of homozygous genome, the heterozygosity of Job’s tears genome is far more complex for the genetic map construction. Therefore, Job’s tear has a more complex marker segregation type which is different from other traditional segregation population. In this study, “Wenyi NO.2” and “Qianyi NO.2” were used as male and female parent, respectively, and the F2 community has been used for genetic map. Besides, the Job’s tears has higher heterozygosity with complexed genome reached up to 1.6 to 1.73 Gb approximately, which makes it harder for high-density genetic map construction[12, 25]. Therefore, it is particularly important to find proper method for high-density linkage map construction of Job’s tear.
Two-step map that optimizes high-density linkage map construction method has been used for accurating high-density genetic map of Job’s tears construction through κ-Nearest neighbor, maximum likelihood algorithm by repeatedly sorted and error correction strategies[26]. Compared with the traditional JoinMap software, Two-step method is more efficient for high-throughput sequencing, more accurate and reliable for linkage maps construction, more efficient for mapping. This mapping strategy has been widely used in the studies of genetic maps such as Mango[18], Laurel[20], Duck Apotheph[21], and peony[27]. And the powerful power of two-step mapping has been further confirmed in this study for high-density lingage maps construction of Job’s tears.
The first high-density genetic map of Job’s tears has been constructed in our study, and 3,605 SLAF markers cluster to 10 genetic linkage groups were obtained withsame nucleus (2n = 20) as the Beijing Job’s tear species[28]. The total length of the map is 1,620.39 cM, with an average genetic distance of 0.45 cM, and 360.5 markers in average of per linkage group. The number of markers on each linkage group are widely variety, some of them are highly clustered in certain region, especially in LG1, LG7 and LG9. This phenomenon migth be caused by factors such as inconsistent molecular marker polymorphism or inconsistent recombination rates in certain chromosome regions of the graph-based parents[19]. Similar phenomena could also be found in plants such aspeony[27], sunflowers[29]. Ma et al. considered that cluster aggregation of molecules in the linkage group is related to the chromosomal filament or hesochtine region[30].
Segregation distortion is widespread in nature, which is considered to be one of the important drivers of species evolution, and reasons of segregation distortion are still doubts and controversy[31]. Faure et al. considers that the segregation distortion is formed due to biological factors including the selection of gamete and zygote, non-homogenous recombination, translocation on chromosomes or non-homogenous sites, low homozygosity of parents for mapping[32]. Previous studies have found that environmental factors, experimental errors, the ability of offspring separation and the loss of chromosomes might also be the reason for marker segregation distortion[27, 33–35]. In this study, about 16.45% of 593 markers showed significant segregation distortion (P < 0.05) in the target group. The high segregation distortion rate further indicated that the genetic diversity of their parents is high. And all 593 segregation distortion markers form 10 SDRs, and cluster distributed in LGs. The aggregation of segregation distortion might be due to the selection of gametophyte and sporophyte[36]. And this phenomenon is widely found in plants[13, 16, 35]. In addition, the use of segregation distortion markers to build a linkage map can increase the genomic coverage of the genetic map[16, 33], and may be conducive to quantitative site (QTL) positioning[37, 38].
High-density genetic linkage map has been constructed in this study which has been provided an effective way for important character analysis through QTL, Map-based cloning, and molecular Marker assisted selection on Job’s tear breeding. The 3,605 SNP markers about 93.78% in total map are the common dominant gene sequence labels which could give helps in comparative genomic researches[39] and association mapping[40]. And more important is that the molecular markers of high-density genetic map through SLAF is developed on the genome-wide level.
This study has demonstrated that the application of SLAF-seq technology on large-scale genetic markers of Job’s tears, and HighMap is an effective tool on molecular marker developing and high-density linkage map constructing by using the high-throughput sequencing data. This work provided an important molecular biology basis for genetic diversity, relationship, variety identification, QTL location of phenotypic matters, and genome function structure analysis of Job’s tears.