Construction of a high-density genetic map using specific-locus amplified fragments in sorghum

Sorghum (Sorghum bicolor) is one of the five dominant crops in the world including corn (Zea mays), wheat (Triticum aestivum), rice (Oryza sativa) and barley (Hordeum vulgare) (http://www.fao.org). With the advantages of high yielding, good adaptability, drought, salt and alkali tolerance, it is one of the most valuable energy crops for the future [1, 2]. Sorghum is a typical C₄ crop and mainly used as a human food and beverage source. Sorghum grain is the main ingredient of top-grade alcohols and its stem can be used as fodder. Both grain and stem including all plant are stock for bioethanol and other bio-industrial products.

The genome size of sorghum (750 Mb) is 3–4 times smaller than corn, thus it was regarded as a diploid model crop for energy plants like polyploidy sugarcane and Miscanthus [3, 4]. A genetic map is a foundation for quantitative and qualitative gene mapping and cloning, and plays a key role in marker-assisted breeding program. High-density genetic maps of sorghum can be used for genome comparison, useful gene mining and gene mapping. The genes for disease and insect-resistance, stress tolerance, sugar concentrations and biological yield can be identified by comparing homology in different plant species, and they can also be located on chromosomes by mapping, which lays a foundation for gene cloning and application. High-density genetic mapping has great importance in increasing statistical power and precision of detecting genes and QTLs.

Genetic map construction of sorghum began in 1990s. The early linkage maps of sorghum were constructed mainly by using labor-intensive or dominant markers such as RFLP (Restriction fragment length polymorphism), AFLP (Amplified fragment length polymorphism) and RAPD (Random amplified polymorphic DNA) [5–10]. These maps have played important role in sorghum gene (QTL) mapping, comparative genomics and genetics studies. However, these genetic marker systems have limited marker numbers, dominant expression, and not repeatable in different maps. More informative marker types can effectively overcome the disadvantages mentioned above are required. Due to the quick development of sequencing and genotyping technologies, simple sequence repeat (SSR) with features of high reproducibility, co-dominant inheritance, multi-allelic variation and abundance in the genome, have replaced dominant markers for constructing linkage maps. SSR markers were first used for polymorphism detecting and linkage group identification [11, 12], then were used to construct sorghum genetic maps with the development of a large amount of SSR markers [13, 14]. Several linkage maps with SSR markers or mainly based on SSR markers have been developed and have been using in sorghum gene (QTL) mapping, genome evolution, molecular genetics and marker-assisted breeding [4, 15–18].

However, the above technologies such as RAPD, RFLP, AFLP and SSR to determine genetic fingerprints have limitations to cover full genome which requires the identification of a large number of polymorphic markers. With these technologies this is a step by step approach that is labor intensive and plagued by process variation. Diversity Arrays Technology (DArT) was initially used to detect a large number of genetic differences between plant and animal varieties. Recently this technology was introduced for sorghum map construction. DArT markers were integrated into a sorghum consensus map which consisted of a total of 1997 markers mapped to 2029 unique loci (1190 DArT loci and 839 other loci) spanning 1603.5 cM and with an average marker density of 1 marker/0.79 cM [19].

Great progress has been made in the sequencing technologies and bioinformatics at an exponentially reduced cost, which led to a revolution in the field of genotyping technologies. Restriction associated DNA sequencing (RAD-seq) and genotyping by sequencing (GBS) have emerged as powerful genotyping platforms, which are capable of identifying, sequencing, and genotyping thousands of markers across almost any genome of interest and number of individuals in a population [20]. The next generation sequencing can directly determine differences in DNA sequence with high accuracy, thus it has been widely used for plant and animal genetic analysis. SLAF (specific-locus amplified fragments) markers, which has been used for genetic investigation, have the properties of being present in large amount, being evenly distributed and avoiding repeated sequences [21]. These markers have been used for crop genetic analysis such as sesame, millet, rice and soybean [22–26], especially in the applications of high-density genetic map construction and functional genes verification. Exploiting this approach to scan the whole sorghum genome has great importance for high density marker development and gene mining for sorghum breeding.

The purpose of this study is to construct a high-density linkage map with SNPs through next generation sequencing technology. The map can provide information and serve as a reference for effective gene exploration and lay a foundation for marker assisted breeding. Further, it can benefit the development of biological energy resources.

In the present study an F₂ mapping population from a cross between a sweet and a grain sorghum variety was employed to construct a sorghum linkage map. The great character variations between the two parents benefited the marker polymorphism discovery. The high-throughput sequencing technology used in the present study has greatly enhanced the identification and guaranteed the quantity and quality of markers. Therefore a high density genetic map was successfully constructed. Some existing sorghum maps are unsatisfactory for gene identification because of lacking adequate markers from the whole genome, and broken chromosome segments. In the present study a dense genetic map was generated in which the whole sorghum genome sequence was surveyed, high quality markers were identified and uniformly distributed on 10 chromosomes. Each chromosome contains 123–318 markers and its length ranged from 132.8 to 300.4 cM. This high density SNP-based linkage map for sorghum can serve as a reference map for cultivated sorghum species and will be useful in genetic mapping.

DNA marker distribution is not random with some clear marker-dense regions and some marker deserts. In the present map, marker deserts (gaps) were observed with varied sizes. Most (97.0%) of the gaps on every linkage group are less than and/or equal to 5.0 cM. In total only 11 gaps larger than 5.0 cM were detected in all chromosomes except chr 9 suggesting that such gaps are not restricted to a particular chromosome. Gaps larger than 10.0 cM were found on chromosomes 1, 4, 6 and 7. The longest one was 15.7 cM on the distal end of chr 7. The presence of these gaps may have negative effects on the application of mapped DNA markers, for example, genomic regions that lack DNA markers will make detection of quantitative trait loci (QTL) difficult [27]. Therefore, more comparable markers between different sorghum maps are needed to fill in the gaps to obtain a more complete coverage of the sorghum genome.

Segregation distortion is a common phenomenon in which the genotypic frequency of a marker deviates from a typical Mendelian ratio. Previous studies have showed that a large number of segregation distortions and SDRs occur in many species, such as maize [28], barley [29] potato [30], sesame [24], peanut [31] and sorghum [3, 19]. The genetic basis of segregation distortion is still under debate, and gametophyte and/or zygotic selection and chromosomal rearrangements may be the main cause of this phenomenon [3]. However, some studies found segregation distortion in a non-random and consistent distribution pattern suggested that distorted segregation is due to the elimination of gametes or zygotes by a lethal factor located in a neighboring region of the marker [19]. On a sorghum consensus map, chr 1 has the highest proportion of chromosomal regions associated with skewed segregation (67%). Two other chromosomes (chr 4 and chr 8) also have over 50% of the chromosomal regions associated with skewed segregation (51.6% and 54.1%, respectively) [19]. In the present study, an F₂ mapping population was employed to construct a linkage map, among the 2246 assigned markers, 1192 markers (53.1%) showed significant segregation distortion. All the skewed markers clustered into segregation distortion regions. Although it is not exactly the same, chr 1 has the highest proportion (71.5%) of skewed markers and chr 4 has the biggest number (14) of SDRs in this final map, which indicates that there may be similar mechanism of skewed segregation phenomenon between the two studies. Further, studies have proved that the presence of segregation distortion markers will not affect the use of linkage maps for applications such as QTL mapping [32, 33].

Genomic approaches such as high-throughput sequencing and large-scale genotyping technologies have been used in genetic linkage mapping. The SLAF-seq method provided significant advantages to generate enough polymorphic markers for high-density genetic map construction. The high density map is sufficient to ensure adequate polymorphic marker coverage in regions of interest and can be used as a reference map for sorghum genetic studies.

A high density sorghum map was constructed in this study by employing SLAF markers developed from high-throughput sequencing technology. The total map length is 2158.1 cM covering sorghum 10 chromosomes with a total of 2246 SLAF markers. The construction of this map can provide detailed information for gene localization, cloning and application of marker-assisted breeding.