Development of A High Sugar Trait-Related SCAR Marker in Sweet Sorghum.
To effectively screen and characterize high sugar traits in sweet sorghum we developed a sequence-characterized amplified regions (SCAR) marker which had a 3.25 cM of linkage distance with the high sugar trait in sorghum. A total of 210 random primers was used to screen the polymorphism between a pair of parent lines sweet sorghum LTR102 (as the female in this experiment) and common grain sorghum 654 (as the male) wherein 48 primers could produce at least one polymorphic fragment. The primer S336 which also produced a polymorphic fragment between the high sugar bulks and normal bulks of F5 progenies was further analyzed. The genomic DNA of LTR102 was amplified by S336 to produce an 1119 bp of fragment. We sequenced this DNA and synthesized a pair of SCAR primers (named as S3361119) specific to the DNA. The co- segregation between the SCAR marker and the high sugar trait was analyzed and a 3.33% of recombinant rate was detected in F5 individuals corresponding a 3.25 cM of linkage distance. This would provide an assistant means for sweet sorghum breeding. Copyright 2014 Friends Science Publishers
Keywords: Sweet sorghum; Bulked segregant analysis; Random amplified polymorphic DNA; Sequence characterized amplified regions (SCAR); Marker-assisted selection.
Sorghum (Sorghum bicolor L. Moench) is a major cereal crop mainly cultivated in the arid and semi-arid regions of the world (Smith and Frederiksen 2000). The name sweet sorghum" is used to distinguish those varieties of sorghum which accumulate high concentrations of soluble sugars like sucrose glucose and fructose in the stem juice from the common grain sorghum (Rao et al. 2013). Although sweet sorghum varieties have important agronomic traits and invaluable economic values the high sugar metabolism- related genes have been little known so far. Thus development of molecular markers tagged the high sugar trait is significant to the selection of fine varieties and clone of high sugar-related genes.DNA molecular marker technology has been documented to be a very powerful tool in crop genetic modification especially for the multiple gene-driven as well as the recessive gene-controlled traits (Winter and Kahl1995). For instance the markers of restriction fragment length polymorphism (RFLP) random amplified polymorphism DNA (RAPD) have been successfully applied in sorghum genomic marking (Bhattramakki et al.2000; Haussmann et al. 2002). In a previous study we developed a SCAR marker which successfully signed the head smut resistance in sorghum (Li et al. 2012).In this study to search for polymorphic fragments we employed a group of RAPD primers to amplify the genomic DNA of sweet sorghum and common line as well as their F5 near-isogenic lines and obtained a primer which produced an 1119 bp of polymorphic DNA in the parents and F5 progenies. Based on the resulted DNA sequence a pair of SCAR primers were designed and synthesized and then successfully tagged the high sugar trait of sorghum with a 3.25 cM of linkage map distance. This may provide an alternative strategy for the selection of new sweet sorghum varieties and cloning of high sugar-related genes.
Materials and Methods
Sweet sorghum LTR102 (as the female parent in this study) and common grain sorghum 654 (as the male parent) were supplied by Liaoning Academy of Agricultural Sciences. To establish near-isogenic lines we created F5 plants from the cross of LTR102 A- 654. Bulked segregant analysis (BSA) was used to identify putative markers tagged the high sugar trait. For the high sugar bulk DNA was extracted and pooled from F5 individuals with high sugar contents and for the low sugar bulk DNA was mixed from low sugar F5 individuals. In this study the high sugar individual was defined as the total sugar content in stalk juice over 16% and the low sugar plant as the sugar content below 12%. The sugar contents in plants were determined according to the method described by Yuan et al. (2008).For polymorphic analysis a total of 210 RAPD primers obtained from the company of Shanghai Shenggong China were used to amplify the parents and two sets of bulks. The genomic DNA was extracted from leaf tissues of parent lines and F5 individuals using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). The EN 61010-1 DNA Thermal Cycler (USA) was used to perform PCR which was established by mixing 17 L of ddwater 2.5 L of 10 A- buffer 2.5 L of25 mM MgCl2 0.5 L of 10 mM dNTP 0.5 L of 5 U/L Taq enzyme 1 L of 10 M primer and 1 L of DNA extract (500 ng/L). The thermal cycles were programmed for an initial denaturation of 3 min at 94C followed by 35 cycles of 30 s at 94C 20 s at 38C and 80 s at 72C; and a final 10 min extension at 72C. PCR products were detected in 1.4% agarose gels.For SCAR marker creation firstly the genomic DNAof LTR102 was amplified by PCR with primer S336 using the same reaction mixture and the same program as above mentioned. RAPD primer S336 is TCCCCATCAC. The resulted DNA fragment was extracted from the gel using DNA recovery kit (Sunbiotech Beijing China) and sequenced (performed in Takara Biotechnology Co. LtdDalian China). Based on the DNA sequence we designedSCAR primer S3361119 forward 5'-CTGTGTGATGTAG- CCTC-3' and reverse 5'-ATCCTAAACCCATCAGTG-3'. Using these primers the co-segregation analysis between the SCAR marker and the high sugar trait was conducted on the F5 near-isogenic populations including 87 high sugar individuals and 33 low sugar individuals by PCR. The reaction program was the same as above mentioned and the reaction mixture was composed as follow: 17 L dd water2.5 L 10 A- buffer 2.5 L 25 mM MgCl2 0.5 L 10 mM dNTP 0.5 L 5 U/L Taq enzyme 0.25 L 10 M forward primer 0.25 L 10 M reverse primer and 0.3 L DNA templates (100 ng/L). PCR products were separated in1.4% agarose gel. The recombinant rates and map distanceswere calculated according to the Kosambi mapping function (Kosambi 1944). The percentage of recombination (r)=recombinant individuals/total tested individuals A- 100. Linkage map distance (cM) = 1/4 A- ln (1+2r)/(1-2r).
The RAPD primers were tested on a survey of polymorphic DNA production in the parent lines and F5 bulks. Among them 48 primers amplified at least one polymorphic DNA fragment between the parent lines as well as the two F5 bulks (data not shown). We selected the primer S336 which produced an 1119 bp of polymorphic DNA fragment (Fig.1) to create SCAR markers. Firstly this DNA was sequenced (data not shown) then a pair of SCAR primers were synthesized based on the DNA sequence in the principle avoiding hairpin structure and appropriate G+C content.The co-segregation analysis in a total of 120 F5individuals was performed using the SCAR primers. One high sugar and 3 low sugar individuals respectively in 87 high sugar group and 33 low sugar group displayed recombination (Table 1) and corresponding linkage map distance was 3.25 cM.To confirm the identity of SCAR marker the genomic DNA of parent LTR 102 654 and representative F5 plants were amplified by primer S3361119 and as expected the marker well tagged the high sugar trait (Fig. 2).
Due to the quantitative trait (QTL) controlled by complex multiple genes the high sugar metabolism-related genes in sweet sorghum have been little known so far. However increasing studies by molecular marker-assisted techniques on the sugar-related QTLs have been emerging in sweet sorghum. For instance a genetic map of sugar components marked with AFLP (amplified fragment length polymorphism) SSR (simple sequence repeat) and EST (expressed sequence tag)-SSR markers was developed and several QTLs were detected using composite interval mapping (CIM) (Shiringani et al. 2010). Based on the RFLP AFLP and SSR markers Bain et al. (2006) constructed a genetic linkage map spanning 983.5 cM and aligning to the 10 linkage groups of sorghum in which two brix-related QTLs were sited on SBI-04 and SBI-10. Another nice stem sugar-related linkage map was constructed by Ritter et al. (2008) where QTL alleles responsible for enhanced sucrose and sugar contents were located on SBI-01 SBI-05 and SBI-06. Using AFLP technology differential markers between sweet and grain sorghum were found to mainly distribute on SBI-08 and SBI-10 (Ritter et al. 2007). Natoli et al. (2002) used the methods of simple interval mapping (SIM) and CIM to fix 1 and 2 sucrose-related QTLs on SBI-03 and SBI-05 respectively. These works and other related works are helpful to understand the genetic mechanism associated with the sugar metabolism in sorghum and the increasing identification of QTLs may accelerate sweet sorghum breeding by marker-assisted selection.However it may be more convenient and efficient for fine variety selection using a simple molecular marker tightly linked to the purpose trait-related major gene. In this context the SCAR marker developed in the present study may be useful to sweet sorghum selection and breeding due to its tight linkage with the high sugar trait as well as the more faithful stable repeatable easy-to-use and inexpensive compared to other molecular markers. Furthermore since the completed genome sequence of sorghum was published (Paterson et al. 2009) the studies associated with the molecular marker-related events have been more effective because the marker identification and the primer design have been easier. Based on the genomic sequence in theory any valuable SCAR markers can be easily mapped to a corresponding linkage group which will greatly aid in the map-based cloning of genes of interest.
Table 1: Co-segregation of the S3361119-amplified polymorphic fragment in F5
Primer###F5 individuals###Total plants tested###Polymorphic fragment###Percentage of recombination %
In conclusion we developed a SCAR marker with a 3.25 cM of linkage map distance with the high sugar trait in sorghum. To our knowledge this is the first SCAR marker tagging the high sugar property in sorghum.
This research was supported by the Natural Science Foundation of Liaoning Province (No. L2012387 201210166003) and National Natural Science Foundation (30870205 31270446).
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|Title Annotation:||sequence-characterized amplified regions|
|Publication:||International Journal of Agriculture and Biology|
|Date:||Jun 30, 2014|
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