Linkage and mapping analyses of the no glue egg gene Ng in the silkworm ( Bombyx mori L.) using simple sequence repeats (SSR) markers

In the silkworm, Bombyx mori, no glue egg is mainly controlled by Ng (No glue) gene, which is located on the 12th chromosome. Owning to a lack of crossing over in females, reciprocal backcrossed F 1 (BC 1 ) progenies were used for linkage analysis and mapping of the Ng gene based on the simple sequence repeats (SSR) linkage map using silkworm strains H9 and P50, which are Ng mutant and normal to egg, respectively. The Ng gene was found to be linked to three SSR markers. Using a reciprocal BC 1 M cross, we constructed a linkage map of 36.4 cM, with Ng mapped at 15.9 cM and the nearest SSR marker at a distance of 7.4 cM. Based on fine genome map of domesticated silkworm ( B. mori ), the result of Kaikoblast show that the physical distance between the near markers (containing Ng gene) is 181.7 Kb. Further analysis show that BGIBMGA005833, BGIBMGA005835 and BGIBMGA005836 are closer to Ng , and the BGIBMGA005835 is nearest to Ng, which physical distance is 44 Kb.


INTRODUCTION
The mulberry silkworm, Bombyx mori, is an agriculturally important insect that has been domesticated for silk production for an estimated 5,000 years (Xiang, 1995).With the development of biotechnology, B. mori is the model organism for Lepidoptera, the second most numerous order of insects and has been used as an important bioreactor for the production of recombinant proteins (Tomita et al., 2003).It has many mutations, more than 400 of them have been identified and more than 200 visible and biochemical mutations have been placed on silkworm linkage maps (Fujii et al., 1998;Miao et al., 2007).These mutations affect many fundamental *Corresponding author.E-mail: cyschx@163.com.
#Both authors contributed equally to this work.aspects of the insect life cycle, including egg and egg shell formation, early embryonic pattern formation, development and diapause, larval feeding behavior, and molting (Nagaraju and Goldsmith, 2002).Mutation at the mucus gland in B. mori produces no glue eggs which are mainly controlled by Ng gene.The Ng gene was mapped at 28.0 of the silkworm classical genetic linkage group 12 (Xiang, 1995).
In recent years, molecular biology has made considerable progress in silkworm.Molecular linkage maps were constructed employing random amplified polymorphic DNA (RAPD), restriction fragment length polymorphic (RFLP), an amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSRs) (Tan, 2001;Miao et al., 2005;Yamamoto et al., 2006;Seyed, 2009), which lay a solid foundation for the location of silkworm.RAPDs, AFLPs and RFLPs are convenient tools for constructing molecular linkage maps.However, their disadvantages restrain its application in the relevant field (Sharma et al., 1996).The SSR technique is a convenient and reliable tool to generate highly polymorphic molecular markers which greatly facilitate building linkage maps (Litt and Luty, 1989;Weber and May, 1989;Bornet and Branchard 2001;Schlötterer, 2004).Microsatellites, or SSRs are randomly repeated units of one to six nucleotides and are abundant in prokaryotic and eukaryotic genomes (Santana et al., 2009).Now, because of polymerase chain reaction (PCR)based, highly reproducible and polymorphic, generally co dominant and abundant in animal and plant genomes, SSRs have become the most widely used of second generation molecular markers (Yoshiaki, 2000;Gupta and Varshney, 2000;Li, 2002;Viruel, 2004).Miao et al. (2005) constructed a genetic linkage map employing 518 SSR markers for B. mori, which covered all 28 chromosomes of silkworm, the greatest progress in the research of SSR markers in silkworm or even Lepidoptera.Li et al. (2006) located the densonucleosis non-susceptible gene nsd-Z on chromosome 15 using SSR markers.In addition, the silkworm genome research team of Southwest University (China, 2008) successfully completed the first fine genome map of domesticated silkworm (B.mori) in the world, it provides a great help to fine map and clone mutant gene.In this study, we located the No glue (Ng) gene using SSR markers, then calculated and analyzed genetic distance and physic distance of Ng in accordance with the silkworm Database.

Silkworm strains and genetic crosses
The P50 strain, which lay glue eggs, and the H9 strain, which lay no glue eggs (Ng mutant), were obtained from the Silkworm Genetics and Breeding Laboratory, School of Life Sciences, Anhui Agricultural University.A single-pair cross between a female (H9) and male (P50) produced the F1 offspring.Owing to lack of crossing over in females, reciprocal backcrossed F1 (BC1) progeny were used for linkage analysis and mapping of the Ng gene.For linkage analysis, BC1 progeny from the cross (H9 × P50)♀ × P50♂, together with a second backcross, BC1 progeny from P50♀ × (H9 × P50)♂, were used for recombination mapping.

DNA extraction
DNA samples were extracted from the single pupae of parents and the individuals of backcross population as described by Sambrook et al. (1989).The pupae was grounded with a mechanical homogenizer in a microcentrifuge tube and suspended in DNA extraction buffer (10 mmol/L Tris-HCl pH 8.0; 0.1 mol/L EDTA pH8.0; 0.5% SDS) that contained 100 µg/mL proteinase K.After digestion with proteinase K at 56°C for 3 to 5 h, phenol-chloroform extraction was carried out, and the DNA was recovered by isopropanol precipitation.The purified DNA was dissolved in TE buffer (pH 8.0).The DNA concentration was measured by spectrophotometry and the samples were diluted to a concentration of 10 ng/µl.

Linkage and recombination analysis
The primer pairs of SSR markers that showed polymorphism between H9 and P50 from 20 SSR markers were used for linkage analysis.We performed PCR amplification of genomic DNA from 22 offspring of BC1F progeny (11 individuals of no glue egg and 11 individuals of glue egg) and detected the primers which linked to Ng gene.To obtain recombination rate, PCR amplification of genomic DNA of 161 offspring of BC1M were performed.These products were detected for analyzing genotypes by acryl amide gels electrophoresis.

Analysis of genetic distance and physical distance between Ng and SSR markers
The genome sequences of markers from the result of data analysis were blasted to analyze the genetic and physical distance between Ng and SSR markers in the silkworm Database (http:// silkworm.Genomics.Org.cn).

Data analysis
The genetic relationship among markers was determined by maximum likelihood analysis, and the segregation pattern of marker data was analyzed using MAPMAKER version 3.0 (Lander et al., 1987) with the backcross data as an input file.The Kosambi mapping function was used to calculate the distances between markers loci in cM.

Polymorphism of SSR markers screening
Figure 1 shows the result of some electrophoretogram of polymorphism screening between H9 and P50.To find  polymorphic primers, the products from individual PCRs using the same primer pair in different DNA templates (H9 and P50) were resolved on PAGE, and the samples were pointed adjacently.The result of some primer pairs through PCR amplification is unsatisfactory and cannot judge polymorphism between H9 and P50 by repeating several times, so we abandoned these primers to avoid influencing subsequent experiment.Finally, we obtained 10 polymorphic markers from 20 SSR markers, including S1201, S1202, S1203, S1205, S1209, S1211, S1217, S1218, S1219 and S1220.

Linkage analysis of SSR markers with Ng gene
In B. mori, linkage analysis can be carried out efficiently because no crossing over occurs in females.The polymorphic markers which were found from H9 and P50 by polymorphism screening were used to analyze the genetic inheritance pattern in 22 offspring from BC 1 F progeny.Because SSRs are inherited in a Mendelian fashion and exhibit co dominant alleles, we screened SSR markers for linkage to Ng by comparing the pattern shown by groups of 22 individuals in BC 1 F progeny with the F 1 and parent P50 pattern.If the individuals of no glue eggs were all the same F 1 pattern (heterozygous, Ng+ Ng ), the SSR marker was confirmed to be linked to Ng gene; otherwise, the SSR marker was discarded (Figure 2).We continued with this type of analysis until we had analyzed all the polymorphic markers.Thus, a total of 3 SSR markers (S1202, S1203 and S1211) were identified to be linked to Ng (Table 2, Figures 3 and 4).

Linkage map construction of Ng gene with SSR markers
Since there is no crossing over in silk moth females, the initial analysis was only done in the SSR markers that were linked to the mutation.To determine the marker order and relative distance with respect to Ng, we carried out genotyping of the 3 SSR markers that were linked to Ng using 161 surviving BC 1 M progeny.As shown in Figures 5 and 6, we scored individual offspring in which the inheritance pattern showed either the same P50 pattern (homozygous, designated 1) or the same F 1 pattern (heterozygous, designated 2) for each SSR marker.The location of each marker was initially established through a 3-point comparative analysis of the number of recombinants in the sorted data and the recombination fractions were then calculated from the whole data set using Mapmaker 3.0.
The BC1M progeny generated the recombination map shown in Figure 7.The order of the SSR markers in the linkage group was established as follows: S1203, Ng, S1202 and S1211.The linkage map was 36.4 cM in length, and Ng was mapped at 15.9 cM.S1202, which is the SSR marker identified as being most closely linked to     Ng, was mapped at a distance of 7.4 cM from Ng.

Prediction of ORFs and analysis of physical distance between Ng and SSR markers
According to the result of genetic map, we blasted the genome sequences which are mostly close to Ng.The result of Kaikoblast show that the physical distance between S1202 and S1203 is 181.7 Kb (Table 3); If one ORF has 100 amino acids at least, with ATG as start codon, we tested 95 ORFs by the Clonemanager software.Meanwhile, the S1203 and S1202 were located in the nscaf2839 in fine genome map of domesticated silkworm.Through further analysis of the up and downstream sequences of S1202 and S1203 in the silkworm   8).

DISCUSSION
In the present study, the Ng gene was found to be linked to 3 SSR markers.A linkage map of 36.4 cM for linkage group was constructed, with Ng mapped at 15.9 cM, and with the closest marker being S1202.The order of the SSR markers in the linkage group is as follows: S1203, Ng, S1202 and S1211 (Figure 7).Based on fine genome map of domesticated silkworm (B.mori), the result of Kaikoblast shows that the physical distance between S1202 and S1203 is 181.7 Kb.Further analysis show that BGIBMGA005833, BGIBMGA005835 and BGIBMGA-005836 are closer to Ng, and the BGIBMGA005835 is nearest to Ng, with physical distance of 44 Kb.Ten polymorphic markers were screened from 20 SSR markers between H9 and P50 in this experiment, but, possibly, because the genetic relationship is too close between parents or small allele fragment which cannot be segregated by PAGE, some SSR markers have no polymorphism or are not able to judge polymorphism.In addition, we also found that some markers have polymorphism in parents, but no linkage to Ng when analysis of linkage was performed using individuals of BC 1 F progeny.The order of S1203 showed from the linkage map is different from the SSR linkage map (Miao et al., 2005).We conclude that the backcross population is too small or occur as double crossover.
In this study, the Ng gene was mapped using a small backcross population.However, fine mapping and cloning of the gene are not completed.Meanwhile, the regulatory mechanism of producing no glue egg which is caused by Ng gene is not yet known.Thus, mapping of the Ng gene will result in a better understanding of the mechanism of producing no glue egg in B. mori.The identification of these markers represents an important step towards positional cloning of the gene with these markers as tags.Now, a near-isogenic line which is set up using Ng mutant strain H9 and normal strain P50 is in progress in our

Figure 2 .
Figure 2. Inheritance pattern of simple sequence repeat (SSR) markers and Ng: a, one SSR marker in H9; b, one SSR marker in P50.

Figure 7 .
Figure 7. Mapping of Ng gene with SSR markers determined by analysis of the BC1M progeny of the cross P50♀ × (H9 × P50)♂.

Figure 8 .
Figure 8.The physical distance between Ng and molecular markers.

Table 1 .
Phenotypes of progeny from reciprocal backcross breeding.

Table 2 .
The SSR loci linked to Ng.

Table 3 .
The result of Kaikoblast in the silkworm Database.