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Article

Isolation and Characterization of Microsatellite Loci in the Asian Rice Gall Midge (Orseolia oryzae) (Diptera: Cecidomyiidae)

1
Directorate of Rice Research, Rajendranagar, Hyderabad 500030, India
2
Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500001, India
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2011, 12(1), 755-772; https://doi.org/10.3390/ijms12010755
Submission received: 10 December 2010 / Revised: 1 January 2011 / Accepted: 13 January 2011 / Published: 20 January 2011

Abstract

:
Microsatellite loci were isolated from the genomic DNA of the Asian rice gall midge, Orseolia oryzae (Wood-Mason) using a hybridization capture approach. A total of 90 non-redundant primer pairs, representing unique loci, were designed. These simple sequence repeat (SSR) markers represented di (72%), tri (15.3%), and complex repeats (12.7%). Three biotypes of gall midge (20 individuals for each biotype) were screened using these SSRs. The results revealed that 15 loci were hyper variable and showed polymorphism among different biotypes of this pest. The number of alleles ranged from two to 11 and expected heterozygosity was above 0.5. Inheritance studies with three markers (observed to be polymorphic between sexes) revealed sex linked inheritance of two SSRs (Oosat55 and Oosat59) and autosomal inheritance of one marker (Oosat43). These markers will prove to be a useful tool to devise strategies for integrated pest management and in the study of biotype evolution in this important rice pest.

1. Introduction

The Asian rice gall midge, Orseolia oryzae (Wood-Mason) (Diptera: Cecidomyiidae), a major pest of rice, Oryza sativa L. [1], forms leaf-sheath gall called silver shoot. The maggot, hatching from the egg, crawls down the leaf sheath, feeds on the apical meristem and induces formation of gall, which renders the tiller sterile resulting in grain yield loss. It is the third most economically important pest of rice in India causing an average annual yield loss worth US$ 80 million [2]. Host plant resistance is the most effective, eco-friendly and cost efficient means of managing the pest to alleviate crop loss [3]. Resistance in plant is controlled by a single, generally, dominant gene. To date, 11 resistance genes have been identified [4]. But the resistance in the commercial rice varieties is short lived due to the ability of the insect to rapidly evolve virulent populations, called biotypes. So far, seven distinct biotypes of the pest have been characterized in India [5]. Pest populations, however, are not homogeneous in biotype composition in time and space [6]. In order to understand the process of evolution of biotypes characterization of diverse biotypes and studies on gene flow among populations is essential. Molecular markers have proven invaluable in such studies. Behura et al. [7] used SCAR/RAPD markers for biotype distinction. AFLP markers have been used in the study of biodiversity of the gall midge populations from 15 sites across five Asian countries [8]. However, these markers lacked reproducibility or are difficult to use. In our earlier studies [5], we devised methods for differentiating the biotypes and screened for different biotypes, which resulted in establishment of a pure culture of three biotypes. Hence, we attempted to develop reliable and easy to use PCR-based simple sequence repeat (SSR) markers for this insect species. In the current work we have, for the first time, identified simple sequence repeats from the Asian rice gall midge genome. The sequence information from these repeat regions was used to develop 90 SSR markers to reveal polymorphism between the three biotypes and sexes and studied inheritance of three markers.

2. Results and Discussion

2.1. Microsatellite Loci and Polymorphism

Out of the 1635 recombinant clones screened, 1309 (80%) clones had the insert of desired size (≥500 bp). Among these, 170 (13%) clones had repeats of varying lengths. From these, 90 microsatellite loci were selected and primers were designed using Primer 3 software ( http://frodo.wi.mit.edu/cgi-bin/primer3) [9]. Of the 90 loci studied, the majority contained dinucleotide repeat motifs (72%), followed by tri- (15.3%), and more complex (12.7%) repeat motifs. Dinucleotide repeats mostly consisted of GT (17.7%) repeats, followed by GA repeats (12.2%). The higher percentage of dinucleotide repeats found was in contrast to the report of SSRs found in another midge, the Hessian fly [10], which had an abundance of trinucleotide repeats.
After standardization, 15 loci (Table 1) were found to be polymorphic between the biotypes and sexes, whereas 75 loci were found monomorphic (Supplementary Table 1). Number of alleles of these 15 markers ranged from two (Oosat21, Oosat46 and Oosat78) to 11 (Oosat59) per locus. All the loci had expected heterozygosity of more than 0.5 in different biotypes and therefore these markers can be considered as good for parentage analysis. The range of observed and expected heterozygosity was calculated to be 0 to 1 and 0.304 to 0.910, respectively. Polymorphic information content (PIC), a measure of informativeness of a marker, calculated using Cervus v2.0 [11], ranged from 0.305 to 0.877; 14 markers with >0.5 PIC are considered highly informative in terms of their suitability for diversity analysis. Oosat26, Oosat55 and Oosat83 were most informative, while Oosat21 the least, against all the three biotypes. Despite multiple alleles, frequency of alleles varied in different biotypes (Table 2). Allele frequency of the predominat allele for each of the biotypes varied from 0.15 to 0.8. Some markers showed high allele frequency for some alleles, therefore fixation tendency of all markers was analysed. Fixation table (Table 3) was generated with following fixation values; FIS, to measure the deviation of genotypic frequencies from panmictic frequencies in terms of heterozygous deficiency or excess, FST, reduction in heterozygosity in a subpopulation due to genetic drift and FIT, overall inbreeding coefficient of an individual relative to the total population. For three loci (Oosat24, Oosat35 and Oosat46) negative FIS values indicated heterozygote excess (outbreeding). For other locus, positive values indicated heterozygote deficiency (inbreeding). FST values up to 0.05 indicate negligible genetic differentiation within the population which was observed for locus Oosat21, Oosat83 and Oosat88. For all the polymorphic loci, the Ewens-Watterson Test for Neutrality was also performed and it was observed that except for the locus Oosat88, all the loci were found neutral. Oosat88 was not behaving neutral in GMB4M population (at 95% confidence level).
Linkage disequilibrium studies (using Fisher’s method) after sequential Bonferroni correction revealed that Oosat26, Oosat36 and Oosat88 were significantly associated with each other. Also the markers Oosat24 and Oosat46, Oosat35 and Oosat21 were associated with each other, whereas other markers segregated independently in the population. P-value (HWE) mentioned in Table 1, was calculated according to Guo and Thompson [12]. Observed deviation from Hardy Weinberg equilibrium for some markers (Oosat3, Oosat21, Oossat35, Oosat43, Oosat79 and Oosat88; P > 0.05) may be the result of inbreeding, natural selection or genetic drift. Null alleles were also detected using Microchecker v.2.2.3 [13] for markers Oosat16, Oosat26, Oosat36, and Oosat83, and these could be as a result of mutations occurring in the flanking regions, preventing one or both of the primers from binding. Other polymorphic markers did not show null alleles. Markers without null alleles, give a better estimate of allele frequencies.
BLASTX analysis was performed using the sequence information of the 15 polymorphic loci which revealed that Oosat78 showed homology to tyrosyl-dna phosphodiesterase of Culex quinquefasciatus (E value: 1 × 10−10). The SSR repeat sequence was found to be present in the intron region of the gene. The gene has been reported to be from the phopholipase D family, which includes diverse groups of enzymes involved in phopholipid metabolism, a bacterial toxin, viral envelop proteins, and bacterial nucleases [14]. BLASTN analysis, and also BLASTX analysis with Hessian fly sequences showed insignificant similarity.

2.2. Inheritance of SSR Markers

The inheritance pattern of three of the labeled markers, Oosat43, Oosat55 and Oosat59, was studied in F2 families of GMB4M through pedigreed crosses. Inheritance of two of the markers—Oosat55 and Oosat59—proved to be sex linked. The male progeny inherited alleles only from the female parent (Figures 1 and 2); but female progeny inherited alleles from both the male and female parents. However, the pattern of inheritance of alleles of Oosat43 suggested an autosomal pattern as the alleles were inherited from both the male and female parents (Figure 3). Inheritance pattern of these markers confirmed existence of sexual dimorphism [15] and the abnormal chromosomal cycle typical of Cecidomyiidae [16,17]. Sex linked markers may be useful in tagging virulence alleles that are often sex linked [18]. Further, studies on inheritance of the markers also suggested some degree of instability with the appearance of novel non-parental alleles appearing in the offspring (Figures 1 and 2). Thus, the present study reports and confirms the usefulness of SSR markers for the rice gall midge.

3. Experimental Section

3.1. Insect Colonies

Colonies of gall midge biotypes (GMB) GMB1, GMB4 and GMB4M are being maintained in the greenhouse at the Directorate of Rice Research under physical isolation and on appropriate differential rice genotypes [5]. Iso-female families were initiated with two founding pairs, since a single female will produce either all male (androgenic) or all female (gynogenic) progeny. Eight to ten F1 pairs were mated separately to obtain F2 adults, which were then pooled to initiate the family. At least 10 generations were reared before using these insects for DNA extraction and for inheritance studies.

3.2. Isolation of Microsatellite Loci

DNA was extracted from the iso-female families of adult midges. The insects were crushed in an extraction buffer (0.1 M NaCl; 0.1 M Tris-HCl, pH 9.1; 0.05 M EDTA; 0.05% SDS), and extracted once with phenol:chloroform:isoamyl alcohol (25:24:1), and then once with chloroform:isoamyl alcohol (24:1). The purified genomic DNA was ethanol precipitated and resuspended in sterile distilled water after rinsing the pellet in 75% alcohol. The pooled DNA from the three biotypes was used for the purpose of library generation.
The library was constructed using hybridization capture approach of Glenn and Schable [19]. Genomic DNA was digested with the restriction enzymes Rsa I and Xmn I and ligated with the super SNX double stranded linkers on both sides to provide the primer binding site for subsequent PCR steps. They also provided sites for cloning in the vectors. Dynabead enrichment for Microsatellite containing DNA was performed. From the hybridized DNA + probe mixture, the DNA fragments with microsatellite repeat were captured using Dynabeads (Dynal, Oslo, Norway) under magnetic field. The amount of eluted DNA was increased by using the PCR enrichment to recover enriched DNA fragments using the super SNX forward primer with the appropriate PCR components under the conditions: 95 °C for 2 min; then, 25 cycles of 95 °C for 20 s, 60 °C for 20 s, 72 °C for 1.5 min; then 72 °C for 30 min. The enriched PCR product was directly cloned into TA vector (Invitrogen, U.S.). Plasmids were isolated and colony PCR was performed. The plasmids containing insert sizes of above 500 bp were selected and diluted to 100 ng/μL, and sequenced using automated sequencer (ABI prism 3700). The sequences were screened for the presence of microsatellites using the software MICAS ( www.cdfd.org.in/micas/) after removal of redundant sequences. Primers were designed from the flanking region of the repeats of the non-redundant sequences using Primer3 software [9].

3.3. Detection of Polymorphism and Data Analysis

DNA was isolated individually from 10 female and 10 male adults from the iso-female families each of GMB1, GMB4 and GMB4M by Hot Shot protocol [20]. These DNA samples were used as templates directly for PCR with the optimized primers and PCR conditions. Amplified product was visualized on 10% PAGE stained with ethidium bromide, which resulted in the identification of 15 polymorphic markers. Of the 15 polymorphic markers, 10 markers were labeled with FAM fluorescent dye and genotyped in 3730 DNA Analyzer with HiDi formamide and GeneScanTM- 500LIZ®Size Standard (Applied Biosystems, U.S.). The results were analyzed with GeneMapper v4.0 software (Applied Biosystems, U.S.) to calculate the allele size and number of alleles. Genetic analysis was performed using Arlequin 3.1 [21], Genepop v4.0 [22] and Cervus v2.0 [11].

3.4. Inheritance of SSR Markers

Three markers (Oosat55, Oosat59 and Oosat43) were selected based on the observed polymorphism between males and females. These markers were selected for inheritance study in the iso-female families of GMB4M. Pedigreed crosses were made to obtain F1 and F2 progeny while insects after mating, were preserved, for genotyping with these markers. At least two parental pairs were used to generate F1 females that produced male and female progeny.

4. Conclusions

In conclusion, we report, for the first time, the development of 15 polymorphic microsatellite markers that can be used for efficient genetic studies, for example linkage analysis, and construction of molecular linkage maps. We also discovered markers that have sex linked inheritance in the gall midge. These markers are currently being screened, in a mapping population, to ascertain linkage with virulence alleles in the insect. This study could pave the way for identification of virulence genes in the insect. Further, these markers will be a good tool for developing strategies for the management of the rice gall midge.

Supplementary Material

Supplementary Table 1. Microsatellite loci and primer sequences for the 75 SSR markers found to be monomorphic in three biotypes of the Asian rice gall midge, Orseolia oryzae (Wood-Mason) (Diptera: Cecidomyiidae).
Supplementary Table 1. Microsatellite loci and primer sequences for the 75 SSR markers found to be monomorphic in three biotypes of the Asian rice gall midge, Orseolia oryzae (Wood-Mason) (Diptera: Cecidomyiidae).
Accession Number (GeneBank)LocusRepeat MotifPrimerAnn. temp.MgCl2 conc.Alleles Size
HM804497Oosat01(GA)10F:TCATCAAAAGGCAATGAGAAA
R:GAAGACAACACACCGCACAT
59 °C1.5 mM159
HM804498Oosat02(TTAAAAT)2 N….(ATTTTA)4F:TGCACAAAAATAACGCAGGA
R:ATAACCCAACCAAACCACGA
60 °C2.0 mM263
HM804500Oosat04(TC)10…(TC)3F:TGCACAAAAATTGCGATTCTAC
R:CCCATATTGGGCAGCATCT
59 °C3.0 mM340
HM804501Oosat05(CA)6F:ATGATCATGCTGCTGTGCTC
R:TGCGCTATTCTCCCCAGTAG
61 °C3.0 mM134
HM804502Oosat06(GT)4…(CG)2...(GT)4...(GT)5F:ATCTCAATCTTGGCGCTGTT
R:TGCGAGCAATGAAACAAAAG
59 °C1.5 mM157
HM804503Oosat07(GT)27F:TGCAGAATTCGGCTTAGTGA
R:GGGCAAATTCTCTGTCTCGT
50 °C2.0 mM124
HM804504Oosat08(CA)8F:GGCTTACTGCATCAGACTCTTTT
R:AGCAGAATCGCTCTTTACGG
52 °C2.0 mM109
HM804505Oosat09(GT)8...(TG)2(CT)2F:CGAATTATATGATCGCGGAAA
R:AAAATGTGTTTTGGGTCGAGA
58 °C1.5 mM152
HM804506Oosat10(CA)8…(CA)6….(CA)5F:TGACGTCAATAGCGACAACG
R:GGCTTAGTGAGTGAGTGTGTGAG
56 °C2.0 mM164
HM804507Oosat11(GA)5F:TCTCACAGTCGCATGTTATC
R:ATGCTGAGAGCATCTGAAAT
56 °C3.0 mM110
HM804508Oosat12(TC)9F:TATGCAAAGTGCGCGATATT
R:TGTGCAGCCATTTACTGTGC
56 °C2.0 mM361
HM804509Oosat13(GA)23F:GGGAAACGATGATGATAATG
R:TTCAGTTGGTGAGTATTCATGT
52 °C1.5 mM145
HM804510Oosat14(TA)3...(GT)4...(GT)2F:GTTCAGGCACCATGATATTT
R:GTAGATTTTTCTTCGCAAGG
54 °C1.5 mM284
HM804511Oosat15(AG)13...(AG)8F:TAATAAAAAGGGCTGTCTGC
R:GCATACAGACAGAAAAATCAA
52 °C3.0 mM114
HM804513Oosat17(TA)6F:CGAAATCGAACACAAACTTC
R:GTTGAGCAGTTCAGGAGATT
55 °C1.5 mM115
HM804514Oosat18(CAA)8F:GCCAGATAATGAAGCCGAGA
R:GCCGAAATGCATTAATGGTC
52 °C1.5 mM255
HM804515Oosat19(CG)4(GC)3F:AGGTGGAAGTGTTCGACGAG
R:GAAGAACTCACGGTCGATGG
57 °C2.5 mM218
HM804516Oosat20(CA)27F:TTTCGAAACGAAATCGAAAT
R:GCTAGCAGAGTGGATGAGCA
52 °C2.0 mM111
HM804518Oosat22(GT)13F:TTTGGGCCATGTATGAGAGC
R:TGGAAGACTGAAGGGAAGACA
55 °C2.0 mM234
HM804519Oosat23(CA)66F:TTTGGGCCATGTATGAGAGC
R:TGGAAGACTGAAGGGAAGACA
59 °C2.0 mM206
HM804521Oosat25(GA)6…(GA)3F:GTTGGTATCTGGTCGAGACG
R:ACGCGCTTACCTGTTCAAAT
53 °C2.5 mM131
HM804523Oosat27(GA)14F:TCGAAAATCAGCTGAACGAA
R:AACTCTTACACCCACACATATTCA
52 °C3.0 mM129
HM804524Oosat28(GTT)3 ATT (GTT)3F:CGCCTTTTTGCAAATTCTCT
R:TGGATATTTGGTGTAAGGCAGA
51 °C3.0 mM115
HM804525Oosat29(ATT)3(ATG)3…(CAT)2F:CGTTCATGTGAATTGGTTGG
R:TGTATACGAAGGTGGCGATG
51 °C3.0 mM137
HM804526Oosat30(GTT)7F:GCCGAAATGCATTAATGGTC
R:TTCGATTATGGCATGGTTCA
50 °C2.5 mM140
HM804527Oosat31(TCCGTT)2…(GAATT)2
(TCCGTT)2..…(TCCGTT)2
F:ATCCGCGATCAATTATTCTG
R:TCCAAGTCCGTGAAATCAAA
48 °C3.0 mM292
HM804528Oosat32(GA)9F:CATGACACCATCCGATGAAT
R:CACATAAACAACCAGGCACAA
50 °C1.5 mM105
HM804529Oosat33(AC)6..(AC)6..(AC)3..(AC)7..
(AC)3..(AC)4
F:CGACACACACGAAACACACA
R:TTTCGGGCACCACTTTACTC
55 °C2.5 mM233
HM804530Oosat34(CAA)12F:TGAGGCAGAATGAAAGAGCA
R:CCATGGCACACGATAACAAT
51 °C1.5 mM156
HM804533Oosat37(GT)10F:TTCGACCGACTGACTGAGTG
R:GAGACGTCGGTCGTGATTTT
53 °C1.5 mM137
HM804534Oosat38(CTT)6F:AACGGTTATAGAGTCGCGATG
R:CGTGTGTTTCCTCACTAGAATCG
53 °C1.5 mM106
HM804535Oosat39(GT)3...(TG)5F:AACTGGCCACGGTCATTATC
R:AATACGTCGACGGAAGAACG
53 °C1.5 mM121
HM804536Oosat40(GT)(AT)(GT)4F:GACCCAATCCACTTTGATCCT
R:TGTCATCTAAAAGTATGTGCAACTGA
55 °C1.5 mM156
HM804537Oosat41(CT)13F:TCGTTGGAATAGCACATTCG
R:TGACGTGTCTATGCCATGTG
54 °C1.5 mM167
HM804538Oosat42(TAA)7(TAG)(TAA)3F:GAGAGCAATTTTGATTCGACTTG
R:GGGCCGAATGAAACAACTAC
54 °C1.5 mM150
HM804540Oosat44(GT)17F:GAAAAGCCGTTCGTTGAATC
R:TTTCCACCAAATAAGAAAACCA
50 °C2.0 mM193
HM804541Oosat45(GA)8…(GA)6F:GAGTGAAAGAAGTCACGCACA
R :GGCATCCACAGTCGAAGAT
50 °C2.0 mM145
HM804543Oosat47(GT)21F:CGAAGTGAATGTTTAATGGTTT
R:CCGGTTTGTATAATTGTGAA
56 °C2.5 mM128
HM804544Oosat48(GA)14…(GA)8F:ACGCTGATCAAAAGAGTTCAG
R:CCCTTGATAACAGAAAGTGAGAAC
52 °C2.5 mM116
HM804545Oosat49(CT)10GT(CT)2CA(GT)2AT
(GT)4GA(GT)20
F:CAACGTCCCATAGTCTGCATT
R:AGCGGCAGTGTTTTCTCTTC
55 °C1.5 mM168
HM804546Oosat50AG (AC)2 (TC)2 ACF:TGAGATGATATGTTCCTTTTTGTC
R:GCAGTTCCGAGATGTTTGTG
53 °C1.5 mM162
HM804547Oosat51(AT)3…(TA)5…(AT)4F:GGTTTGACGGGCACTGTAT
R:CGGCCACTGTATCTATAGGC
50 °C2.0 mM269
HM804548Oosat52(ACT)3(TGT)4F:AACTTGGAATGAAGCGTTCG
R:CGAGGTCTACCTCTACCCATAGAT
55 °C1.5 mM141
HM804549Oosat53(ATG)4…ATGF:GAGTTGCTTTGAAACGATTGC
R:ATCGTCGGATGAGTGTTTGA
54 °C1.5 mM110
HM804550Oosat54(GT)8F:CTTGGCGTTTCGTTTATCTCA
R:CCAATAAAGCAAGCACGTGTAA
54 °C1.5 mM149
HM804552Oosat56(GAAC)4…(GAAC)4 (GAAC)4F:TGCGAACATTCAACGACCTA
R:ACCACGCATACGTCAGGACT
56 °C1.5 mM138
HM804553Oosat57(TTG)8F:CGATGTAGGCAACATTTTCG
R:CCAATGAACATTCCCATCAA
52 °C1.5 mM100
HM804554Oosat58ACA (CAA)6 ACAF:GTTCGGTCGGTGTCTTTTTC
R:AAAGACCACACGCTGAAAGG
48 °C2.0 mM111
HM804556Oosat60(TA)4TC(TA)2TA TC
(TA)2TT(AT)6
F:CCAGTGATTTGAGCATCGAG
R:TCTTGGTTTTACGACCATTTCA
54 °C1.5 mM186
HM804557Oosat61(AGC)2N5(CAG)2ACA
..(GAC)2
F:GTCTGACTGGCATCACCAGA
R:CGGCTATTTCCTGTCGGTAG
55 °C1.5 mM152
HM804558Oosat62(CA)3(CT)2(CA)14F:GGAACCATTAAACACTCACTTCG
R:GGAAATTCATGGTCCGAAAA
53 °C1.5 mM129
HM804559Oosat63(TG)16N4(CT)6F:GAAGCACTGCAACAACCAAA
R:TGTTCGCTCACACCGTTTAG
54 °C1.5 mM147
HM804560Oosat64(CA)3 AG (AC)2F:TGAGACAGTTTTCGACTCCTTG
R:TAGAGGGCTTTTTCGACTGC
54 °C1.5 mM131
HM804561Oosat65(GTT)3Nn(AT)CA(TC)2 (CA)2F:TTCCTAGAATGTGGCGTTTG
R:TTGAACGCAGGTTTAATTGC
50 °C2.0 mM194
HM804562Oosat66(ACA)3(TC)2CT(TC)13N3(CA)14F:TGCATTTCCGACAGGTTTTA
R:CACCTATCGTCTTAAAGGAAATGA
53 °C1.5 mM167
HM804563Oosat67(AT)2(TA)3F:CTGTGCACACTTTGCCATTC
R:ACTCCGTGTATGCGGAAAAG
55 °C1.5 mM202
HM804564Oosat68(TTTC)3F:CGCATGAAATTTGGATCAGC
R:ATCGTGCCAAAAGTGACTGA
54 °C1.5 mM101
HM804565Oosat69(TC)7CATA(CA)3CTCAF:CCGGATAGATAGCCGTGTTT
R:CTCACTTGGTGGGTGAGTACC
56 °C2.0 mM115
HM804566Oosat70(GA)3TA(GA)3Nn(GA)4F:ATTTGGCCATGGCTATTTGA
R:GCTGGGGGCTAATCTCTCTC
54 °C1.5 mM103
HM804567Oosat71(AT)2(AC)3 (CCA)3F:GTGTGCGCACTTTACTGGTG
R:TGTGGTGGATTTGCTTTTTG
55 °C1.5 mM199
HM804568Oosat72(CAT)N2 (CAT)4F:TCGTCGGATGAGTGTTTGAT
R:CAGAGGAGTTGCTTTGAAACG
54 °C1.5 mM114
HM804569Oosat73(TG)12F:GGAAAACATGTCGGCAGAA
R:TGCACATGGTGTTGTTGTTG
62 °C2.0 mM123
HM804570Oosat74(AG)9F:TGAACATTGATACAGTGCGACA
R:TGTGTCCGGGCCAATCTA
55 °C1.5 mM123
HM804571Oosat75(CT)4 N3 (TC)7 N2 (TC)9 N2 (CT)7F:CAGTTTCGGTTCGTTTTTCA
R:CTTGCCATCCATTCATCAGA
56 °C2.0 mM127
HM804572Oosat76(GTT)5F:GGAAATTTTATTTCGGGAATTCAT
R:ACCAAAGCTTTTCAACAACAG
52 °C1.5 mM190
HM804573Oosat77(GTC)4 GTT (GTC)2F:CGAATTCAGCACGAACACTG
R:ACGTTTTCGATCACCGTTTC
55 °C1.5 mM180
HM804576Oosat80(TC)16F:TTGAAAAGTGAGGCTGATG
R:TTAAACGTCCATCAAGTGAG
55 °C1.5 mM237
HM804577Oosat81(AG)15F:TAAGCGATGTTGCTTGC
R:CGATTTTGTCGTTGTGC
55 °C1.5 mM188
HM804578Oosat82(GT)17F:AAATGAAAAGCCGTTCG
R:TGCTAGCAGTTTCATTTCC
52 °C1.5 mM211
HM804580Oosat84(GT)16F: CAATCGTTTCAGTTCCTTT
R:CACCCAAAATTCAATCG
54 °C1.5 mM160
HM804581Oosat85(TC)16F: CTAGCAGAATCACATTGA
R:CAAATCATGCTCATAGTTCC
55 °C1.5 mM358
HM804582Oosat86(AG)15F:TAAGCGATGTTGCTTGC
R:GCGATTTGTCGTTGTGC
55 °C1.5 mM188
HM804583Oosat87(AC)15F:TCCACCAAATACAGAAAACC
R:AAATGAAAAGCCGTTCG
52 °C1.5 mM192
HM804585Oosat89(TG)16F:GAAGCACTGCAACAACC
R:GATCTGTTCGCTCACACC
55 °C1.5 mM151
HM804586Oosat90(TG)16F: GAACATTATATTTTGAAAG
R: AATGAAGCCTGAAGAAAGC
55 °C1.5 mM206

Acknowledgements

This work was supported by the Department of Biotechnology, Government of India through a grant (F.No.BT/AB/03/AR/2005) under Functional Genomics of Rice. We sincerely thank Suresh Nair, Staff Research Scientist, International Centre for Genetic Engineering and Biotechnology, New Delhi, P. Rajendrakumar, Senior Scientist, Directorate of Sorghum Research, Hyderabad for their critical review of the manuscript and suggestions. We also thank the Directors and staff of Centre for DNA Fingerprinting and Diagnostics, Hyderabad and Directorate of Rice Research, Hyderabad for their encouragement and assistance.

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Figure 1. Pedigreed crosses revealing inheritance of Oosat55 in gall midge biotype 4M.
Figure 1. Pedigreed crosses revealing inheritance of Oosat55 in gall midge biotype 4M.
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Figure 2. Pedigreed crosses revealing inheritance of Oosat59 in gall midge biotype 4M.
Figure 2. Pedigreed crosses revealing inheritance of Oosat59 in gall midge biotype 4M.
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Figure 3. Pedigreed crosses revealing inheritance of Oosat43 in gall midge biotype 4M.
Figure 3. Pedigreed crosses revealing inheritance of Oosat43 in gall midge biotype 4M.
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Table 1. Microsatellite loci and primer sequences for the 15 SSR markers observed to be polymorphic in the three different biotypes of the Asian rice gall midge, Orseolia oryzae (Wood-Mason).
Table 1. Microsatellite loci and primer sequences for the 15 SSR markers observed to be polymorphic in the three different biotypes of the Asian rice gall midge, Orseolia oryzae (Wood-Mason).
Accession Number (GeneBank)LocusRepeatPrimerAnn. Temp (°C)MgCl2 (mM)Size RangenBiotypeHeHoPICHWE (P)
HM804499Oosat03(TG)12*F:TTGATTGTCCCAAGGAGCAT601.5135–148 44GMB10.5830.3500.4750.0126
GMB40.4500.3500.4010.0020
R:ATTCGCGTTGTGGATTGTTTGMB4M0.6400.5000.5720.0550
HM804512Oosat16(TG)15 (GAGT)6*F:TGTTCAGCTTGTTCAGC551.5153–1628GMB10.8030.2000.7540.0000
GMB40.5600.1000.4780.0000
R:CATTGGAACGAAATTAGTGGGMB4M0.8100.5000.7700.0000
HM804517Oosat21(TA)6 (TG)18F:CCGATTTCACTCGATGTTGTT533.0136–1502GMB10.5180.0000.3650.0000
GMB40.5150.3570.3740.3150
R:TTCTAACTTGAACTCCTCATTCGGMB4M0.5080.2860.3740.1320
HM804520Oosat24(AC)11(CA)5F:CCTCGGTCGCATCTCATATT523.0160–2004GMB10.6341.0000.5010.0040
GMB40.5181.0000.4450.0000
R:CCATTCAACAGATTTGGCGTAGMB4M0.6451.0000.5480.0010
HM804522Oosat26(GT)15*F:TGTCAGGTGGAACAGTAAATTG533.0214–2369GMB10.8000.2000.7640.0000
GMB40.7500.4500.6860.0020
R:GCCTGAAGAAAGCTGAATGAAGMB4M0.7000.2000.6480.0000
HM804531Oosat35(CA)11 (GA) (GA)2F:GCCCGTTGATTGCTTTGTAT511.5185–2205GMB10.7960.9280.7040.0200
GMB40.7350.7140.6740.0000
R:TATCGTTGTCGTCGTCTTCGGMB4M0.3040.3570.2471.0000
HM804532Oosat36(GT)14*F:CAGTTCCTTTTGTATATGCGTGAG511.5145–17410GMB10.7800.8000.7380.0100
GMB40.7600.6000.6960.0070
R:GCACCCAAAATTCAATCGTTGMB4M0.8100.4500.7690.0001
HM804539Oosat43(CT)9*F:TCGTTGGAATAGCACATTCG541.5165–1887GMB10.7000.4500.6330.0200
GMB40.3800.4000.3051.0000
R:TGACGTGTCTATGCCATGTGGMB4M0.6200.5000.5570.0040
HM804542Oosat46(GA)19F:AAATTGGCAGAGCGGAAGTA442185–2503GMB10.4940.7850.3590.0360
GMB40.6481.0000.5530.0000
R:TTTCACGGCCATCACATAAGGMB4M0.6451.0000.5480.0010
HM804551Oosat55(CA)2 (CA)20*F:CGTCGCCTTGTTGTAATATGTAAG551.5103–13510GMB10.7800.5000.7440.0000
GMB40.7900.4000.7510.0000
R:ACAGCCAATTGTGTTGCTTGGMB4M0.9000.6500.8680.0000
HM804555Oosat59(CA)20*F:CGTCGCCTTGTTTAATATG551.578–10711GMB10.8800.3000.8520.0000
GMB40.5800.1500.5340.0000
R:CCAATTGTGTTGCTTGAGMB4M0.9100.3000.8770.0000
HM804574Oosat78CAG (CAA)2 (CAG)6 CAAF:CCCAGCTCTTCGAATTCTATTG562190–2003GMB10.4760.0000.3050.0000
GMB40.6770.0000.5480.0000
R:CCCGAATCATTTTGCATTGTGMB4M0.3490.0000.3050.0000
HM804575Oosat79(TG)11*F:CGCCCTAAAGAGTCGTGAAG551.5118–1285GMB10.3500.4000.3291.0000
GMB40.6000.5500.5110.5940
R:GAACCGGATGATTTGAATGGGMB4M0.6800.6000.6110.8100
HM804579Oosat83(AG)15*F:GCGAGTCAAAACACACG551.5105–1209GMB10.8400.6000.8030.0000
GMB40.7100.5000.6460.0008
R:ACACACACATATGCTCTTCCGMB4M0.7900.2500.7420.0000
HM804584Oosat88(TC)15*F:ACAGAAGGTAGAAGGAGAGC551.5184–1926GMB10.7700.7000.7090.0200
GMB40.7100.6500.6650.2210
R:AGTTGGCGATTGAGTGAGGMB4M0.7600.6000.6930.0500
n: total number of alleles; He: Expected heterozygosity; Ho: Observed heterozygosity;
PIC: Polymorphic information content; HWE (P-value): Hardy Weinberg equilibrium
*FAM-labeled.
Table 2. Allele frequencies (most frequent alleles) of the polymorphic SSR markers amplified in different gall midge biotypes.
Table 2. Allele frequencies (most frequent alleles) of the polymorphic SSR markers amplified in different gall midge biotypes.
S/No.LociFrequency of Most Frequent Alleles * (Size, bp) and Number of Alleles in Each Biotype
GMB1GMB4GMB4M

Frequency (Size)No. of AllelesFrequency (Size)No. of AllelesFrequency (Size)No. of Alleles
1Oosat030.475 (148)40.674 (148)40.525 (148)4
2Oosat160.350 (153)60.600 (155)50.325 (155)8
3Oosat210.600 (136)20.525 (150)20.525 (136)2
4Oosat240.500 (180)40.475 (160,180)40.500 (190)3
5Oosat260.350 (220)90.350 (219)60.500 (222)6
6Oosat350.375 (190)50.375 (200)40.825 (200)2
7Oosat360.350 (150)80.350 (153)50.375 (152)10
8Oosat430.425 (188)70.750 (168)20.550 (166)5
9Oosat460.625 (185)20.500 (250)30.500 (185)3
10Oosat550.400 (112)80.400 (125)90.175 (125)10
11Oosat590.200 (83,84)110.625 (97)60.150 (79,84)11
12Oosat780.750 (200)20.500 (210)30.750 (200)2
13Oosat790.800 (122)50.500 (122)30.425 (122)5
14Oosat830.250 (115,116)90.450 (116)50.350 (116)6
15Oosat880.325 (192)50.475 (184)60.300 (184)4
*N = 60 (10 male and 10 female adults of the three biotype screened).
Table 3. F-Statistics and gene flow for all loci.
Table 3. F-Statistics and gene flow for all loci.
S/No.LocusFISFITFSTNm
1Oosat030.26660.30550.05314.4625
2Oosat160.62380.65970.09552.3669
3Oosat210.45850.46430.010623.3289
4Oosat24−0.7118−0.39860.18301.1162
5Oosat260.61470.66180.12221.7961
6Oosat35−0.10950.06250.15501.3624
7Oosat360.19650.28510.11032.0168
8Oosat430.19220.34660.19121.0578
9Oosat46−0.6058−0.48080.07792.9611
10Oosat550.36080.41780.08922.5526
11Oosat590.67690.71640.12231.7948
12Oosat781.00001.00000.17721.1610
13Oosat790.03800.12960.09522.3753
14Oosat830.41140.43480.03966.0628
15Oosat880.11160.15510.04894.8598
FIS: fixation index (inter-individual); FST: fixation index (subpopulations); FIT: fixation index (total population).
Nm = Gene flow estimated from FST = 0.25(1 – FST)/FST.

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Bentur, J.S.; Sinha, D.K.; Padmavathy, C.; Revathy, C.; Muthulakshmi, M.; Nagaraju, J. Isolation and Characterization of Microsatellite Loci in the Asian Rice Gall Midge (Orseolia oryzae) (Diptera: Cecidomyiidae). Int. J. Mol. Sci. 2011, 12, 755-772. https://doi.org/10.3390/ijms12010755

AMA Style

Bentur JS, Sinha DK, Padmavathy C, Revathy C, Muthulakshmi M, Nagaraju J. Isolation and Characterization of Microsatellite Loci in the Asian Rice Gall Midge (Orseolia oryzae) (Diptera: Cecidomyiidae). International Journal of Molecular Sciences. 2011; 12(1):755-772. https://doi.org/10.3390/ijms12010755

Chicago/Turabian Style

Bentur, Jagadish S., Deepak Kumar Sinha, Ch. Padmavathy, Charagonda Revathy, Mayandi Muthulakshmi, and Javaregowda Nagaraju. 2011. "Isolation and Characterization of Microsatellite Loci in the Asian Rice Gall Midge (Orseolia oryzae) (Diptera: Cecidomyiidae)" International Journal of Molecular Sciences 12, no. 1: 755-772. https://doi.org/10.3390/ijms12010755

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