CACTA and MITE transposon distributions on a genetic map of rice using F15 RILs derived from Milyang 23 and Gihobyeo hybrids

Up to 35% of the rice genome consists of various kinds of transposons, and CACTA and MITE are two of the major class 2 DNA transposons in the genome. We have employed the consensus sequences of Rim2/Hipa CACTA, Stowaway MITE Pangrangja, and Tourist MITE Ditto for transposon display (TD) analysis to locate them on a genetic map, with 58 SSR markers used to anchor them. The TD analysis produced a high profile of the polymorphisms between the parental lines, Oryza sativa var. Gihobyeo/O. sativa var. Milyang, in intraspecific F15 RIL lines, locating 368 markers of Rim2/Hipa CACTA, 78 markers of Tourist MITE Ditto, and 22 markers of Stowaway MITE Pangrangja. In the segregation analysis, non-parental segregating bands and segregation distortion bands were observed. The recombinant genetic map spans 3023.9 cM, with 5.7 cM the average distance between markers. The TD markers were distributed unequally on the chromosomes because many TD markers were located in pericentric chromosomal regions except in the cases of chromosomes 2, 3, 6 and 9. Although the number of transposon markers was not sufficient to include all rice class 2 transposons, the current map of CACTA and MITE transposons should provide new insight into the genome organization of rice since no previous DNA transposon map is available.     

<Emphasis Type="Italic">Mutator</Emphasis>-Based Transposon Display: A Genetic Tool for Evolutionary and Crop-Improvement Studies in Maize

Transposable elements account for up to 85% of the maize genome and have significant implications in crop-improvement and evolutionary analyses. The Mutator (Mu) transposon superfamily, a class of DNA transposons, comprises the most complex and active elements in the maize genome, suggesting a special role in plant evolution. Here, we designed a set of Mu-specific primers based on terminal invert repeats and used a transposon display (TD) method for genotyping. We analyzed the distribution pattern of Mu insertions in teosinte (wild relative), sorghum (distant relative), and domesticated maize accessions (dent, sweet, and waxy). The MU-TD analysis suggested the presence of high polymorphic insertions among the species and subspecies, indicating the utility of the method in studying genetic variation and species relationships. Furthermore, we analyzed 80 maize recombinant inbred line populations. Mu-TD generated an average of 60% Mu-anchored polymorphic fragments in which insertions appeared to be segregating in significantly high numbers. The amplification profile was highly reproducible, confirming the utility of Mu elements as a new set of TD markers for developing high-density genetic maps.     

CACTA transposon-derived Ti-SCARs for cultivar fingerprinting in rapeseed

Transposon display (TD) is a technique for analyzing polymorphic insertions and excisions of transposable elements. We used the CACTA transposon to develop cultivar-specific transposon insertion-sequence characterized amplified regions (Ti-SCARs) for rapeseed (Brassica napus). Using 16 combinations of TD primers, we detected 19 cultivar-specific fragments among six rapeseed cultivars. Of the 16 primer pairs, 12 successfully amplified targets in B. napus and six amplified novel, cultivar-specific markers. Cultivar-specific markers can be used for cultivar fingerprinting and marker-assisted selection in the rapeseed breeding program. Moreover, because Ti-SCARs are based on transposon insertions, Ti-SCAR markers may be used in reverse genetic techniques for isolating novel genes in plants.     

Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 1. Map construction and localization of loci showing distorted segregation

Microsatellites have become the most important class of markers for mapping procedures. Primarily based on restriction fragment length polymorphism (RFLP) markers, several molecular genetic maps of maize have been developed, mainly using temperate inbred maize lines. To characterize the level of polymorphism of microsatellite loci and construct a genetic map in tropical maize, two elite inbred lines, L-08-05F and L-14-4B, were crossed to produce 400 F(2) individuals that were used as a mapping population. A survey of 859 primer pair sequences of microsatellites was used. The polymorphism screens of each microsatellite and genotype assignment were performed using high-resolution agarose gels. About 54 % of the primer sets gave clearly scorable amplification products, 13 % did not amplify and 33 % could not be scored on agarose gels. A total of 213 polymorphic markers were identified and used to genotype the mapping population. Among the polymorphic markers, 40 showed loci deviating from expected Mendelian ratios and clusters of deviating markers were located in three chromosome regions. Non-Mendelian scoring was present in 19 markers. The final genetic map with 117 markers spanned 1634 cM in length with an average interval of 14 cM between adjacent markers.     

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

Linkage group alignment of sorghum RFLP maps using a RIL mapping population.

Several molecular maps have been constructed in sorghum (Sorghum bicolor L. Moench) using a variety of probes from different grass species such as sorghum, maize, sugarcane, rice, oat, and barley. In order to enhance the utility of the existing mapping information by the sorghum research community, alignment and integration of all major molecular maps is necessary. To achieve this objective, a genetic map of 214 loci with a total map distance of 1200 cM was constructed using 98 F7 sorghum recombinant inbred lines (RILs) from a cross between two inbred lines, B35 and Tx7000. Few cDNA clones of sorghum and maize related to photosynthesis and drought stress were mapped on this map for the first time. Five major restriction fragment length polymorphism (RFLP) maps independently developed in this species were used for alignment purpose. The distributions of previously mapped markers were compared with their respective sorghum maps to align each of the linkage groups. In general, consistent linear order among markers was maintained in all the linkage maps. The successful alignment of these RFLP maps will now allow selection of a large number of markers for any region of the sorghum genome with many potential applications ranging from fine mapping and marker-assisted selection to map-based cloning for the improvement of sorghum and related species.     

Near-isogenic lines for measuring phenotypic effects of DIMBOA-Glc methyltransferase activity in maize

Three O-methyltransferases (BX10a, b, c) catalyze the conversion of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) to 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) in maize (Zea mays). Variation in benzoxazinoid accumulation and resistance to Rhopalosiphum maidis (corn leaf aphid) was attributed to a natural CACTA family transposon insertion that inactivates Bx10c. Whereas maize inbred line B73 has this transposon insertion, line CML277 does not. To characterize the phenotypic effects of DIMBOA-Glc methyltransferase activity, we created near-isogenic lines derived from B73 and CML277 that do or do not contain the transposon insertion. Bx10c inactivation causes high DIMBOA-Glc, low HDMBOA-Glc, and decreased aphid reproduction relative to near-isogenic lines that have a functional Bx10c gene. These results confirm the importance of this locus in maize aphid resistance. The availability of Bx10c near-isogenic lines will facilitate further research on the function of different benzoxazinoids and DIMBOA-Glc methyltransferase activity in maize defense against herbivores and pathogens.     

Genetic variation analysis of the Bali street dog using microsatellites

Background

Transposons constitute the major fractions of repetitive sequences in eukaryotes, and have been crucial in the shaping of current genomes. Transposons are generally divided into two classes according to the mechanism underlying their transposition: RNA intermediate class 1 and DNA intermediate class 2. CACTA is a class 2 transposon superfamily, which is found exclusively in plants. As some transposons, including the CACTA superfamily, are highly abundant in plant species, and their nucleotide sequences are highly conserved within a family, they can be utilized as genetic markers, using a slightly modified version of the conventional AFLP protocol. Rim2 /Hipa is a CACTA transposon family having 16 bp consensus TIR sequences to be present in high copy numbers in rice genome. This research was carried out in order to develop a Rim2/Hipa CACTA-AFLP or Rim2/Hipa CACTA-TD (transposon display, hereafter Rim2/Hipa -TD) protocol for the study of genetic markers in map construction and the study of genetic diversity in rice.

Results

Rim2/Hipa -TD generated ample polymorphic profiles among the different rice accessions, and the amplification profiles were highly reproducible between different thermocyclers and Taq polymerases. These amplification profiles allowed for clear distinction between two different ecotypes, Japonica and Indica, of Oryza sativa. In the analysis of RIL populations, the Rim2/Hipa -TD markers were found to be segregated largely in a dominant manner, although in a few cases, non-parental bands were observed in the segregating populations. Upon linkage analysis, the Rim2/Hipa -TD markers were found to be distributed in the regions proximal to the centromeres of the chromosomes. The distribution of the Rim2/Hipa CACTA elements was surveyed in 15 different Oryza species via Rim2/Hipa -TD. While Rim2/Hipa -TD yielded ample amplification profiles between 100 to 700 bp in the AA diploid Oryza species, other species having BB, CC, EE, BBCC and CCDD, profiles demonstrated that most of the amplified fragments were larger than 400 bp, and that our methods were insufficient to clearly distinguish between these fragments. However, the overall amplification profiles between species in the Oryza genus were fully distinct. Phenetic relationships among the AA diploid Oryza species, as evidenced by the Rim2/Hipa -TD markers, were matched with their geographical distributions.

Conclusion

The abundance of the Rim2/Hipa TIR sequences is very informative since the Rim2/Hipa -TD produced high polymorphic profiles with ample reproducibility within a species as well as between species in the Oryza genus. Therefore, Rim2/Hipa -TD markers can be useful in the development of high-density of genetic map around the centromeric regions. Rim2/Hipa -TD may also prove useful in evaluations of genetic variation and species relationships in the Oryza species.     

利用RFLP、SSR、AFLP和RAPD标记分析玉米自交系遗传多样性的比较研究

利用 ＲＦＬＰ、ＳＳＲ．ＡＦＬＰ和ＲＡＰＤ ４种分子标记方法研究了 １５个玉米（Ｚｅａ ｍａｙｓ Ｌ．）自交系的遗传多样性,同时对４种标记系统进行比较。在供试材料中筛选到具多态性的ＲＦＬＰ探针酶组合５６个,６６对ＳＳＲ引物,２０个ＲＡＰＤ引物和９个ＡＦＬＰ引物组合,分别检测到多态性带１６７、２０１、８７和１０８条。ＳＳＲ标记位点的平均多态性信息量（ＰＩＣ）最大（０．５４）,ＡＦＬＰ标记位点最小（０．３６）,但ＡＦＬＰ标记具有最高的多态性检测效率（Ａｉ,３２．２）。４种分子标记所得遗传相似系数相关性显著,比较相关系数表明 ＲＡＰＤ可靠性较低。依据 ４种分子标记结果将 １５个供试自交系划分为塘四平头、旅大红骨、兰卡斯特、瑞德和ＰＮ共５个类群,与系谱分析基本一致。认为ＳＳＲ和ＲＦＬＰ两种分子标记方法适合进行玉米种质遗传多样性的研究。     

Analysis of genetic diversity and relationships among waxy maize inbred lines in Korea using SSR markers

Information regarding the genetic diversity and genetic relationships among elite inbred lines is necessary to improve new cultivars in maize breeding programs. In this study, genetic diversity and genetic relationships were investigated among 84 waxy maize inbred lines using 50 SSR markers. A total of 269 alleles were identified at all the loci with an average of 5.38 and a range between 2 and 13 alleles per locus. The gene diversity values varied from 0.383 to 0.923 with an average of 0.641. The cluster tree generated using the described SSR markers recognized two major groups at 32% genetic similarity. Group I included 33 inbred lines while group II included 51 inbred lines. The clustering patterns of most of the waxy maize inbred lines did not clearly agree with their source, pedigree or geographic location. The average GS among all inbred lines was 35.7 ± 10.8. Analysis of waxy maize inbred lines collected from Korea and China at 50 SSR loci revealed higher values of average number of alleles (4.9) and gene diversity (0.638) in Korean inbred lines as compared to Chinese inbred lines (3.5 and 0.563, respectively). The information obtained from the present studies would be very useful for maize breeding programs in Korea.     

Sequence-based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet [<Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv.]

The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar ‘Prasad’, 690 clones were sequenced which generated 112.95 kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F₂ mapping population. Out of 172 pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30–0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.     

白菜EST-SSR标记的通用性

EST-SSR是从表达序列标签(expressedsequencetag,EST)中开发的新型简单序列重复(simplesequencerepeat,SSR)标记。根据白菜EST设计了15对SSR引物,对白菜、油菜、玉米、高粱、水稻和茶树等进行了PCR,研究了白菜的EST-SSR标记在不同物种间的通用性。所设计的引物对不同白菜品种、近缘种油菜和远缘种玉米、高粱、水稻和茶树的扩增成功率分别为100%、93.3%、80%、93.3%、93.3%和86.7%。在15对引物中,有11对在远缘种中都有扩增产物,而且一些引物可显示多态性,多态性引物分别占了可扩增引物的33.3%、28.6%、28.6%和61.5%。这些结果表明,白菜EST-SSR引物具有较高的通用性,这对于比较基因组学研究有重要意义。     

Genetic dissection of intermated recombinant inbred lines using a new genetic map of maize

A new genetic map of maize, ISU-IBM Map4, that integrates 2029 existing markers with 1329 new indel polymorphism (IDP) markers has been developed using intermated recombinant inbred lines (IRILs) from the intermated B73xMo17 (IBM) population. The website http://magi.plantgenomics.iastate.edu provides access to IDP primer sequences, sequences from which IDP primers were designed, optimized marker-specific PCR conditions, and polymorphism data for all IDP markers. This new gene-based genetic map will facilitate a wide variety of genetic and genomic research projects, including map-based genome sequencing and gene cloning. The mosaic structures of the genomes of 91 IRILs, an important resource for identifying and mapping QTL and eQTL, were defined. Analyses of segregation data associated with markers genotyped in three B73/Mo17-derived mapping populations (F2, Syn5, and IBM) demonstrate that allele frequencies were significantly altered during the development of the IBM IRILs. The observations that two segregation distortion regions overlap with maize flowering-time QTL suggest that the altered allele frequencies were a consequence of inadvertent selection. Detection of two-locus gamete disequilibrium provides another means to extract functional genomic data from well-characterized plant RILs.     

Molecular tagging of gene conferring leaf blight resistance using microsatellites in sorghum [Sorghum bicolor (L.) Moench

Resistance to leaf blight in sorghum [Sorghum bicolor (L.) Moench] accession G-118 was found to segregate as a single dominant trait in a cross to susceptible cultivar, HC-136. Molecular marker(s) linked to the locus for disease resistance was identified using simple sequence repeat (SSR) markers coupled with bulk segregant analysis. Genomic DNA from the parental cultivars and bulks were screened by PCR amplification with 50 simple sequence repeat primer pairs. Out of these, 38 SSR primers produced polymorphism between parents. After screening of these 38 SSRs with resistant and susceptible bulk, one SSR primer, Xtxp 309 produced a unique band of approximately 700 bp only in resistant parent and resistant bulk and a unique band of 450 bp only in susceptible parent and susceptible bulk. Upon screening with individual resistant and susceptible recombinant inbred lines (RILs), marker Xtxp 309 produced amplification in 23 of the 26 resistant RILs and no amplification was produced in any of the 25 susceptible RILs. The same marker Xtxp 309 produced amplification in 21 of the susceptible RILs and 3 of the resistant RILs of 450 bp band. This was found to be located at a distance of 3.12 cM away from the locus governing resistance to leaf blight which was considered to be closely linked and 7.95 cM away from the locus governing susceptibility to leaf blight. This marker may prove useful in MAS for gene introgression, plant genetic diagnostics and gene pyramiding for resistance via genetic transformation for disease resistance in plants.     

Structure and evolution of the genomes ofsorghum bicolor andZea mays

Cloned maize genes and random maize genomic fragments were used to construct a genetic map of sorghum and to compare the structure of the maize and sorghum genomes. Most (266/280) of the maize DNA fragments hybridized to sorghum DNA and 145 of them detected polymorphisms. The segregation of 111 markers was analyzed in 55 F₂ progeny. A genetic map was generated with 96 loci arranged in 15 linkage groups spanning 709 map units. Comparative genetic mapping of sorghum and maize is complicated by the fact that many loci are duplicated, often making the identification of orthologous sequences ambiguous. Relative map positions of probes which detect only a single locus in both species indicated that multiple rearrangements have occurred since their divergence, but that many chromosomal segments have conserved synteny. Some sorghum linkage groups were found to be composed of sequences that detect loci on two different maize chromosomes. The two maize chromosomes to which these loci mapped were generally those which commonly share duplicated sequences. Evolutionary models and implications are discussed.     

Molecular marker-based genetic diversity assessment of Striga-resistant maize inbred lines

Striga-resistant maize inbred lines are of interest to maize breeding programs in the savannas of Africa where the parasitic weed is endemic and causes severe yield losses in tropical maize. Assessment of the genetic diversity of such inbred lines is useful for their systematic and efficient use in a breeding program. Diversity analysis of 41 Striga-resistant maize inbred lines was conducted using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers to examine the genetic relationships among these lines and to determine the level of genetic diversity that exists within and between their source populations. The two marker systems generated 262 and 101 polymorphic fragments, respectively. Genetic similarity (GS) values among all possible pairs of inbred lines varied from 0.45 to 0.95, with a mean of 0.61±0.002 for AFLPs, and from 0.21 to 0.92, with a mean of 0.48±0.003, for SSRs. The inbred lines from each source population exhibited a broad range of GS values with the two types of markers. Both AFLPs and SSRs revealed similar levels of within population genetic variation for all source populations. Cluster and principal component analysis of GS estimates with the two markers revealed clear differentiation of the Striga-resistant inbred lines into groups according to their source populations. There was clear separation between early- and late-maturing Striga-resistant inbred lines. Considering the paucity of germplasm with good levels of resistance to Striga in maize, the broad genetic diversity detected within and among source populations demonstrates the genetic potential that exists to improve maize for resistance to Striga.     

Analysis of genetic distance by SSR in waxy maize

We examined the genetic diversity of 80 inbred waxy maize lines using 22 SSR molecular markers that could be used to achieve heterosis in waxy maize. Eighty inbred waxy maize lines with different phenotypes, 40 yellow, 25 white, 13 black, and two red lines were analyzed by SSR molecular marker fingerprint and cluster analysis. Using a standard genetic distance of 0.55, the 80 waxy maize inbred lines were clustered into nine groups. Among them, group II, group V, groups VII and VIII, and group IX were divided into three subgroups at a genetic distance of 0.46, into two subgroups at 0.49, into two subgroups at 0.46, and into four subgroups at 0.493, respectively. All but one of the yellow waxy maize inbred lines were clustered in groups VI, VII, VIII, and IX. Group IX (30 lines) contained 28 yellow lines; the other 11 yellow lines were distributed among groups VI, VII and VIII. Among the 25 white lines, 21 were clustered in groups III, V, VI and the third subgroup of group II. The black line N72 was in a group of its own. The black lines N75, N76 and N78 were distributed in groups VII, VIII and IX, respectively. The other nine black lines were clustered in group II. The red lines were distributed in the second subgroup of group II and there was no difference in genetic distance between them. In conclusion, there were considerable genetic differences among waxy maize inbred lines of different colors. The mean genetic distance of inbred lines of the same color was significantly less than that of lines of different colors. Therefore, we concluded that it was more accurate to determine the difference between the populations using the highly stable DNA genetic markers.     

Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

Microsatellite marker-based genetic diversity assessment among exotic and native maize inbred lines of Bangladesh

Hybrid development is basically dependent on the variability among available genetic resources. Polymorphism among the maize inbreds is essentially needed for maize hybridization. This study aimed at the assessment of diversity among 22 maize inbreds by 18 microsatellite markers. The study identified 187 alleles at 18 SSR loci. The amplified allele frequency per microsatellite locus was 10.4 and the highest allele per locus was 17 in SSR primer pair phi026. SSR primer set p-umc1292, phi074 and phi090 showed the lowest 6 alleles per genotype per locus. The locus phi026 showed the highest degree of gene diversity (0.92), and the locus p-umc1292 had the lowest of gene diversity (0.77) with a mean value of 0.862 among the microsatellites. At each site, the most prevalent allele varied between 0.14 (bnlg371) and 0.36. (p-umc1292). At any given locus, an average of 0.22 out of the 22 selected maize inbred lines had a common major allele. The average value of the polymorphic information content (PIC) was 0.85, within the range of 0.74 at the lowest to 0.92 at the highest. The higher PIC values of phi026 and nc013 established them to be the best markers for maize inbred lines. The UPGMA clustering generated seven distinct groups having 12.5% of similarity coefficient. The results revealed that inbred lines E10, E27, E19, E34, E35, E4, E43, E28, E11, E21, E17, E38, E25, E34, E14, E16, E39 and E3 were more diversified. These lines are promising to be used as parent materials for hybrid maize development in the future.