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Published online 23 February 2005
Published in Crop Sci 45:717-721 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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Introgression of Perennial Teosinte Genome into Maize and Identification of Genomic In Situ Hybridization and Microsatellite Markers

Qilin Tanga, Tingzhao Ronga,*, Yunchun Songb, Junpin Yangc, Guangtang Pana, Wanchen Lia, Yubi Huanga and Moju Caoa

a Maize Research Inst., Sichuan Agriculture Univ., Ya'an, Sichuan 625014, P.R. China
b The Key Lab. of the Ministry of Education of Plant Developmental Biology, Wuhan Univ., Wuhan, 430072, P.R. China
c Sichuan Crop Research Inst., Chengdu, 625000, P.R. China

* Corresponding author (rongtz{at}sicau.edu.cn)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To transfer desirable characters from perennial teosinte [Zea perennis (Hitchc.) Reeves & Mangelsd.] into maize (Z. mays L.), we have generated an interspecific hybrid and its backcross generations (BC1F3). The maize x perennial teosinte BC1F3 (MZI202) resembled maize, with chromosome number 2n = 20. MZI202 was studied by multicolor genomic in situ hybridization (McGISH) and simple-sequence repeat (SSR) microsatellites markers. The McGISH experiments provided direct evidence that MZI202 was a maize–perennial teosinte substitution line with introgression of three alien chromosomes from perennial teosinte. The SSR assay further confirmed that a single chromosome 6 and the pair of chromosome 10 of maize were replaced by perennial teosinte chromosomes in MZI202.

Abbreviations: DAPI, 4',6-diamidino-2-phenylindole • GISH, genomic in situ hybridization • McGISH, multicolor genomic in situ hybridization • PBS, phenobarbital sodium • PCR, polymerase chain reaction • SSC, standard saline citrate • SSR, simple-sequence repeat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INTROGRESSION OF USEFUL GENES from wild relatives into maize has been frequently conducted (Iltis et al., 1979; Galinat, 1985). Zea perennis is a perennial species with 2n = 40. It contains a number of novel genes and traits, such as resistance to economically important diseases and pests. Perennial teosinte is a potential new gene source for maize improvement (Wilkes, 1967; Mangelsdorf, 1974; Galinat, 1985). Interspecific crosses between perennial teosinte and maize resulted in hybrids with a somatic chromosome number of 2n = 30 (Molina and Naranjo, 1987; Poggio and Naranjo, 1990; Poggio et al., 1999). As early as 1924, the meiotic configurations and chromosome pairing of F1 hybrids between the 2n = 20 species of genus Zea and perennial teosinte were intensively studied, and the meiotic configuration was determined by using conventional cytogenetic techniques (Molina and Naranjo, 1987; Poggio and Naranjo, 1990; Naranjo and Poggio, 1994; Molina and Garcia, 1999; Poggio et al., 1999). These previous studies were the basis for the classification of genus Zea and polyploidy. However, there are very few reports on the exploitation of perennial teosinte in maize improvement. One reason is that the interspecific cross between maize and perennial teosinte can produce the F1 hybrid of maize x perennial teosinte (2n = 30), but this interspecific hybrid shows abnormal meiosis behaviors and infertility due to the inherent genetic barriers (Wilkes, 1967; Mangelsdorf, 1974; Molina and Naranjo, 1987; Poggio and Naranjo, 1990; Naranjo and Poggio, 1994; Molina and Garcia, 1999; Poggio et al., 1999). This strong breeding barrier of incompatibility in the F1 was the major constraint to be overcome for the introgression of the valuable traits of perennial teosinte into the modern maize varieties.

To overcome the strong incompatibility barrier of F1 maize x perennial teosinte, several methods have been exploited, including developing bridge lines and doubling the chromosome number of the F1 interspecific hybrid plants. Furthermore, environmental factors may exert a large influence on the cross incompatibility of the maize x perennial teosinte F1 plants.

The objectives of this study were to (i) identify the number of alien perennial teosinte chromosomes, and (ii) assess the individual substituted chromosomes of maize in BC1F3 by using the analyses of genomic in situ hybridization (GISH) and SSR markers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials
The pollen parent perennial teosinte (P2) (Accession 9475; 2n = 40) was provided by the International Maize and Wheat Improvement Center (CIMMYT). This perennial accession was chosen for its resistance to important diseases and pests (Fig. 1A) . The recurrent parent (P1) was the maize inbred 48-2 (2n = 20). The interspecific cross between maize and perennial teosinte produced viable F1 seeds. The maize x perennial teosinte (2n = 30) F1 plants resembled perennial teosinte (Fig. 1B), but the F1 was highly sterile (<5% fertile). Few viable seeds could be found because of the inherent genetic barriers.


Figure 1
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  		Fig. 1. The morphological characters of wild parent and its interspecific hybrid progenies. (A) Perennial teosinte, (B) the F1 of maize x perennial teosinte, (C) the F2 of maize x perennial teosinte, (D) the BC1F3 of maize x perennial teosinte (MZI202).

 
Anyhow, a few F2 seeds could be obtained in the greenhouse after using special technical treatments such as short light treatment (10-h photostage and 14-h darkness each day) and the application of 2.14 x 10–6 mol L–1 gibberellic acid on the F1 silk during self-pollination. F2 seeds have a low germination rate. One F2 maize-like plantlet was obtained, although its growth and development was weak (Fig. 1C). We backcrossed the F2 individual with maize pollen, and obtained several F2 x P1 seeds. The F2 x P1 plants were self-pollinated to produce BC1F3 progeny (Fig. 1D). Because of the limitation of materials and the difficulty for obtaining a good cytological preparation, one BC1F3 plant named MZI202 was selected for tested material in the present study. The total genomic DNA of maize, perennial teosinte, and the MZI202 were isolated from adult leaves by following the protocol of Kynast et al. (2001).

Genomic In Situ Hybridization
Chromosome Preparation
The root tip preparation was done according to Benabdelmouna et al. (2001) and Song et al. (1994). Root tips were collected and immediately pretreated with saturated {alpha}-bromonaphthalene solution for 3 hr at 27°C. After rinsing in tap water, the pretreated root tips were rinsed with distilled water or 0.075 M KCl for 30 min, followed by fixing in a fresh fixative (3:1 methanol to glacial acetic acid ratio) and stored overnight at 4°C. Spikes of the F1 were collected from the early stage to midboot stage and directly fixed in Carnoy's solution (chloroform–ethanol–acetic acid, 6:3:1) for at least 2 d before making meiotic preparations. The fixed materials were incubated in the enzyme mixture of 2% cellulase (SERVA) and 2% pectinase (SERVA) at 28°C for approximately 120 to 150 min. The softened materials were again rinsed in distilled water and fixed in the fresh fixative (3:1 methanol to glacial acetic acid ratio) for 30 min at the room temperature. Finally, the treated materials were smashed onto a clean slide and dried over a flame (Song et al., 1994). The chromosomes were observed on slides under a phase-contrast microscope and were kept at –20°C before proceeding with the GISH procedure.

Probe Preparation and Hybridization
The total genomic DNA of maize was labeled with DIG-dUTP by nick translation. Genomic DNA of perennial teosinte was labeled with bio-dATP by nick translation. The slide preparations were pretreated with 100 µg mL–1 ribonuclease in 2 x SSC (standard saline citrate: 0.3 M sodium chloride plus 0.03 M sodium citrate) at 37°C for 1 h. Chromosomal DNA was then denatured by immersing the slides in the 70% deionized formamide in 2 x SSC at 70°C for 3 to 5 min. After dehydration in an ice-cold 70, 95, and 100% ethanol series, a 50-µL hybridization mixture with 50% deionized formamide (Sigma Chemical Co., St. Louis, MO), 10% sodium dextran sulfate (Sigma), 2 x SSC, 10 µg of salmon sperm DNA, and 20 to 40 ng of labeled probe mixture was prepared. The hybridization mixture was denatured at 100°C for 10 min, then chilled on ice for 10 min. The hybridization was performed overnight at 37°C. The slides were then washed in 20% formamide, 2 x SSC, 0.1 x SSC, at 42°C for 15 min, and subsequently washed in 0.1% of Triton X-100, phenobarbital sodium (PBS) at room temperature for 5 min. For hybridized probe detection, the slide was covered with streptavidin-Cy3 (Sigma) and antidigoxigenin rhodamineat conjugation in 37°C for half an hour, and washed with PBS. Then the slides were counterstained with 1 µg mL–1 4',6-diamidino-2-phenylindole (DAPI). Chromosomes were observed with an Olympus (Lake Success, NY) BX60 fluorescence microscope equipped with Sensys 1401E CCD camera (Photometrics Limited, Tucson, AZ). Red, green, and blue images were captured in black and white with different filters. The images were combined and pseudo colored in the computer by using V++ Image Analysis Software (Digital Optics Corporation, Charlotte, SC).

Microsatellite Markers, PCR Conditions, and PCR Product Separation
The appropriate genomic DNA sample was polymerase chain reaction (PCR) amplified in the presence of primers for maize chromosome-specific SSR markers and the SSR reactions program selected from the Maize Genetics and Genomics Database (http://www.maizegdb.org/, verified 4 Aug. 2004) and Kynast et al. (2001). From the Maize Genetics and Genomics Database, 93 maize chromosome-specific SSR markers, covering all 10 maize chromosomes, were initially used to screen for the specific polymorphisms in maize or perennial teosinte. The primers that generated only clear bands specific to the maize parent or perennial teosinte were used to analyze the MZI202. Consequently, 20 SSR primers were identified, with two primers for each maize chromosome (Table 1). The method reported by Kynast et al. (2001) for SSR analysis was applied in this study.


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  		Table 1. Twenty SSR primers cover with 10 maize chromosomes revealed the specific polymorphism between maize and perennial teosinte associated with the introgression chromosomes examining for MZI202.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Perennial Teosinte Chromosome by GISH
The MZI202 (BC1F3 of maize x perennial teosinte) had the same chromosome number as maize (2n = 20). To verify the introgression of perennial teosinte chromosome segments into MZI202, maize genomic DNA was digoxigenin labeled and its signals were detected with FITC. Perennial teosinte genomic DNA was biotin labeled and its signals were detected with Cy-3. We hybridized the mixture of the two kinds of labeled probes onto the mitotic chromosomes of maize (inbred line 48-2). The results showed that the yellow signals covered the chromosome pairs 1, 7, 8, 9, and 10, and yellow-green signals covered the chromosome pairs 2, 3, 4, 5, and 6 (Fig. 2A) . The same mixture of the two kinds of labeled DNA was hybridized on mitotic chromosomes of MZI202. The GISH results of MZI202 were different from those of maize. Three chromosomes appeared strongly labeled in red and they were obviously separated from the maize genome (Fig. 2C,D). This data suggested that the three alien perennial teosinte chromosomes had replaced three maize chromosomes in MZI202. And, according to the morphology of the maize chromosomes, only one, maize chromosome 6, was observed in MZI202 (Fig. 2B, 2C).


Figure 2
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  		Fig. 2. Multicolor GISH images of the mitotic metaphase chromosomes of the parent maize and MZI202. Their chromatins were probed by the probes mixture containing the digoxigenin-labeled maize genomic DNA and the biotin-labeled perennial teosinte genomic DNA, and the nuclei were counterstained with DAPI (blue). (A) The metaphase chromosomes of maize showing strong yellow signals or yellow-green band signals, red arrows showing maize chromosome 6. (B) 20 metaphase chromosomes of MZI202, red arrow showing one maize chromosome 6 in MZI202. (C, D) the metaphase chromosomes of MZI202, the arrows showing three perennial teosinte chromosomes with red signals.

 
Detection of Perennial Teosinte Chromosome by SSR Markers
The SSR microsatellites assay was used to confirm the substituted maize chromosome(s) in MZI202. The SSR survey comparing its parents with MZI202 revealed that the specific polymorphism of p-bnlg249 and p-nc009 on maize chromosome 6 could be detected in both maize and perennial teosinte. The specific polymorphism of p-phi071 on maize chromosome 10 could be detected only in perennial teosinte. With the exceptions of chromosomes 6 and 10, none of the specific polymorphisms of perennial teosinte were detected in the remaining chromosomes of MZI202 (Table 1 and Fig. 3) . On the basis of these analyses, it was possible to suggest that a single chromosome 6 and the pair of chromosome 10 of maize were replaced by perennial teosinte chromosomes.


Figure 3
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  		Fig. 3. Identification of substituted maize chromosomes by SSRs. (C) The PCR products of genomic DNA of MZI202 plant, (A) maize, and (B) perennial teosinte were shown after electrophoresis in 4.5% (w/v) agarose gel. The arrows showed the specific polymorphism where one member of chromosome 6 and two members of chromosome 10 were possibly replaced by perennial teosinte chromosomes in MZI202.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The McGISH protocol has been used successfully to detect three whole-chromosome introgressions in MZI202. The results of this study indicated that three maize chromosomes were substituted by perennial teosinte chromosomes. The SSR assay successfully confirmed the species-specificity of three substituted chromosome segments, with one member of maize chromosome 6 and two homologs of maize chromosome 10. It is apparent that the combined use of SSR and GISH techniques is effective and much more reliable in examining the chromatin introgression from perennial teosinte to maize. Similar explanations were proposed for introgression in oat (Avena sativa L.) and maize crosses (Kynast et al., 2001), potato (Solanum tuberosum L.) (+) tomato (Lycopersicon esculentum Mill.) fusion hybrids (Jacoben and De Jong, 1995), wheat (Triticum aestivum L.) x jointed goatgrass (Aegilops spp.) (Wang et al., 2002), barley (Hordeum vulgare L.) and wheat crosses (Malysheva et al., 2003), and wheat–rye (Secale cereale L.) addition and substitution lines (Alkhimova et al., 1999). MZI202 was a maize–perennial teosinte substituted material with three alien chromosomes from perennial teosinte, it could be a new progenitor for evolution analysis. MZI202 was maize-like plant, with vigorous growth and disease resistance. These features would represent a favorable starting point toward further investigation of the specific genes or alleles for contributing to maize improvement. Furthermore, MZI202 and its progenies have the potential to serve as a bridge connecting maize and its wild relatives, such as perennial teosinte.

In MZI202 we have observed many mitotic cells. Most of the green signal labeled maize chromosomes could pair based on the arm ratio and relative length of their homologs except chromosome 6. Therefore, we thought one of the three missing maize chromosomes was chromosome 6. Whether the three substitution chromosomes from perennial teosinte contain satellite chromosome remains to be further confirmed, because the karyotype of perennial teosinte was less known than that of maize. Maize is a tetraploid (Moore et al, 1995). There are many duplicated sequences in its genome; for example, many molecular markers existing in chromosome 2 might appear in chromosome 7 or 10 (Donty and Helentjaris, 1992). Therefore, we think the duplicated sequences in the maize genome may explain why MZI202 produces composite SSR banding patterns rather than perennial teosinte patterns for markers p-phi071 and p-phi063 (Fig. 3). Although a SSR assay could provide some useful information, it was not completed. Further studies with cloned repetitive sequences by FISH or FISH location of rDNA should further confirm the substitution lines.


   ACKNOWLEDGMENTS
 
This research was supported by the National 863 Research Program of China (Grant No. 2001AA241051), the Maize Genetic and Breeding Research Program of Sichuan Educational Bureau, and the National Natural Science Foundation of China (Grant No. 39870423).

Received for publication March 6, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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Right arrow Crop Cytology

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