Microsatellite and RAPD polymorphisms in Ontario corn hybrids are ...

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Abstract. Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic re- lationships among 37 Ontario corn ...
Molecular Breeding 7: 13–24, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Microsatellite and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings G.L. Sun, M. William, J. Liu, K.J. Kasha & K.P. Pauls∗ Biotechnology Division, Department of Plant Agriculture, University of Guelph, Guelph, Ontario, N1G 2W1 Canada; Author for correspondence (e-mail: [email protected]) Received 15 November 1999; accepted in revised form 10 July 2000

Key words: Genetic diversity, Corn hybrids, Microsatellite, RAPD, Maturity rating, Cluster analysis

Abstract Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic relationships among 37 Ontario corn hybrids. Almost all (95%) of the 160 RAPD fragments and all of the 79 microsatellite alleles were polymorphic across the 37 hybrids. Similarity values among the hybrids ranged from 31% to 86% when based on the RAPD data. The similarities based on microsatellite markers ranged from 12% to 77%. The genetic diversity revealed by microsatellite marker analysis was higher than that obtained from RAPD analysis. The similarity matrices for the microsatellite data and the RAPD data were moderately correlated (0.43). Cluster analyses based on either type of marker showed that most of the hybrids from the same company were closely related to each other. Both dendrograms clustered similar pairs or groups of hybrids. A principal component analysis, based on the combined RAPD and microsatellite data, yielded a good separation of the hybrids with Ontario Corn Heat Unit (OCHU) values 2800. Seventeen RAPD markers and 5 microsatellite markers were significantly associated with the OCHU ratings of the hybrids.

Introduction Knowledge of the genetic diversity and relationships among corn hybrids is important for planning breeding strategies, hybrid identification, and corn germplasm conservation. It is also important that farmers have the opportunity to make informed choices about which hybrids have the potential to maximize profitability but allow them to spread their climate, pest- or diseaserelated risks across different genotypes (Troyer et al. 1983). Different descriptors have been used to characterize corn germplasm diversity including: morphological traits, pedigree data (Duvick 1984; Darrah and Zuber 1986), inter-inbred hetrosis (e.g. Troyer et al. 1988; Smith and Smith 1989), isozymes (Smith 1984), zein chromatographic profiles (Smith 1988) and DNAbased markers such as restriction fragment length polymorphisms (RFLPs) (Smith and Smith 1991). RFLPs are considered to be the most useful type of descriptors from this list due to the high degree of

polymorphism that is detected by this technique in corn (Smith and Smith 1991). However, RFLP analyses are labor-intensive, time consuming and require relatively large DNA samples. Recently, there has been a shift to the use of polymerase chain reaction (PCR)-based markers, such as random-amplified polymorphic DNA (RAPD) markers (Williams et al. 1990), because they are easy to produce and analyze. RAPD analysis has become an important technique for population genetic studies because the amplified DNA products are derived from coding and non-coding regions across the whole genome. RAPD markers have been used to examine both interspecific and intraspecific variation in a number of plant species (Kazan et al. 1993; Liu et al. 1994; Rus-Kortekaas et al. 1994; Thormann et al. 1994; Abo-elmafa and Shimada 1995; Brummer et al. 1995; Sun et al. 1997) and to characterize corn hybrids (Stojsin et al. 1996). In addition, Thormann et al. (1994) found a high correlation (0.88) between RAPD- and RFLP-based genetic similarity values in cruciferous

14 Table 1. The hybrids used for this study. No.

Code

Company

OCHU∗

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

DK1 DK2 DK3 DK4 DK5 DK6 DK7 DK8 DK9 DK10 DK11 DK12 DK13 P3902 P3921 P3905 P3860 P3733 P3951 P3751 PK4405 PK7105 PK2733 PK3652 PK776 PK4949 PK2626 PK2405 PK2525 PK2408 EXEL PK4774 HL2241 CAR1877 NK2555 PRIDEK115 MYCO2380

Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Dekalb Pioneer Hi-Bred Pioneer Hi-Bred Pioneer Hi-Bred Pioneer Hi-Bred Pioneer Hi-Bred Pioneer Hi-Bred Pioneer Hi-Bred Pickseed Pickseed Pickseed Pickseed Pickseed Pickseed Pickseed Pickseed Pickseed Pickseed Pickseed Pickseed Hyland Cargill N.K. Pride Mycogen

2400–2500 2500–2600 2600–2700 2700–2800 2800–2900 2900–3000 3000–3100 3100–3200

2650 2600–2700 2650–2800 2700–2900 3100 2500–2600 3000

2650–2800 2600–2700 2500–2600 2600–2700 2500–2600 2900–3100 2900–3000 2500–2600 2500–2600 2600–2700 2600–2700 2600–2700

∗ : Ontario Corn Heat unit

species. RAPD data can be generated more quickly and with less labor than is required for other molecular markers, such as RFLPs. However, there is some loss of information when RAPD markers are used because they are dominant rather than codominant. Microsatellites are useful molecular markers because they are: (1) abundant, (2) uniformly distributed, (3) highly polymorphic, (4) codominant, (5) rapidly produced by PCR, (6) relatively simple to interpret,

and (7) easily accessed by other laboratories via published primer sequences (Saghai-Maroof et al. 1994). Surveys of DNA sequence databases have revealed an abundance of microsatellite loci in plants, and subsequent studies have demonstrated the informativeness of these markers in several genera. Microsatelliterelated data in plants (especially in crop plants) has increased considerably over the past few years (Condit and Hubbell 1991; Morgante and Olivieri 1993; Kresovich et al. 1995; Brown et al. 1996). The level of microsatellite polymorphism in plant studies has been far greater than that found with RFLPs (Wu and Tanksley 1993; Saghai-Maroof et al. 1994; Rongwen et al. 1995). Microsatellite markers have been used to study genetic diversity, identify germplasm and characterize population structures in plants (Thomas and Scott 1993; Russell et al. 1997; Senior et al. 1998; Sun et al. 1999). The exchange of corn germplasm from one latitudinal to another is limited by differences in the time required for material to mature in different corn growing regions. Maturity is considered to be quantitative, and different studies have identified 2–19 loci in corn that affect this trait (Giesbrecht 1960; Hallawer 1965; Beavis et al. 1991). Recent investigations have used molecular markers to identify quantitative trait loci (QTL) controlling maturity (Koester et al. 1993; Berke and Rocheford 1995; Austin and Lee 1996). RFLP analysis of F2 individuals and F3 families major QTL for days to flowering on chromosome 1, 8 and 10 (Koester et al. 1993). A study to determine the number and chromosomal locations of QTL controlling male anthesis in S1 lines identified sixteen RFLP loci located on eight chromosomal regions (Berke and Rocheford 1995). Austin and Lee (1996) used recombinant inbred (RI) lines to detect and characterize QTL in maize for flowering and other traits. They identified twelve loci, located on chromosomes 1–9, that were significantly associated with anthesis date, ten loci located on chromosomes 1, 2, and 4–10 that were significantly associated with silk emergence, and four loci located on chromosomes 2, 3, 6, and 8 that were significantly associated with the anthesis to silk interval. Khavkin and Coe (http://www.agron.mossouri.edu/mnl/72/02khavkin.html) revised the QTL database for traits related to maturity, and listed more than 240 loci spread over all 10 maize chromosomes in their catalogue. The objectives of the present study were to (1) estimate the genetic diversity among 37 Ontario corn hybrids, (2) compare the genetic relatedness values

15 obtained from RAPD and microsatellite analyses, and (3) identify markers associated with maturity as measured by the Ontario Corn Heat Unit rating scale.

Materials and methods Plant materials and PCR procedure Thirty-seven commercial corn hybrids ranging in maturity from 2400 to 3200 Ontario Corn Heat Units (OCHU) were studied (Table 1). Corn Heat Unit ratings are a relative maturity measure that is calculated from maximum and minimum daily temperatures during the frost-free growing season in each area of the province of Ontario (www.gov.on.ca/OMAFRA/english/crops/facts/93-119. htm). The OCHU rating provides an indexing system to assist farmers to select the most suitable hybrids for their area. Each RAPD reaction mixture contained 0.2 mM of each deoxynucleotide, 2.5 mM MgCl2 1.0 U Taq polymerase, 15 pmol primer and 20 ng template DNA in a volume of 20 µl. The PCR program consisted of 30 cycles of denaturation at 94 ◦ C for 1 min, annealing at 36 ◦ C for 1 min and extension at 72 ◦ C for 2 min. The final cycle had a 3 min extension step at 72 ◦ C. The PCR fragments were electrophoresed through 1.2% agarose gels, stained with ethidium bromide, and visualised with ultraviolet light. The presence or absence of strong DNA fragments was scored for each sample. The PCR reactions for microsatellite analyses contained 0.2 mM of each deoxynucleotide, 2.0 mM MgCl2 1.5 U Taq polymerase, 15 pmol of each primer and 30 ng of template DNA in a reaction volume of 20 µl. PCR performed with a PTC-100TM programmable thermal controller (MJ Research Inc) using a ‘touchdown’ PCR program consisting of 18 cycles of denaturation at 95 ◦ C for 1 min and extension at 72 ◦ C for 2 min. The annealing temperature (1 min) was progressively decreased by 1 ◦ C every third cycle from 65 ◦ C to 55 ◦ C. The PCR reaction continued for 20 additional cycles of 95 ◦ C for 1 min, 55 ◦ C for 1 min and 72 ◦ C for 2 min. The reaction was terminated with a 10 min extension at 72 ◦ C. The PCR products were electrophored through a 12% non-denaturing polyacrylamide gel, and stained with ethidium bromide. Data analysis Data matrices were entered into the NTSYS program (Rohlf 1993). The data were analyzed with

the Qualitative routine to generate Jaccard’s similarity coefficients. Similarity coefficients were used to construct dendrograms using the UPGMA (unweighted pair group method with arithmetic average) and the SHAN (sequential, hierarchical, and nested clustering) routine in the NTSYS program. The relationships between the results from the different marker methods were analyzed with Mantel’s test for correlation between matrices (Mantel 1967). This test compares the elements of two matrices and estimates the degree of correlation between the matrices by means of a test criterion (Z), and a product-moment correlation (r). A principal coordinate analysis (PCA) was conducted with the same program using DCENTER and EIGEN procedures. This multivariate approach was chosen to complement the cluster analysis information, because cluster analysis is more sensitive to closely related individuals, whereas PCA is more informative regarding distances among major groups (Hauser and Crovello 1982). Results Based on results of a previous screen of RAPD primers for polymorphism with corn inbred lines (data not shown), only primers that gave highly reproducible RAPD patterns were used for present study. Twentyfour random 10-mer primers from UBC (University of British Columbia, Canada) were used to amplify fragments from the DNA temples of 37 hybrids. A total of 160 strong bands were scored, giving an average of 6.7 fragments per primer. Of the 160 fragments, 153 (95%) were polymorphic across the 37 hybrids, and gave from 1 to 7 polymorphic fragments with a mean of 6.4 polymorphic fragments per primer. From a screen of 96 microsatellite primers, seventeen pairs of primers were chosen that produced polymorphic patterns with DNA from 5 inbred corn lines (data not shown). Three to eight bands (alleles) were amplified when the 17 primers were used with DNA from the 37 hybrids. A total of 79 bands were scored, and all were polymorphic across this set of hybrids. On average, 4.6 alleles per primer were amplified. Assessment of genetic diversity Two independent genetic similarity matrices were produced for the RAPD and microsatellite data using the Jaccard algorithm (data not shown). The similarities from the RAPD data ranged from 31% common

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Figure 1. Dendrogram for 37 Ontario corn hybrids based a cluster analysis (UPGMA) of genetic similarities (Jaccard coefficient) from RAPD data.

bands for PrideK115 vs. Pickseed hybrid PK4405 to 86% common bands for Dekaleb hybrids DK6 versus DK7. The coefficient of similarity for the microsatellite data ranged from 12% for PK4405 versus P3921 and DK4 versus DK12 to 77% for DK6 versus DK7. Thus, the two methods agreed that the Dekalb hybrids DK6 and K7 were most similar. The largest differences were identified between Pickseed hybrid PK4405 and PrideK115 or P3921. In general, the microsatellite data gave lower similarity values than those based on the RAPD analyses. The correlation between the two genetic similarity matrices using the Mantel’s test (Mantel 1967) was 0.43, suggesting that the estimations of genetic relatedness provided by the two marker systems were only moderately related. Dendrograms based on the similarity values from RAPD and microsatellite data were constructed using UPGMA to illustrate the relationships among the corn hybrids under study. The RAPD-data dendrogram (Figure 1) showed that hybrids from the same company were clustered together. For example, five of

seven Pioneer Hi-bred hybrids were allocated to one group, and hybrids P3733 and P3751 were assigned to a separate group. The thirteen Dekalb hybrids fell into three separate groups with DK1, DK2, DK3 and DK11 in one group, DK4, DK5, DK6, DK7, DK9 and DK10 in another group, and DK12 and DK13 in the third group. The most related Dekalb hybrids were DK6 and DK7. Four Pickseed hybrids (PK776, PK4949, PK2626 and PK2525) were clustered together. The Pickseed varieties Exel and PK4774 were in one group, and PK7150 and PK2408 were in another cluster. The other four Pickseed hybrids were separated from all of these groups and were spread throughout the dendrogram. Hybrid Car1877 was tightly clustered with hybrid PrideK115. The dendrogram (Figure 2) derived from the microsatellite data also clustered hybrids from same company into related groups, but the boundaries between the groups were not as clear as in the RAPDbased dendrogram. Nevertheless, several clusters, which contain hybrids developed by the same com-

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Figure 2. Dendrogram for 37 Ontario corn hybrids based on a cluster analysis (UPGMA) of genetic similarities (Jaccard coefficient) from microsatellite data.

pany, can be recognized. Eight of thirteen Dekalb hybrids (DK4, DK5, DK6, DK7, DK8, DK9, DK10 and DK11) were allocated to one group. Four of seven Pickseed hybrids (PK3652, PK776, PK4949 and PK2525) were grouped together and two other Pickseed hybrids (Exel and PK4774) were grouped. P3902, P3921 and P3860, P3905 and P3951 from Pioneer Hi-bred were also grouped and Car1877 clustered with PrideK115. A cluster analysis based on the combination of the two data sets is shown in Figure 3. The thirteen Dekalb hybrids were separated into three groups with group 1 containing DK1, DK2 and DK3; group 2 containing DK4, DK5, DK6, DK7, DK8, DK9, DK10, DK11; and group 3 containing DK12 and DK13. It is noteworthy that the hybrids with OCHU < 2800 were included in group 1, while the hybrids with OCHU values >2800 were clustered into group 2. Five of the seven Pioneer Hi-bred hybrids with OCHU values

2800 were grouped together and separated from the other Pickseed hybrids that had OCHU values 2800 fell into groups

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Figure 3. Dendrogram for 37 Ontario corn hybrids based on a cluster analysis (UPGMA) of genetic similarities (Jaccard coefficient) from RAPD and microsatellite data.

I and II, and all of the hybrids with OCHU values