International Rice Research Institute, P. 0. Box 933, Manila, Philippines
Concept of marker-aided selection (Tanksley et al. 1989) and recent preliminary reports (Abenes et al. 1993) show that target genes can be identified in a segregating population at early plant growth stage based on linked DNA markers. The linked DNA markers can be identified by RFLP analysis using either Southern hybridization or polymerase chain reaction (PCR). Practical application of marker-aided selection requires that markers be identified with high level of accuracy and efficiency, be cost effective, and easy to use. Southern hybridization requires application of either radiochemicals or very expensive nonradioactive materials and is laborious, time-consuming and relatively large quantities of high quality DNA are needed. PCR overcomes these drawbacks and therefore is a method of choice.
To use PCR in marker-assisted selection, the generation of Specific Amplicon Polymorphism (SAP) such as PCR-based RFLP (PBR) (Williams et al. 1991; Ghareyazie et al. 1993) to follow a target gene is required. In the most desirable cases, Amplicon Length Polymorphism (ALP) between gene donor and recipient can be obtained, and represents the simplest and fastest way of detecting polymorphism. Digestion of PCR products with restriction endonucleases (4- base cutters) has been found to increase the level of polymorphism as well as the number of alleles in rice (Ghareyazie et al. 1993). However it is not clear how often ALP and PBR can be observed in rice for marker aided-selection.
To answer this question we analyzed the data obtained from a rice germplasm consisting of 35 Iranian, 3 indica and 2 japonica varieties. Total genomic DNA was extracted from the rice leaves. The DNA was then amplified with 14 sets of primers synthesized based on mapped RFLP markers. Amplicons were digested with 9 different restriction endonucleases (4-base cutters), regardless of the presence or absence of polymorphism (ALP). We then performed pairwise comparison among the 40 varieties for polymorphism. To calculate the abundance of PBR for marker-aided selection, we assumed the segregating populations for selection in a breeding program derived from pairwise crosses. Alleles in PBR are defined as polymorphisms detected by one enzyme or enzyme combination. For example the PCR products at the waxy locus before digestion are monomorphic (1 allele only), but after digestion with Taq1, three alleles were detected, and after digestion with HinFI, 2 alleles were obtained (data not shown). Combination of these alleles gives rise to 5 different alleles (polymorphism) at this locus. The total number of polymorphic pairs (NP) for each marker locus were then calculated as follows:
NP = n-1Summation`i=1`(Xi*nSummation`j=i+1 Xj)Where, n is the total number of alleles in a given locus, Xi and Xj are the number of varieties carrying the ith and jth alleles respectively.
The abundance of ALP and PBR is shown in Table 1. Out of the 9895 pairwise comparisons we detected ALP in 1277 pairs of varieties (13%). This percentage of ALP in rice is obviously not high enough for general use in marker-aided selection. Additional polymorphism was generated by restriction digestion of PCR products. The number of alleles increased for many marker
Table 1. The abundance of ALP and PCR-based RFLP in 14 rice loci
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Allele Allele
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Marker #Entrya 1 2 3 ALP Total ALP% #Entrya 1 2 3 4 5 6 PBR Totl PBR%
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PTA248 39 24 15 360 741 49 39 21 15 3 360 741 49
RG13 37 22 10 5 380 666 57 26 14 4 4 2 1 1 221 325 68
RG64 40 32 7 1 263 741 34 40 30 7 2 1 263 741 34
RG100 40 40 0 780 0 40 40 0 780 0
RG118 39 39 0 741 0 38 20 11 4 2 1 451 703 64
RG120 39 39 0 741 0 39 39 0 741 0
RG173 39 39 0 741 0 40 36 3 1 147 780 19
RG235 40 35 5 175 780 22 40 35 5 175 780 22
RG241 39 39 0 741 0 34 31 2 1 95 561 17
RG257 40 40 0 780 0 40 38 2 76 780 10
RG329 40 40 0 780 0 22 21 1 21 231 9
RG365 36 33 3 99 630 16 36 33 3 99 630 16
RG386 23 23 0 253 0 26 17 9 153 325 47
Waxy 40 40 0 780 0 39 24 6 4 3 2 440 741 60
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Total 1277 9895 13 2501 8859 28
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a=Number of entries examined for ALP and PBR.
loci. For example, there are 6 alleles for RG13 in PBR while only 3 in ALP.
For Waxy locus a total of 5 alleles were revealed in PBR. Increased number
of alleles gives rise to more polymorphic pairs. Out of 8859 pairwise
comparisons we found 2501 pairs being polymorphic at these loci (28%). Since
the total number of enzymes available is much higher than used in this study,
and considering the fact that studied varieties did not include all
isozyme groups (Glaszmann 1987), we expect more than 28% PBR in rice. Since
marker-aided selection is used to complement phenotype-based selection, we do
not expect to perform marker-aided selection on all target genes. If we
assume that marker-aided selection will be performed on one third or half of
the target genes, we can basically rely on ALP or PBR for marker
aided-selection. Accuracy, ease and speed of ALP and PBR therefore are
attractive and should be included in the plant breeding programs when
marker-aided selection is used.References
Abenes, M. L. P., E. R. Angeles, G. S. Khush and N. Huang, 1993. Selection of bacterial blight resistant rice plants in the F`2` generation via their linkage to molecular markers. RGN 10: 120-123.
Ghareyazie B., N. Huang, G. Second, J. Bennett and G. S. Khush, 1993. Comparison between PCR-based RFLP and Southern-based RFLP as DNA markers for germplasm classification in rice. RGN 10: 129-132.
Glaszmann J. C., 1987. Isozymes and classification of Asian rice varieties. Theor. Appl. Genet. 74: 21-30.
Tanksley S. D., N. D. Young, A. H. Paterson and M. W. Bonierbale, 1989. RFLP mapping in plant breeding: new tools for old sciences. Biotechnology 7: 257-264.
Williams, M. N. V., N. Pande, S. Nair, M. Mohan and J. Bennett, 1991. Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice (Oryza sativa L.) genomic DNA. Theor. Appl. Genet. 82: 489-498.