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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
Agricultural Sciences in China
August 2009
2009, 8(8): 920-930
Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.) ZHENG Da-hao1, YU Yang2, WANG Zhen-ping3 and LI Yan-ru1 Agricultural College, Yanbian University, Longjing 133400, P.R.China Crop Tillage and Cultivation Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, P.R.China 3 Tonghua Academy of Agricultural Sciences, Meihekou 135000, P.R.China 1 2
Abstract Ten temperate and adapted exotic breeding populations of maize were studied with electrophoretic techniques. Three isozyme systems coded by nine allozyme loci were used for evaluating the genetic variability within and among populations. The results revealed that 78.57% of allozyme loci were polymorphic. Low allelic variation with a mean number of 1.84 alleles per locus per population was detected. But, these populations still maintained higher level of heterozygosity; moreover, the exotic populations had greater gene diversity than the temperate populations. All the populations were non-panmictic with negative Wright’s fixation indexes (-0.091- -0.424). The tropical BS16 was typified by maximum allelic richness, percent of polymorphic loci and heterozygosity. More than 93% of the gene diversity maintained within populations, and the genetic differentiation among populations was low (0.002-0.191). Multivariate analysis demonstrated that the tropical BS29 diverged from other populations in the reverse direction. The temperate BS9 and tropical BS16 were divergent each other, and highly differentiated from other temperate and tropical populations, consequently, these two populations would be analogically postulated as potential germplasms to establish new heterotic groups for temperate maize breeding programs. Key words: maize, breeding population, allozyme, diversity structure, multivariate analysis
INTRODUCTION Abundant genetic variation among and within maize germplasms is indispensable for the recognition and exploitation of heterotic patterns among germplasm pools. In addition, for the base and synthetic populations, the breeding values rely on the genetic diversity within initial populations constructed, and how much genetic variation is conserved within their derived populations after cycles of recurrent selection. Allozyme assay is a useful tool to detect genetic variation. Isozyme data have been used in preparation of keys for classifying
maize inbreds and populations, determining allele frequency and heterozygosity/homozygosity at different allozyme loci, identifying rare alleles, accounting for residual variability in developed inbreds. Numerous studies based on isozyme electrophoresis were conducted on identification of cultivars and parental lines (Cardy and Kannenberg 1982; Sanou et al. 1997), analysis of genetic variability among parental lines, cultivars, populations and races (Stuber et al. 1980; Goodman and Stuber 1983; Doebley et al. 1983, 1985; Smith et al. 1985; Kahler et al. 1986; Salanoubat and Pernes 1986; Pflüger and Schlatter 1996). The simplicity of the isozyme assays together with its rapid and lower cost
Received 11 March, 2009 Accepted 24 April, 2009 Correspondence ZHENG Da-hao, Professor, Tel/Fax: +86-433-3261794, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60296-5
Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
still guarantees its use today (Gimenes and Lopes 2000; Mauria et al. 2000; Sanchez et al. 2000; Liu et al. 2001; Lu et al. 2002). In particular, as a functional description of gene expressions, isozymatic data still offer advantages compensating for shortcomings of unselected non-functional DNA markers, such as random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), single nucleotide polymorphism (SNP), etc. The information about allozymatic variability has practical implications for germplasm preservation, and provides an efficient approach for directly comparing the magnitude and distribution of genetic diversity between different populations and species (Lu et al. 2002; Pinto et al. 2003). Zea mays L. contains more than 200 races (Gimenes and Lopes 2000), but only a few have been utilized in maize breeding programs, in particular, not all usable races have practical values in maize hybrid breeding. In addition, the excessive dependence on inbred recycling approach based on limited germplasm sources resulted in a limited genetic diversity within temperate maize germplasm (Darrah and Zuber 1985; Lu and Bernardo 2001; Zheng et al. 2008). In order to overcome the consequent germplasm bottleneck having become a restrictive factor in temperate maize breeding activities (Hallauer 1989), many works have focused on using tropical and subtropical germplasms in temperate hybrid breeding programs. However, because of the deficiency of adaptability in temperate regions, exotic germplasms are commonly crossed with temperate maize to develop new inbreds. Recently, domesticating exotic populations prior to their direct utilizations in temperate maize breeding programs is of importance in practice. And, some important exotic populations have been domesticated for temperate regions. Compared with non-adapted tropical and subtropical germplasms, the adapted exotic populations must have greater practical values in their utilizations in temperate maize breeding programs. The objectives of the this study were to evaluate, at the isozyme level, (1) the genetic variability of temperate and adapted exotic breeding populations of historical and potential importance in developing maize inbreds, (2) the population structure and, (3) the genetic differentiation and gene flow between populations.
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MATERIALS AND METHODS Plant materials Plants from ten open-pollinated breeding populations of maize were analyzed, including adapted exotic materials adapted to the temperate region. All the materials were provided by Dr. K R Lamkey at Iowa State University, USA, in 1999. These populations were maintained in the Maize Research Laboratory at Agricultural College of Yanbian University, China, of which six, Iowa Stiff Stalk Synthetic [BSSS (R) C12], Iowa Corn Borer Synthetic No. 1 [BSCB1 (R) C12], open-pollinated Lancaster Surecrop [BSL (S) C7, LSC], Krug Yellow Dent (BS18, Krug), Iodent (BS30), and the broad-based synthetic BS9 (CB) C5, were typical maize populations of historical or potential importance in the development of inbreds in the Corn Belt. Three, BS16 (CB) C4 (ETO), BS28 (C6) (Tuxpeño), and BS29 (Suwan-1), were the adapted exotic populations. One was BSTL (S) C5, a strain of Lancaster Surecrop (LSC) containing 1/4 of exotic germplasm of the Mexican race and Tuxpeño.
Isozyme electrophoretic analysis About 150 individuals for each of ten populations (one kernel per ear of each individual) were grown at 28°C in the daytime and 25°C in the night for 40-60 h. Approximately 300 mg of coleoptile tissue were collected from each individual, finely homogenized at 0-4°C in 0.6 mL of 0.1 mol L-1 Tris-HCl (pH 8.0), then, centrifuged at 10 000 r/min. The supernatant fluid was mixed with 200 L of 50% glycerol. Enzyme extraction, electrophoresis, gel staining, scoring as well as genotyping of alleles for each locus were performed according to the methods described by Stuber et al. (1988). Finally, 100 individuals from each of populations were genotyped at nine allozyme loci of three enzyme systems, esterase (Est) (EC 3.1.1.1), peroxidase (Per) (EC 1.11.1.7) and α-amylase (Amy) (EC 3.2.1.1).
Data analysis The genetic diversity and structure of populations were
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71.43 85.71 71.43 71.43 71.43 85.71 71.43 100.00 71.43 85.71 78.57 1.71 1.86 1.71 1.71 1.86 2.00 1.71 2.14 1.71 2.00 1.84 0.051 0.052
0.035
0.070
Total
12 13 12 12 13 14 12 15 12 14 12.90 0.740 0.756 0.665 0.690 0.660 0.625 0.531 0.645 0.668 0.625 0.661
B A
0.260 0.244 0.335 0.310 0.340 0.375 0.469 0.355 0.332 0.375 0.340 0.607 0.630 0.565 0.735 0.755 0.725 0.699 0.595 0.675 0.611 0.660
B A
0.393 0.370 0.435 0.265 0.245 0.275 0.301 0.405 0.325 0.389 0.340 0.165 0.215 0.250 0.055 0.425 0.460 0.510 0.420 0.530 0.424 0.345
C B
0.448 0.667 0.625 0.480 0.955 0.580 0.615 0.380 0.650 0.385 0.579
A
B
0.835 0.785 0.750 0.945 0.505 0.540 0.490 0.545 0.470 0.525 0.639
Number of alleles
Amy1 Per5 Per3
0.552 0.333 0.375 0.520 0.045 0.420 0.385 0.620 0.350 0.615 0.422
A B
1.000 0.975 1.000 1.000 1.000 0.975 1.000 0.855 1.000 0.975 0.978 0.025 0.055
0.145
0.025
0.025
A B
1.000 1.000 1.000 1.000 1.000 0.995 1.000 0.500 1.000 1.000 0.950 0.253
0.500
0.005
A
0.582 0.505 0.600 0.484 0.551 0.550 0.520 0.379 0.505 0.645 0.532
B A
0.418 0.495 0.400 0.516 0.449 0.450 0.480 0.621 0.495 0.355 0.468 Mean
Adapted exotic
BSSS BSCB1 BSTL BSL BS9 BS18 BS30 BS16 BS28 BS29 Temperate
Frequency of alleles detected at allozyme locus
Est8 Est4 Est3
One hundred individuals for each of ten populations were analyzed at three isozyme systems. Among the isozyme systems, the α-amylase (Amy) system revealed monolocus polymorphism, i.e., only one locus was identified in this system, whereas the esterase (Est) and peroxidase (Per) systems showed multiple loci polymorphism, i.e., two or more loci were identified in each isozyme system. Across all populations, nine allozyme loci were identified, but only seven loci were polymorphic (Table 1). Among the populations, BS16, BS29, BS18, BSCB1, and BS18 exhibited more number of alleles and higher percentage of polymorphic loci than BSSS, BSTL, BSL, BS28 and BS30. Compared to temperate populations, the adapted exotic populations had slightly greater mean number of alleles and higher percentage of polymorphic loci. These alleles existed at quite different frequencies among populations (Table 1), and 80.6% of alleles were at the frequencies of 0.05 to 0.95. In this
Est2
Allozymatic polymorphism
Population
RESULTS
Table 1 Frequency of alleles detected at seven polymorphic loci and the percentage of polymorphic loci (%) in ten populations
assessed based on mean number of alleles per locus, the percentage of polymorphic loci, the observed heterozygosity (Ho), the expected heterozygosity (He) (Nei 1987), and inbreeding coefficient [f = 1 - (Ho/He)] (Wright 1978). Wright’s fixation index (FIS) was estimated to quantify the deficiency and excess of heterozygotes for each population and each locus (Wright 1978). The gene diversity in the total population (HT) was partitioned into gene diversity within (HS) and between (DST) populations (HT = HS + DST), and the proportion of total gene diversity partitioned between populations was estimated as GST = DST/HT (Nei 1987). The genetic differentiation between populations (FST) was estimated according to Weir and Cockerham (1984), and the gene flow was given by Nm = 0.25 × (1 - FST)/FST (Cockerham and Weir 1993). Based on FST, principal component analysis (PCA) and cluster analysis with the unweighted pair-group method algorithm (UPGMA) were performed. The data were analyzed using the programs TFPGA 1.3 (Miller 1997) and FSTAT 293 (Goudet 1995), except PCA performed with NTSYS 2.10 software (Rohlf and Slice 1992).
ZHENG Da-hao et al. Percentage of polymorphic loci (%) Mean
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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
study, some alleles occurred in only a few populations, e.g., allele Est3-A was detected in population BS16 and BS18, Est4-A in BSCB1, BS16, BS18, and BS29, Per3A in BS9, BS6, and BS29. Nevertheless, these alleles existed at very low frequencies of 0.005 to 0.145. Among the populations, the adapted exotic BS16 was typified by a maximum allelic richness (2.14 alleles on average) with all the alleles detected and percentage of polymorphic loci (100%), indicating that BS16 had the most abundant allozymatic variation.
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The observed heterozygosity (Ho) and expected heterozygosity (He) The heterozygosity could reflect the gene diversity for a population. Across all polymorphic loci, Ho and He values for each population varied from 0.310 and 0.276 for BSL to 0.550 and 0.453 for BS16, and averaged 0.409 and 0.333, respectively (Table 2). The differences in Ho and He values were significant among loci within each population and among populations at most of loci
Table 2 The observed heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient ( f ) at allozyme loci, and Wright’s fixation index (FIS) over all loci for the temperate and adapted exotic breeding populations Temperate populations Est2
Est3
Est4
Est8
Per3
Per-5
Amy1
Over all loci
Ho He f Ho He f Ho He f Ho He f Ho He f Ho He f Ho He f Ho He FIS
Adapted exotic populations
BSSSR
BSCB1
BSTL
BSL
BS9
BS18
BS30
BS16
BS28
BS29
0.835 0.489 -0.714 0.000 0.000 NA 0.000 0.000 NA 0.636 0.498 -0.281 0.330 0.277 -0.193 0.704 0.479 -0.472 0.520 0.387 -0.347 0.432 * 0.304 * -0.424
0.439 0.503 0.127 0.000 0.000 NA 0.050 0.049 -0.021 0.479 0.447 -0.073 0.430 0.339 -0.269 0.740 0.469 -0.584 0.488 0.371 -0.317 0.375 ** 0.311 ** -0.207
0.560 0.482 -0.162 0.000 0.000 NA 0.000 0.000 NA 0.350 0.471 0.258 0.500 0.377 -0.329 0.530 0.494 -0.073 0.650 0.448 -0.455 0.370 * 0.325 ** -0.141
0.448 0.502 0.108 0.000 0.000 NA 0.000 0.000 NA 0.500 0.502 0.003 0.110 0.104 -0.053 0.530 0.392 -0.356 0.580 0.430 -0.351 0.310 * 0.276 * -0.124
0.778 0.497 -0.568 0.000 0.000 NA 0.000 0.000 NA 0.091 0.087 -0.043 0.720 0.562 -0.282 0.410 0.372 -0.103 0.460 0.451 -0.02 0.351 * 0.281 * -0.25
0.720 0.497 -0.451 0.010 0.010 0.000 0.050 0.049 -0.021 0.440 0.490 0.102 0.760 0.499 -0.526 0.510 0.401 -0.274 0.690 0.471 -0.468 0.454 ** 0.345 ** -0.318
0.500 0.502 0.003 0.000 0.000 NA 0.000 0.000 NA 0.230 0.476 0.518 0.796 0.502 -0.589 0.602 0.423 -0.426 0.649 0.501 -0.299 0.397 * 0.343 ** -0.156
0.552 0.474 -0.166 0.440 0.503 0.125 0.290 0.249 -0.165 0.340 0.474 0.283 0.770 0.528 -0.462 0.810 0.484 -0.678 0.650 0.460 -0.415 0.550 ** 0.453 ** -0.216
0.850 0.502 -0.698 0.000 0.000 NA 0.000 0.000 NA 0.440 0.457 0.038 0.657 0.501 -0.313 0.650 0.441 -0.478 0.663 0.446 -0.492 0.466 * 0.335 ** -0.392
0.490 0.460 -0.065 0.000 0.000 NA 0.050 0.049 -0.021 0.190 0.476 0.602 0.545 0.544 -0.002 0.717 0.478 -0.505 0.710 0.471 -0.511 0.386 * 0.354 ** -0.091
* ** , Significance at P < 0.05 and 0.01, respectively. NA indicated that the allele detected at a specific locus for a population had been homogeneous.
excluding Est3 and Est4 loci (P < 0.05) (Table 2 and Fig.1). In addition, adapted exotic populations exhibited higher heterozygosity with Ho of 0.407 and He of 0.331 than temperate populations with Ho of 0.384 and He of 0.312, but the differences in Ho and He were not significant (P > 0.05). Over all loci, all the populations exhibited heterozygote excess with negative Wright’s fixation index (FIS) values of -0.424 for BSSS to -0.091 for BS29, indicating that these breeding populations were not panmictic. In this study, inbreeding coefficient (f) values varied from -0.714 at Est2 for BSSS to 0.602 at Est8 for
Fig. 1 Dendrogram for the observed heterozygosity (Ho), the expected heterozygosity (He) and the Wright’s fixation index (FIS) at each allozyme locus over all populations. ** indicates significance at P < 0.01 for the differences among populations.
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ZHENG Da-hao et al.
tal population. In contrast, heterozygote deficiencies were occurred in the total population at both Est3 and Est8 loci, evidenced by positive Wright’s fixation indexes of 0.526 at Est2 locus and 0.255 at Est8 locus.
BS29 (Table 2). The positive f value implied that the homogeneity and/or heterozygote deficiencies occurred in particular populations and allozyme loci. For example, at Est3 and Est4 loci, the alleles in six out of ten populations, BSSS, BSTL, BSL, BS9, BS28, and BS30, had been homogeneous. In particular, BS29 and BS30 had been overly deficient in heterozygote at Est8 locus with great positive f values of 0.602 and 0.518, respectively. Among the populations, only BS16 and BS18 maintained all the alleles and heterozygotes at all loci examined, particularly the former. Nevertheless, BSSS and BS28 possessed much more heterozygotes with greater F IS values of -0.424 and 0.392, respectively, although both populations had been homogeneous at Est3 and Est4 loci.
Gene diversity within and among populations The gene diversity (HT) for the total population was 0.356 across all loci, and ranged from 0.043 at Est4 locus to 0.498 at Est2 locus (Table 3). The gene diversities within populations (HS) and among populations (DST) were 0.333 and 0.023, respectively. HS and DST values varied from 0.040 at Est4 to 0.490 at Est2 and from 0.003 at Est4 to 0.050 at Est8, respectively. In this study, the proportion of total gene diversity partitioned among populations (GST) was only about 6.5% over all loci, indicating that most of the genetic variation occurred within populations (> 93% over all loci). An exception was observed at Est3 locus with the greatest GST value of 0.427 (42.7%), probably due to that the alleles at this locus were detected in just two populations. Regarding the gene diversity for typical temperate and adapted exotic populations, most of gene diversities also resided within populations (Table 3). Over all loci, the proportions of the gene diversity occurred within populations were greater than 93% for both typical temperate and adapted exotic populations (GST 0.062). It was worth of mentioning that exotic germplasms had greater gene diversity than typical temperate germplasm. Here, the gene diversity in the total exotic population was 0.405, whereas it was 0.323 in the total temperate population. Furthermore, HT value for the total temperate population increased by 3.4%, when
Contribution of allozymatic diversities at various loci to overall heterozygosity The present results revealed a higher overall mean Ho value of 0.410 than Ho value of 0.359 across all populations and all loci, and subsequent negative overall mean FIS value of -0.141. However, FIS value at each locus over all populations varied from -0.381 to 0.526, indicating that each allozyme locus had a different contribution to the genetic diversity for each populations (Fig.1). Among the three isozyme systems tested, the Est isozyme system exhibited a great variability. But, high inbreeding and/or loss of alleles were observed at Est3 and Est4 loci. Over all populations, higher Ho than He were observed at Est2, Est4, Per3, Per5, and Amy1 loci, with negative FIS values of -0.381 to -0.022, reflecting heterozygote excesses at these loci in the to-
Table 3 Nei’s estimates of the gene diversity for the total population as well as the temperate and adapted exotic populations Locus Est2 Est3 Est4 Est8 Per3 Per5 Amy1 Overall
HT
HS
DST
GST
Whole
Tem1
Tem
TS
Whole
Tem1
Tem
TS
Whole
Tem1
Tem
TS
Whole
Tem1
Tem
TS
0.498 0.096 0.043 0.488 0.472 0.449 0.449 0.356
0.496 0.002 0.017 0.469 0.404 0.443 0.429 0.323
0.499 0.000 0.012 0.463 0.365 0.434 0.411 0.312
0.500 0.278 0.107 0.499 0.526 0.468 0.458 0.405
0.490 0.055 0.040 0.438 0.423 0.442 0.443 0.333
0.494 0.002 0.016 0.416 0.359 0.434 0.426 0.307
0.497 0.000 0.012 0.383 0.320 0.427 0.409 0.293
0.478 0.168 0.099 0.470 0.524 0.466 0.458 0.380
0.008 0.041 0.003 0.050 0.049 0.007 0.006 0.023
0.002 0.000 0.000 0.053 0.044 0.009 0.003 0.016
0.002 0.000 0.000 0.080 0.044 0.007 0.002 0.019
0.022 0.11 0.008 0.029 0.002 0.002 0.000 0.025
0.016 0.427 0.070 0.102 0.104 0.016 0.013 0.065
0.004 0.000 0.000 0.113 0.109 0.020 0.007 0.050
0.004 0.000 0.173 0.121 0.016 0.005 0.061
0.044 0.396 0.075 0.058 0.004 0.004 0.000 0.062
H T, HS, DST , and GST indicated that the gene diversity in the total populations, within populations and among populations, and the proportion of total gene diversity partitioned among populations, respectively. Whole, Tem1, Tem, and TS indicated that the total population, the temperate populations including BSTL contained 1/4 of Mexican race and Tuxpeño germplasms, the typical temperate populations (BSSS, BSCB1, BS9, BS18, and BS30), and the exotic populations (BS16, BS28, and BS29), respectively.
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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
BSTL containing 1/4 of tropical Mexican race and Tuxpeño germplasms was included.
Genetic differentiation and gene flow among populations Estimates of the genetic differentiation (FST) and gene flow (Nm) for pairwise populations over all allozyme loci were presented in Table 4. FST values for pairwise populations varied from 0.002 for BS18 vs. BS30 to 0.191 for BS9 vs. BS16, and averaged 0.068. The tropical BS16 was highly differentiated from other populations with FST values of 0.103 to 0.191 excluding BSSS. The temperate synthetic BS9 was particularly and highly divergent from BSSS, BSL, BS16, and BS29 with great FST values of 0.135 to 0.191, whereas BSL was particularly differentiated from BS9, BS16, BS28 and BS30 with FST values of 0.105 to 0.167. Particularly low levels of differentiations were observed between BSCB1 and BSTL, BS18 and BS28, BS18 and BS30, and BS28 and BS30 (FST < 0.007). Except for above, the other pairwise populations revealed medium differentiations (0.016-0.081). The gene flow (Nm) between populations could reflect changes of genetic diversity within and among populations. In this study, a maximum Nm value was detected between two RYD-type BS18 and BS30 (124.75), followed by pairwise populations of BS18 vs. BS28, BS30 vs. BS28, and BSCB1 vs. BSTL (35.4662.25). Surprisingly, gene flows between BSSS and the other two RYD-type populations BS18 and BS30 were quite small (2.92-4.47). In contrast, medium gene flows between BSSS and other non-RYD type germplasms BSCB1, BSL and BSTL were observed (10.62-15.38), equivalent to that between the temper-
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ate BS18 and tropical BS29 (11.66). Except for BS18 and BS30, small amounts of gene flows between other temperate populations and adapted exotic populations were expectedly detected, particularly, between the tropical BS16 and temperate BS9 (1.06-6.33). Among the temperate populations, less gene flow occurred in pairwise populations of BSSS, BSCB1, BSL and BSTL vs. BS9, BS18 and BS30.
Multivariate analysis of populations Based on estimates of genetic differentiation (FST), eight populations clustered as two separate groups, and the other two populations BS9 and BS16 remained ungrouped (Fig.2). Expectedly, two RYD-type populations BS18 and BS30 were clustered together. However, both were clustered with two exotic populations BS28 and BS29, in particular, tightly with BS28. Unexpectedly, BSSS was not clustered with the other two RYD-type populations BS18 and BS30, but with BSL. In addition, both BSSS and BSL were largely grouped with BSCB1 and BSTL. Principal component analysis revealed that the tropical BS16 and the temperate BS9 diverged in the opposite direction, and both were separated from the other populations (Fig.2). The tropical BS28 was positioned adjacent to the temperate RYD-type BS18 and BS30. These three populations were tightly grouped together, and located far from BSSS, BSL, BSCB1, and BSTL. With respect to typical temperate populations, BSSS and BSL were divergent from BSCB1 and BSTL, but these four populations still largely grouped together in cluster analysis. A remarkable observation here was that the tropical BS29 was divergent from all other temperate and tropical populations in a reverse direction.
Table 4 Genetic differentiation (FST) (below diagonal) and gene flow (Nm) (above diagonal) between populations over all loci Temperate
Population Temperate
Adapted exotic
BSSS BSCB1 BSL BSTL BS9 BS18 BS30 BS16 BS28 BS29
Adapted exotic
BSSS
BSCB1
BSL
BSTL
BS9
BS18
BS30
0.023 0.016 0.017 0.155 0.053 0.079 0.056 0.076 0.040
10.62 0.032 0.007 0.074 0.037 0.056 0.134 0.043 0.065
15.38 7.56 0.042 0.167 0.075 0.101 0.150 0.105 0.084
14.46 35.46 5.70 0.081 0.028 0.041 0.130 0.038 0.039
1.36 3.13 1.25 2.84 0.060 0.058 0.191 0.045 0.135
4.47 6.51 3.08 8.68 3.92 0.002 0.108 0.004 0.021
2.92 4.21 2.23 5.85 4.06 124.75 0.116 0.005 0.032
BS16 4.21 1.62 1.42 1.67 1.06 2.07 1.91 0.117 0.103
BS28
BS29
3.04 5.56 2.13 6.33 5.31 62.25 49.75 1.89 0.038
6.00 3.60 2.73 6.16 1.60 11.66 7.56 2.18 6.33 -
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ZHENG Da-hao et al. BS9 0.17
BSCB1 BS16 BSTL
PC3 (17.6%)
0.08
-0.01
BSL BSSS
BS28
) 0.21 .8% (31 0.08 -0.11 2 PC -0.06 -0.19 -0.20 -0.32 -0.31 -0.18 -0.04 PC1 (46.2 %)
BS18 BS30
BS29
0.09
0.23
Fig. 2 Associations among populations revealed by principal component analysis (PCA) based on the genetic differentiation estimates for pairwise populations. PC1, PC2, and PC3 were the first three principal components which together accounted for 95.6% of total variation. The dashed ellipses showed the clustering of the groups on the basis of the unweighted pair group method algorithm (UPGMA).
DISCUSSION Genetic variability within and among populations The heterozygosity is a function of both allele numbers and allele frequencies (Lu et al. 2002), which reflects the genetic differentiation and gene flow between populations (Templeton 2006). In particular, heterozygote excess or deficiency and mating feature for a population can be estimated via the inbreeding coefficient based on allele frequency (Gillespie 1998; Templeton 2006). In this study, a low allelic diversity was detected, but mean Ho and He values for each population and the gene diversity (HT) for the total population were higher than those in previous reports for unselected races of Mexican maize (Doebley et al. 1985), Chinese southwestern landraces (Lu et al. 2002) and Brazilian races (Gimenes and Lopes 2000). Although a limited number of allozyme loci were used here, it seems rather unlikely that a further increase in number of allozyme loci would appreciably improve the allelic richness at a single allozyme locus, because such allozyme loci are not related to the allelic diversity at other loci. So, the present results probably implied that these breeding populations still maintained greater heterozygosities than unselected races, although experienced cycles of the improvement for
agronomic traits. In addition, more genetic diversity was partitioned within advanced-cycle breeding populations (> 93%) than within unselected races, such as Brazilian races (> 83%) (Gimenes and Lopes 2000), Mexican germplasm (> 72%) (Doebley et al. 1985), conifers (> 90%) (Dancik and Yeh 1983), and Chinese southwestern landraces (77%) (Lu et al. 2002). Similar to the previous report by Gimenes and Lopes (2000), no private allele was particularly found in temperate or adapted exotic populations in this study; nevertheless, some alleles were detected just in a few populations at particular allozyme loci with very high frequencies. The populations examined had become homogeneous or highly inbred at particular loci, and exhibited a tendency of losing heterozygotes. Among the populations, only the tropical BS16 and temperate BS18 had all alleles detected. In practice, maize breeding populations are handpollinated with bulk pollen. And, limited numbers of individuals are chosen to be used to re-synthesize a subsequent population. This commonly results in a non-panmictic population, evidenced by present results that 58.9% of allele frequencies were significantly departure from Hardy-Weinberg (HW) equilibrium in most populations (P < 0.05), in particular, all the populations examined had negative FIS values (-0.091 - -0.424). Therefore, loss of heterozygotes and low allelic variability for a population much more likely resulted from a recent founding of a population from a small number of individuals, a recent reduction in the population size (Doebley et al. 1983; Heywood and Fleming 1986). Although current breeding populations are results of improvements for agronomic traits as well as resistances to biotic and abiotic stresses with an increase of the frequency of favorable alleles within populations, the reduction of gene diversity is undesirable. In order to avoid further losing of genetic diversity, our results recommended that future efforts on the breeding populations should be directed at sampling a great number of individuals during the subsequent improvement by recurrent selection. Besides, primal exotic open-pollinated populations should be grown in areas of origin to preserve the maximum genetic variation for future use in temperate maize breeding programs, as suggested by Kahler et al. (1986).
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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
Low level of differentiation and gene flow among populations Genetic differentiation could be expressed as the genetic variability between populations (Templeton 2006), and reflects the genetic distance between populations (Reynolds et al. 1983). Substantial genetic differences among the breeding populations were confirmed by large discrepancies in FST values for pairwise populations. In this study, the maximum FST value was 95.5 times as great as the minimum value. However, the maximum FST value only accounted for 19.1% of the total gene diversity (Table 4). Low level of among-population differentiations was also found in the previous reports, and small FST value between populations was probably due to limited isozyme systems used and/or a weak power of genetic differentiation of allozymes (Zhang et al. 1993; Pogson et al. 1995; Lu et al. 2002; Pressoir and Berthaud 2004). Nevertheless, Dubreuil and Charcosset (1998) observed that allozyme and RFLP markers had a similar relative magnitude of genetic differentiation. In particular, there is a general tendency for outcrossing plant species to show little or no differentiation among populations, whereas the populations from different races were more similar to each other than populations from the same race (Gottlieb 1974; Hamrick and Loveless 1986; Gimenes and Lopes 2000). Gene flow increases genetic variability within a population, but decreases genetic variability between populations (Templeton 2006). In this study, values of genetic differentiation among populations based on allozymatic data were small, but it was extremely influenced by gene flow between populations (Fig.3). While the gene flow increased from 1.06 to 5.31, FST value between populations sharply decreased from 0.191 to 0.053, hereafter no longer changed obviously. This indicated that even a small amount of gene flow could cause a sharp decrease of genetic differentiation and reduce genetic variability between populations.
927
used in temperate maize breeding programs (Fig.2). Reid Yellow Dent (RYD), LSC, Leaming, Minnesota 13 and Krug are the populations having been widely used in the US maize breeding program (Troyer 1999); in particular, RYD and LSC are the most important germplasms in worldwide maize breeding, as well as in China (Liu 1992; Wang et al. 1998; Zheng et al. 2002). In modern temperate maize breeding programs, BSSS is a major source of RYD germplasm (Hallauer et al. 1988; Hagdorn et al. 2003). Numerous previous studies grouped the inbreds with genetic backgrounds of RYD, LSC and BSCB1 in separate heterotic groups (Wang et al. 1998; Hagdorn et al. 2003; Zheng et al. 2008). PCA revealed that BSSS diverged obviously from BSL and BSCB1, although these populations were still largely grouped together at the population level in clustering. Unexpectedly, BSSS exhibited much greater divergences from the other two RYD-type BS18 and BS30 than from non-RYD type BSL, BSCB1 and BSTL, whereas BS18 and BS30 showed closer genetic relations each other (Fig.2). In this study, small amounts of gene flow and genetic differentiation were obtained between BSSS and the other RYD-type populations BS18 and BS30 (Table 4). The great divergence and small amounts of gene flow between BSSS and the other RYD-type BS18 and BS30 were all probably due to the fact that BSSS contained six non-RYD germplasms in its background (Hagdorn et al. 2003). The separation of the population BSTL from the typical LSC (BSL) was in agreement with the fact that BSTL is a strain of LSC contained 1/4 exotic germplasm of
Relationships among base breeding populations Principal component and cluster analyses based on allozymatic data in this study demonstrated that a great genetic variability is present among the breeding populations examined; however, restricted germplasms are
Fig. 3 Associations between genetic differentiation (FST) and gene flow (Nm) for pairwise populations based on allozymatic data.
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928
Mexican race and Tuxpeño in its background (Hallauer 1989). In consideration of the dispersion of RYD and LSC populations in Fig.2, potential tropical-subtropical × temperate heterotic patterns could be inferred from the multivariate analyses based on genetic differentiations. Among these, Suwan-1 × temperate maize pattern has been accepted by Chinese maize breeding activities (Wu et al. 2003; Fan et al. 2008). In PCA, Suwan-1 (BS29) greatly and reversely diverged from other temperate and adapted exotic germplasms, whereas Tuxpeño (BS28) slightly diverged from Krug (BS18) and Iodent (BS30). The divergence of BS16 from BS28 was in consistence with previous studies and breeding practices that ETO (BS16) had strong heterosis over Tuxpeño (BS28) germplasm, and ETO × Tuxpeño was identified as the most important heterotic pattern in the tropical maize breeding program (Hallauer et al. 1988). It was worthy to note that, besides Suwan-1 (BS29), the tropical BS16 (ETO) and temperate BS9 were distinctly divergent from all other breeding germplasms, and consequently, these two populations would be analogically postulated as potential germplasms to establish new temperate × temperate and temperate × tropical heterotic patterns for temperate maize breeding programs. In summary, the representative temperate and adapted exotic breeding populations examined exhibited low allelic richness, and were deficient in heterozygotes at particular loci, implying a tendency of losing the allelic diversity within populations. Nonetheless, these breeding populations still maintained high heterozygosity within populations with negative Wright’s fixation indexes. Among the populations, the temperate BS9 and tropical BS16 (ETO) were highly differentiated from other temperate and adapted exotic populations. And, the tropical BS29 (Suwan-1) diverged from others in the reverse direction in PCA. Despite insufficient allozyme loci used, this study will be still helpful in understanding the current status of the genetic variability of these recurrently improved breeding populations, and establishing the strategy how to construct a new synthetic and preserve sufficient genetic variation promising for further recycling improvements and re-synthesize subsequent populations. Overall, a successful establishment of a base population available
ZHENG Da-hao et al.
to exploit a new heterotic group is based on that the progenitors used to form the synthetic populations with a large variation are broadly based. And, only using sufficient elite individuals and/or lines derived from each population as parents can promise to capture enough genetic variation for recycling breeding programs.
Acknowledgements We truly appreciate Dr. K R Lamkey at Iowa State University, USA, for providing the base populations of maize. This work was supported by Jilin Provincial Science and Technology Department, China ( 20000565).
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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.) ZHENG Da-hao1, YU Yang2, WANG Zhen-ping3 and LI Yan-ru1 Agricultural College, Yanbian University, Longjing 133400, P.R.China Crop Tillage and Cultivation Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, P.R.China 3 Tonghua Academy of Agricultural Sciences, Meihekou 135000, P.R.China 1 2
Abstract Ten temperate and adapted exotic breeding populations of maize were studied with electrophoretic techniques. Three isozyme systems coded by nine allozyme loci were used for evaluating the genetic variability within and among populations. The results revealed that 78.57% of allozyme loci were polymorphic. Low allelic variation with a mean number of 1.84 alleles per locus per population was detected. But, these populations still maintained higher level of heterozygosity; moreover, the exotic populations had greater gene diversity than the temperate populations. All the populations were non-panmictic with negative Wright’s fixation indexes (-0.091- -0.424). The tropical BS16 was typified by maximum allelic richness, percent of polymorphic loci and heterozygosity. More than 93% of the gene diversity maintained within populations, and the genetic differentiation among populations was low (0.002-0.191). Multivariate analysis demonstrated that the tropical BS29 diverged from other populations in the reverse direction. The temperate BS9 and tropical BS16 were divergent each other, and highly differentiated from other temperate and tropical populations, consequently, these two populations would be analogically postulated as potential germplasms to establish new heterotic groups for temperate maize breeding programs. Key words: maize, breeding population, allozyme, diversity structure, multivariate analysis
INTRODUCTION Abundant genetic variation among and within maize germplasms is indispensable for the recognition and exploitation of heterotic patterns among germplasm pools. In addition, for the base and synthetic populations, the breeding values rely on the genetic diversity within initial populations constructed, and how much genetic variation is conserved within their derived populations after cycles of recurrent selection. Allozyme assay is a useful tool to detect genetic variation. Isozyme data have been used in preparation of keys for classifying
maize inbreds and populations, determining allele frequency and heterozygosity/homozygosity at different allozyme loci, identifying rare alleles, accounting for residual variability in developed inbreds. Numerous studies based on isozyme electrophoresis were conducted on identification of cultivars and parental lines (Cardy and Kannenberg 1982; Sanou et al. 1997), analysis of genetic variability among parental lines, cultivars, populations and races (Stuber et al. 1980; Goodman and Stuber 1983; Doebley et al. 1983, 1985; Smith et al. 1985; Kahler et al. 1986; Salanoubat and Pernes 1986; Pflüger and Schlatter 1996). The simplicity of the isozyme assays together with its rapid and lower cost
Received 11 March, 2009 Accepted 24 April, 2009 Correspondence ZHENG Da-hao, Professor, Tel/Fax: +86-433-3261794, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60296-5
Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
still guarantees its use today (Gimenes and Lopes 2000; Mauria et al. 2000; Sanchez et al. 2000; Liu et al. 2001; Lu et al. 2002). In particular, as a functional description of gene expressions, isozymatic data still offer advantages compensating for shortcomings of unselected non-functional DNA markers, such as random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), single nucleotide polymorphism (SNP), etc. The information about allozymatic variability has practical implications for germplasm preservation, and provides an efficient approach for directly comparing the magnitude and distribution of genetic diversity between different populations and species (Lu et al. 2002; Pinto et al. 2003). Zea mays L. contains more than 200 races (Gimenes and Lopes 2000), but only a few have been utilized in maize breeding programs, in particular, not all usable races have practical values in maize hybrid breeding. In addition, the excessive dependence on inbred recycling approach based on limited germplasm sources resulted in a limited genetic diversity within temperate maize germplasm (Darrah and Zuber 1985; Lu and Bernardo 2001; Zheng et al. 2008). In order to overcome the consequent germplasm bottleneck having become a restrictive factor in temperate maize breeding activities (Hallauer 1989), many works have focused on using tropical and subtropical germplasms in temperate hybrid breeding programs. However, because of the deficiency of adaptability in temperate regions, exotic germplasms are commonly crossed with temperate maize to develop new inbreds. Recently, domesticating exotic populations prior to their direct utilizations in temperate maize breeding programs is of importance in practice. And, some important exotic populations have been domesticated for temperate regions. Compared with non-adapted tropical and subtropical germplasms, the adapted exotic populations must have greater practical values in their utilizations in temperate maize breeding programs. The objectives of the this study were to evaluate, at the isozyme level, (1) the genetic variability of temperate and adapted exotic breeding populations of historical and potential importance in developing maize inbreds, (2) the population structure and, (3) the genetic differentiation and gene flow between populations.
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MATERIALS AND METHODS Plant materials Plants from ten open-pollinated breeding populations of maize were analyzed, including adapted exotic materials adapted to the temperate region. All the materials were provided by Dr. K R Lamkey at Iowa State University, USA, in 1999. These populations were maintained in the Maize Research Laboratory at Agricultural College of Yanbian University, China, of which six, Iowa Stiff Stalk Synthetic [BSSS (R) C12], Iowa Corn Borer Synthetic No. 1 [BSCB1 (R) C12], open-pollinated Lancaster Surecrop [BSL (S) C7, LSC], Krug Yellow Dent (BS18, Krug), Iodent (BS30), and the broad-based synthetic BS9 (CB) C5, were typical maize populations of historical or potential importance in the development of inbreds in the Corn Belt. Three, BS16 (CB) C4 (ETO), BS28 (C6) (Tuxpeño), and BS29 (Suwan-1), were the adapted exotic populations. One was BSTL (S) C5, a strain of Lancaster Surecrop (LSC) containing 1/4 of exotic germplasm of the Mexican race and Tuxpeño.
Isozyme electrophoretic analysis About 150 individuals for each of ten populations (one kernel per ear of each individual) were grown at 28°C in the daytime and 25°C in the night for 40-60 h. Approximately 300 mg of coleoptile tissue were collected from each individual, finely homogenized at 0-4°C in 0.6 mL of 0.1 mol L-1 Tris-HCl (pH 8.0), then, centrifuged at 10 000 r/min. The supernatant fluid was mixed with 200 L of 50% glycerol. Enzyme extraction, electrophoresis, gel staining, scoring as well as genotyping of alleles for each locus were performed according to the methods described by Stuber et al. (1988). Finally, 100 individuals from each of populations were genotyped at nine allozyme loci of three enzyme systems, esterase (Est) (EC 3.1.1.1), peroxidase (Per) (EC 1.11.1.7) and α-amylase (Amy) (EC 3.2.1.1).
Data analysis The genetic diversity and structure of populations were
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71.43 85.71 71.43 71.43 71.43 85.71 71.43 100.00 71.43 85.71 78.57 1.71 1.86 1.71 1.71 1.86 2.00 1.71 2.14 1.71 2.00 1.84 0.051 0.052
0.035
0.070
Total
12 13 12 12 13 14 12 15 12 14 12.90 0.740 0.756 0.665 0.690 0.660 0.625 0.531 0.645 0.668 0.625 0.661
B A
0.260 0.244 0.335 0.310 0.340 0.375 0.469 0.355 0.332 0.375 0.340 0.607 0.630 0.565 0.735 0.755 0.725 0.699 0.595 0.675 0.611 0.660
B A
0.393 0.370 0.435 0.265 0.245 0.275 0.301 0.405 0.325 0.389 0.340 0.165 0.215 0.250 0.055 0.425 0.460 0.510 0.420 0.530 0.424 0.345
C B
0.448 0.667 0.625 0.480 0.955 0.580 0.615 0.380 0.650 0.385 0.579
A
B
0.835 0.785 0.750 0.945 0.505 0.540 0.490 0.545 0.470 0.525 0.639
Number of alleles
Amy1 Per5 Per3
0.552 0.333 0.375 0.520 0.045 0.420 0.385 0.620 0.350 0.615 0.422
A B
1.000 0.975 1.000 1.000 1.000 0.975 1.000 0.855 1.000 0.975 0.978 0.025 0.055
0.145
0.025
0.025
A B
1.000 1.000 1.000 1.000 1.000 0.995 1.000 0.500 1.000 1.000 0.950 0.253
0.500
0.005
A
0.582 0.505 0.600 0.484 0.551 0.550 0.520 0.379 0.505 0.645 0.532
B A
0.418 0.495 0.400 0.516 0.449 0.450 0.480 0.621 0.495 0.355 0.468 Mean
Adapted exotic
BSSS BSCB1 BSTL BSL BS9 BS18 BS30 BS16 BS28 BS29 Temperate
Frequency of alleles detected at allozyme locus
Est8 Est4 Est3
One hundred individuals for each of ten populations were analyzed at three isozyme systems. Among the isozyme systems, the α-amylase (Amy) system revealed monolocus polymorphism, i.e., only one locus was identified in this system, whereas the esterase (Est) and peroxidase (Per) systems showed multiple loci polymorphism, i.e., two or more loci were identified in each isozyme system. Across all populations, nine allozyme loci were identified, but only seven loci were polymorphic (Table 1). Among the populations, BS16, BS29, BS18, BSCB1, and BS18 exhibited more number of alleles and higher percentage of polymorphic loci than BSSS, BSTL, BSL, BS28 and BS30. Compared to temperate populations, the adapted exotic populations had slightly greater mean number of alleles and higher percentage of polymorphic loci. These alleles existed at quite different frequencies among populations (Table 1), and 80.6% of alleles were at the frequencies of 0.05 to 0.95. In this
Est2
Allozymatic polymorphism
Population
RESULTS
Table 1 Frequency of alleles detected at seven polymorphic loci and the percentage of polymorphic loci (%) in ten populations
assessed based on mean number of alleles per locus, the percentage of polymorphic loci, the observed heterozygosity (Ho), the expected heterozygosity (He) (Nei 1987), and inbreeding coefficient [f = 1 - (Ho/He)] (Wright 1978). Wright’s fixation index (FIS) was estimated to quantify the deficiency and excess of heterozygotes for each population and each locus (Wright 1978). The gene diversity in the total population (HT) was partitioned into gene diversity within (HS) and between (DST) populations (HT = HS + DST), and the proportion of total gene diversity partitioned between populations was estimated as GST = DST/HT (Nei 1987). The genetic differentiation between populations (FST) was estimated according to Weir and Cockerham (1984), and the gene flow was given by Nm = 0.25 × (1 - FST)/FST (Cockerham and Weir 1993). Based on FST, principal component analysis (PCA) and cluster analysis with the unweighted pair-group method algorithm (UPGMA) were performed. The data were analyzed using the programs TFPGA 1.3 (Miller 1997) and FSTAT 293 (Goudet 1995), except PCA performed with NTSYS 2.10 software (Rohlf and Slice 1992).
ZHENG Da-hao et al. Percentage of polymorphic loci (%) Mean
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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
study, some alleles occurred in only a few populations, e.g., allele Est3-A was detected in population BS16 and BS18, Est4-A in BSCB1, BS16, BS18, and BS29, Per3A in BS9, BS6, and BS29. Nevertheless, these alleles existed at very low frequencies of 0.005 to 0.145. Among the populations, the adapted exotic BS16 was typified by a maximum allelic richness (2.14 alleles on average) with all the alleles detected and percentage of polymorphic loci (100%), indicating that BS16 had the most abundant allozymatic variation.
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The observed heterozygosity (Ho) and expected heterozygosity (He) The heterozygosity could reflect the gene diversity for a population. Across all polymorphic loci, Ho and He values for each population varied from 0.310 and 0.276 for BSL to 0.550 and 0.453 for BS16, and averaged 0.409 and 0.333, respectively (Table 2). The differences in Ho and He values were significant among loci within each population and among populations at most of loci
Table 2 The observed heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient ( f ) at allozyme loci, and Wright’s fixation index (FIS) over all loci for the temperate and adapted exotic breeding populations Temperate populations Est2
Est3
Est4
Est8
Per3
Per-5
Amy1
Over all loci
Ho He f Ho He f Ho He f Ho He f Ho He f Ho He f Ho He f Ho He FIS
Adapted exotic populations
BSSSR
BSCB1
BSTL
BSL
BS9
BS18
BS30
BS16
BS28
BS29
0.835 0.489 -0.714 0.000 0.000 NA 0.000 0.000 NA 0.636 0.498 -0.281 0.330 0.277 -0.193 0.704 0.479 -0.472 0.520 0.387 -0.347 0.432 * 0.304 * -0.424
0.439 0.503 0.127 0.000 0.000 NA 0.050 0.049 -0.021 0.479 0.447 -0.073 0.430 0.339 -0.269 0.740 0.469 -0.584 0.488 0.371 -0.317 0.375 ** 0.311 ** -0.207
0.560 0.482 -0.162 0.000 0.000 NA 0.000 0.000 NA 0.350 0.471 0.258 0.500 0.377 -0.329 0.530 0.494 -0.073 0.650 0.448 -0.455 0.370 * 0.325 ** -0.141
0.448 0.502 0.108 0.000 0.000 NA 0.000 0.000 NA 0.500 0.502 0.003 0.110 0.104 -0.053 0.530 0.392 -0.356 0.580 0.430 -0.351 0.310 * 0.276 * -0.124
0.778 0.497 -0.568 0.000 0.000 NA 0.000 0.000 NA 0.091 0.087 -0.043 0.720 0.562 -0.282 0.410 0.372 -0.103 0.460 0.451 -0.02 0.351 * 0.281 * -0.25
0.720 0.497 -0.451 0.010 0.010 0.000 0.050 0.049 -0.021 0.440 0.490 0.102 0.760 0.499 -0.526 0.510 0.401 -0.274 0.690 0.471 -0.468 0.454 ** 0.345 ** -0.318
0.500 0.502 0.003 0.000 0.000 NA 0.000 0.000 NA 0.230 0.476 0.518 0.796 0.502 -0.589 0.602 0.423 -0.426 0.649 0.501 -0.299 0.397 * 0.343 ** -0.156
0.552 0.474 -0.166 0.440 0.503 0.125 0.290 0.249 -0.165 0.340 0.474 0.283 0.770 0.528 -0.462 0.810 0.484 -0.678 0.650 0.460 -0.415 0.550 ** 0.453 ** -0.216
0.850 0.502 -0.698 0.000 0.000 NA 0.000 0.000 NA 0.440 0.457 0.038 0.657 0.501 -0.313 0.650 0.441 -0.478 0.663 0.446 -0.492 0.466 * 0.335 ** -0.392
0.490 0.460 -0.065 0.000 0.000 NA 0.050 0.049 -0.021 0.190 0.476 0.602 0.545 0.544 -0.002 0.717 0.478 -0.505 0.710 0.471 -0.511 0.386 * 0.354 ** -0.091
* ** , Significance at P < 0.05 and 0.01, respectively. NA indicated that the allele detected at a specific locus for a population had been homogeneous.
excluding Est3 and Est4 loci (P < 0.05) (Table 2 and Fig.1). In addition, adapted exotic populations exhibited higher heterozygosity with Ho of 0.407 and He of 0.331 than temperate populations with Ho of 0.384 and He of 0.312, but the differences in Ho and He were not significant (P > 0.05). Over all loci, all the populations exhibited heterozygote excess with negative Wright’s fixation index (FIS) values of -0.424 for BSSS to -0.091 for BS29, indicating that these breeding populations were not panmictic. In this study, inbreeding coefficient (f) values varied from -0.714 at Est2 for BSSS to 0.602 at Est8 for
Fig. 1 Dendrogram for the observed heterozygosity (Ho), the expected heterozygosity (He) and the Wright’s fixation index (FIS) at each allozyme locus over all populations. ** indicates significance at P < 0.01 for the differences among populations.
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ZHENG Da-hao et al.
tal population. In contrast, heterozygote deficiencies were occurred in the total population at both Est3 and Est8 loci, evidenced by positive Wright’s fixation indexes of 0.526 at Est2 locus and 0.255 at Est8 locus.
BS29 (Table 2). The positive f value implied that the homogeneity and/or heterozygote deficiencies occurred in particular populations and allozyme loci. For example, at Est3 and Est4 loci, the alleles in six out of ten populations, BSSS, BSTL, BSL, BS9, BS28, and BS30, had been homogeneous. In particular, BS29 and BS30 had been overly deficient in heterozygote at Est8 locus with great positive f values of 0.602 and 0.518, respectively. Among the populations, only BS16 and BS18 maintained all the alleles and heterozygotes at all loci examined, particularly the former. Nevertheless, BSSS and BS28 possessed much more heterozygotes with greater F IS values of -0.424 and 0.392, respectively, although both populations had been homogeneous at Est3 and Est4 loci.
Gene diversity within and among populations The gene diversity (HT) for the total population was 0.356 across all loci, and ranged from 0.043 at Est4 locus to 0.498 at Est2 locus (Table 3). The gene diversities within populations (HS) and among populations (DST) were 0.333 and 0.023, respectively. HS and DST values varied from 0.040 at Est4 to 0.490 at Est2 and from 0.003 at Est4 to 0.050 at Est8, respectively. In this study, the proportion of total gene diversity partitioned among populations (GST) was only about 6.5% over all loci, indicating that most of the genetic variation occurred within populations (> 93% over all loci). An exception was observed at Est3 locus with the greatest GST value of 0.427 (42.7%), probably due to that the alleles at this locus were detected in just two populations. Regarding the gene diversity for typical temperate and adapted exotic populations, most of gene diversities also resided within populations (Table 3). Over all loci, the proportions of the gene diversity occurred within populations were greater than 93% for both typical temperate and adapted exotic populations (GST 0.062). It was worth of mentioning that exotic germplasms had greater gene diversity than typical temperate germplasm. Here, the gene diversity in the total exotic population was 0.405, whereas it was 0.323 in the total temperate population. Furthermore, HT value for the total temperate population increased by 3.4%, when
Contribution of allozymatic diversities at various loci to overall heterozygosity The present results revealed a higher overall mean Ho value of 0.410 than Ho value of 0.359 across all populations and all loci, and subsequent negative overall mean FIS value of -0.141. However, FIS value at each locus over all populations varied from -0.381 to 0.526, indicating that each allozyme locus had a different contribution to the genetic diversity for each populations (Fig.1). Among the three isozyme systems tested, the Est isozyme system exhibited a great variability. But, high inbreeding and/or loss of alleles were observed at Est3 and Est4 loci. Over all populations, higher Ho than He were observed at Est2, Est4, Per3, Per5, and Amy1 loci, with negative FIS values of -0.381 to -0.022, reflecting heterozygote excesses at these loci in the to-
Table 3 Nei’s estimates of the gene diversity for the total population as well as the temperate and adapted exotic populations Locus Est2 Est3 Est4 Est8 Per3 Per5 Amy1 Overall
HT
HS
DST
GST
Whole
Tem1
Tem
TS
Whole
Tem1
Tem
TS
Whole
Tem1
Tem
TS
Whole
Tem1
Tem
TS
0.498 0.096 0.043 0.488 0.472 0.449 0.449 0.356
0.496 0.002 0.017 0.469 0.404 0.443 0.429 0.323
0.499 0.000 0.012 0.463 0.365 0.434 0.411 0.312
0.500 0.278 0.107 0.499 0.526 0.468 0.458 0.405
0.490 0.055 0.040 0.438 0.423 0.442 0.443 0.333
0.494 0.002 0.016 0.416 0.359 0.434 0.426 0.307
0.497 0.000 0.012 0.383 0.320 0.427 0.409 0.293
0.478 0.168 0.099 0.470 0.524 0.466 0.458 0.380
0.008 0.041 0.003 0.050 0.049 0.007 0.006 0.023
0.002 0.000 0.000 0.053 0.044 0.009 0.003 0.016
0.002 0.000 0.000 0.080 0.044 0.007 0.002 0.019
0.022 0.11 0.008 0.029 0.002 0.002 0.000 0.025
0.016 0.427 0.070 0.102 0.104 0.016 0.013 0.065
0.004 0.000 0.000 0.113 0.109 0.020 0.007 0.050
0.004 0.000 0.173 0.121 0.016 0.005 0.061
0.044 0.396 0.075 0.058 0.004 0.004 0.000 0.062
H T, HS, DST , and GST indicated that the gene diversity in the total populations, within populations and among populations, and the proportion of total gene diversity partitioned among populations, respectively. Whole, Tem1, Tem, and TS indicated that the total population, the temperate populations including BSTL contained 1/4 of Mexican race and Tuxpeño germplasms, the typical temperate populations (BSSS, BSCB1, BS9, BS18, and BS30), and the exotic populations (BS16, BS28, and BS29), respectively.
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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
BSTL containing 1/4 of tropical Mexican race and Tuxpeño germplasms was included.
Genetic differentiation and gene flow among populations Estimates of the genetic differentiation (FST) and gene flow (Nm) for pairwise populations over all allozyme loci were presented in Table 4. FST values for pairwise populations varied from 0.002 for BS18 vs. BS30 to 0.191 for BS9 vs. BS16, and averaged 0.068. The tropical BS16 was highly differentiated from other populations with FST values of 0.103 to 0.191 excluding BSSS. The temperate synthetic BS9 was particularly and highly divergent from BSSS, BSL, BS16, and BS29 with great FST values of 0.135 to 0.191, whereas BSL was particularly differentiated from BS9, BS16, BS28 and BS30 with FST values of 0.105 to 0.167. Particularly low levels of differentiations were observed between BSCB1 and BSTL, BS18 and BS28, BS18 and BS30, and BS28 and BS30 (FST < 0.007). Except for above, the other pairwise populations revealed medium differentiations (0.016-0.081). The gene flow (Nm) between populations could reflect changes of genetic diversity within and among populations. In this study, a maximum Nm value was detected between two RYD-type BS18 and BS30 (124.75), followed by pairwise populations of BS18 vs. BS28, BS30 vs. BS28, and BSCB1 vs. BSTL (35.4662.25). Surprisingly, gene flows between BSSS and the other two RYD-type populations BS18 and BS30 were quite small (2.92-4.47). In contrast, medium gene flows between BSSS and other non-RYD type germplasms BSCB1, BSL and BSTL were observed (10.62-15.38), equivalent to that between the temper-
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ate BS18 and tropical BS29 (11.66). Except for BS18 and BS30, small amounts of gene flows between other temperate populations and adapted exotic populations were expectedly detected, particularly, between the tropical BS16 and temperate BS9 (1.06-6.33). Among the temperate populations, less gene flow occurred in pairwise populations of BSSS, BSCB1, BSL and BSTL vs. BS9, BS18 and BS30.
Multivariate analysis of populations Based on estimates of genetic differentiation (FST), eight populations clustered as two separate groups, and the other two populations BS9 and BS16 remained ungrouped (Fig.2). Expectedly, two RYD-type populations BS18 and BS30 were clustered together. However, both were clustered with two exotic populations BS28 and BS29, in particular, tightly with BS28. Unexpectedly, BSSS was not clustered with the other two RYD-type populations BS18 and BS30, but with BSL. In addition, both BSSS and BSL were largely grouped with BSCB1 and BSTL. Principal component analysis revealed that the tropical BS16 and the temperate BS9 diverged in the opposite direction, and both were separated from the other populations (Fig.2). The tropical BS28 was positioned adjacent to the temperate RYD-type BS18 and BS30. These three populations were tightly grouped together, and located far from BSSS, BSL, BSCB1, and BSTL. With respect to typical temperate populations, BSSS and BSL were divergent from BSCB1 and BSTL, but these four populations still largely grouped together in cluster analysis. A remarkable observation here was that the tropical BS29 was divergent from all other temperate and tropical populations in a reverse direction.
Table 4 Genetic differentiation (FST) (below diagonal) and gene flow (Nm) (above diagonal) between populations over all loci Temperate
Population Temperate
Adapted exotic
BSSS BSCB1 BSL BSTL BS9 BS18 BS30 BS16 BS28 BS29
Adapted exotic
BSSS
BSCB1
BSL
BSTL
BS9
BS18
BS30
0.023 0.016 0.017 0.155 0.053 0.079 0.056 0.076 0.040
10.62 0.032 0.007 0.074 0.037 0.056 0.134 0.043 0.065
15.38 7.56 0.042 0.167 0.075 0.101 0.150 0.105 0.084
14.46 35.46 5.70 0.081 0.028 0.041 0.130 0.038 0.039
1.36 3.13 1.25 2.84 0.060 0.058 0.191 0.045 0.135
4.47 6.51 3.08 8.68 3.92 0.002 0.108 0.004 0.021
2.92 4.21 2.23 5.85 4.06 124.75 0.116 0.005 0.032
BS16 4.21 1.62 1.42 1.67 1.06 2.07 1.91 0.117 0.103
BS28
BS29
3.04 5.56 2.13 6.33 5.31 62.25 49.75 1.89 0.038
6.00 3.60 2.73 6.16 1.60 11.66 7.56 2.18 6.33 -
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ZHENG Da-hao et al. BS9 0.17
BSCB1 BS16 BSTL
PC3 (17.6%)
0.08
-0.01
BSL BSSS
BS28
) 0.21 .8% (31 0.08 -0.11 2 PC -0.06 -0.19 -0.20 -0.32 -0.31 -0.18 -0.04 PC1 (46.2 %)
BS18 BS30
BS29
0.09
0.23
Fig. 2 Associations among populations revealed by principal component analysis (PCA) based on the genetic differentiation estimates for pairwise populations. PC1, PC2, and PC3 were the first three principal components which together accounted for 95.6% of total variation. The dashed ellipses showed the clustering of the groups on the basis of the unweighted pair group method algorithm (UPGMA).
DISCUSSION Genetic variability within and among populations The heterozygosity is a function of both allele numbers and allele frequencies (Lu et al. 2002), which reflects the genetic differentiation and gene flow between populations (Templeton 2006). In particular, heterozygote excess or deficiency and mating feature for a population can be estimated via the inbreeding coefficient based on allele frequency (Gillespie 1998; Templeton 2006). In this study, a low allelic diversity was detected, but mean Ho and He values for each population and the gene diversity (HT) for the total population were higher than those in previous reports for unselected races of Mexican maize (Doebley et al. 1985), Chinese southwestern landraces (Lu et al. 2002) and Brazilian races (Gimenes and Lopes 2000). Although a limited number of allozyme loci were used here, it seems rather unlikely that a further increase in number of allozyme loci would appreciably improve the allelic richness at a single allozyme locus, because such allozyme loci are not related to the allelic diversity at other loci. So, the present results probably implied that these breeding populations still maintained greater heterozygosities than unselected races, although experienced cycles of the improvement for
agronomic traits. In addition, more genetic diversity was partitioned within advanced-cycle breeding populations (> 93%) than within unselected races, such as Brazilian races (> 83%) (Gimenes and Lopes 2000), Mexican germplasm (> 72%) (Doebley et al. 1985), conifers (> 90%) (Dancik and Yeh 1983), and Chinese southwestern landraces (77%) (Lu et al. 2002). Similar to the previous report by Gimenes and Lopes (2000), no private allele was particularly found in temperate or adapted exotic populations in this study; nevertheless, some alleles were detected just in a few populations at particular allozyme loci with very high frequencies. The populations examined had become homogeneous or highly inbred at particular loci, and exhibited a tendency of losing heterozygotes. Among the populations, only the tropical BS16 and temperate BS18 had all alleles detected. In practice, maize breeding populations are handpollinated with bulk pollen. And, limited numbers of individuals are chosen to be used to re-synthesize a subsequent population. This commonly results in a non-panmictic population, evidenced by present results that 58.9% of allele frequencies were significantly departure from Hardy-Weinberg (HW) equilibrium in most populations (P < 0.05), in particular, all the populations examined had negative FIS values (-0.091 - -0.424). Therefore, loss of heterozygotes and low allelic variability for a population much more likely resulted from a recent founding of a population from a small number of individuals, a recent reduction in the population size (Doebley et al. 1983; Heywood and Fleming 1986). Although current breeding populations are results of improvements for agronomic traits as well as resistances to biotic and abiotic stresses with an increase of the frequency of favorable alleles within populations, the reduction of gene diversity is undesirable. In order to avoid further losing of genetic diversity, our results recommended that future efforts on the breeding populations should be directed at sampling a great number of individuals during the subsequent improvement by recurrent selection. Besides, primal exotic open-pollinated populations should be grown in areas of origin to preserve the maximum genetic variation for future use in temperate maize breeding programs, as suggested by Kahler et al. (1986).
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Structure of Allozymatic Diversity of Ten Temperate and Adapted Exotic Breeding Populations of Maize (Zea mays L.)
Low level of differentiation and gene flow among populations Genetic differentiation could be expressed as the genetic variability between populations (Templeton 2006), and reflects the genetic distance between populations (Reynolds et al. 1983). Substantial genetic differences among the breeding populations were confirmed by large discrepancies in FST values for pairwise populations. In this study, the maximum FST value was 95.5 times as great as the minimum value. However, the maximum FST value only accounted for 19.1% of the total gene diversity (Table 4). Low level of among-population differentiations was also found in the previous reports, and small FST value between populations was probably due to limited isozyme systems used and/or a weak power of genetic differentiation of allozymes (Zhang et al. 1993; Pogson et al. 1995; Lu et al. 2002; Pressoir and Berthaud 2004). Nevertheless, Dubreuil and Charcosset (1998) observed that allozyme and RFLP markers had a similar relative magnitude of genetic differentiation. In particular, there is a general tendency for outcrossing plant species to show little or no differentiation among populations, whereas the populations from different races were more similar to each other than populations from the same race (Gottlieb 1974; Hamrick and Loveless 1986; Gimenes and Lopes 2000). Gene flow increases genetic variability within a population, but decreases genetic variability between populations (Templeton 2006). In this study, values of genetic differentiation among populations based on allozymatic data were small, but it was extremely influenced by gene flow between populations (Fig.3). While the gene flow increased from 1.06 to 5.31, FST value between populations sharply decreased from 0.191 to 0.053, hereafter no longer changed obviously. This indicated that even a small amount of gene flow could cause a sharp decrease of genetic differentiation and reduce genetic variability between populations.
927
used in temperate maize breeding programs (Fig.2). Reid Yellow Dent (RYD), LSC, Leaming, Minnesota 13 and Krug are the populations having been widely used in the US maize breeding program (Troyer 1999); in particular, RYD and LSC are the most important germplasms in worldwide maize breeding, as well as in China (Liu 1992; Wang et al. 1998; Zheng et al. 2002). In modern temperate maize breeding programs, BSSS is a major source of RYD germplasm (Hallauer et al. 1988; Hagdorn et al. 2003). Numerous previous studies grouped the inbreds with genetic backgrounds of RYD, LSC and BSCB1 in separate heterotic groups (Wang et al. 1998; Hagdorn et al. 2003; Zheng et al. 2008). PCA revealed that BSSS diverged obviously from BSL and BSCB1, although these populations were still largely grouped together at the population level in clustering. Unexpectedly, BSSS exhibited much greater divergences from the other two RYD-type BS18 and BS30 than from non-RYD type BSL, BSCB1 and BSTL, whereas BS18 and BS30 showed closer genetic relations each other (Fig.2). In this study, small amounts of gene flow and genetic differentiation were obtained between BSSS and the other RYD-type populations BS18 and BS30 (Table 4). The great divergence and small amounts of gene flow between BSSS and the other RYD-type BS18 and BS30 were all probably due to the fact that BSSS contained six non-RYD germplasms in its background (Hagdorn et al. 2003). The separation of the population BSTL from the typical LSC (BSL) was in agreement with the fact that BSTL is a strain of LSC contained 1/4 exotic germplasm of
Relationships among base breeding populations Principal component and cluster analyses based on allozymatic data in this study demonstrated that a great genetic variability is present among the breeding populations examined; however, restricted germplasms are
Fig. 3 Associations between genetic differentiation (FST) and gene flow (Nm) for pairwise populations based on allozymatic data.
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928
Mexican race and Tuxpeño in its background (Hallauer 1989). In consideration of the dispersion of RYD and LSC populations in Fig.2, potential tropical-subtropical × temperate heterotic patterns could be inferred from the multivariate analyses based on genetic differentiations. Among these, Suwan-1 × temperate maize pattern has been accepted by Chinese maize breeding activities (Wu et al. 2003; Fan et al. 2008). In PCA, Suwan-1 (BS29) greatly and reversely diverged from other temperate and adapted exotic germplasms, whereas Tuxpeño (BS28) slightly diverged from Krug (BS18) and Iodent (BS30). The divergence of BS16 from BS28 was in consistence with previous studies and breeding practices that ETO (BS16) had strong heterosis over Tuxpeño (BS28) germplasm, and ETO × Tuxpeño was identified as the most important heterotic pattern in the tropical maize breeding program (Hallauer et al. 1988). It was worthy to note that, besides Suwan-1 (BS29), the tropical BS16 (ETO) and temperate BS9 were distinctly divergent from all other breeding germplasms, and consequently, these two populations would be analogically postulated as potential germplasms to establish new temperate × temperate and temperate × tropical heterotic patterns for temperate maize breeding programs. In summary, the representative temperate and adapted exotic breeding populations examined exhibited low allelic richness, and were deficient in heterozygotes at particular loci, implying a tendency of losing the allelic diversity within populations. Nonetheless, these breeding populations still maintained high heterozygosity within populations with negative Wright’s fixation indexes. Among the populations, the temperate BS9 and tropical BS16 (ETO) were highly differentiated from other temperate and adapted exotic populations. And, the tropical BS29 (Suwan-1) diverged from others in the reverse direction in PCA. Despite insufficient allozyme loci used, this study will be still helpful in understanding the current status of the genetic variability of these recurrently improved breeding populations, and establishing the strategy how to construct a new synthetic and preserve sufficient genetic variation promising for further recycling improvements and re-synthesize subsequent populations. Overall, a successful establishment of a base population available
ZHENG Da-hao et al.
to exploit a new heterotic group is based on that the progenitors used to form the synthetic populations with a large variation are broadly based. And, only using sufficient elite individuals and/or lines derived from each population as parents can promise to capture enough genetic variation for recycling breeding programs.
Acknowledgements We truly appreciate Dr. K R Lamkey at Iowa State University, USA, for providing the base populations of maize. This work was supported by Jilin Provincial Science and Technology Department, China ( 20000565).
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