Genetic diversity in South African maize (Zea mays L.) genotypes as determined with microsatellite markers

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1 African Journal of Biotechnology Vol. 12(2), pp , 9 January, 2013 Available online at DOI: /AJB ISSN Academic Journals Full Length Research Paper Genetic diversity in South African maize (Zea mays L.) genotypes as determined with microsatellite markers Mienie, CMS 1 * and Fourie, AP 2 1 North West University, Private Bag X6001, Potchefstroom 2520, South Africa. 2 Agricultural Research Council-Grain Crops Institute, Private Bag X1251, Potchefstroom 2520, South Africa. Accepted 27 October, 2012 One thousand and forty three (1043) maize genotypes including white and yellow maize inbred lines as well as hybrids from the public germplasm collection were characterized with 80 microsatellite markers distributed throughout the genome. A total of 1874 alleles were amplified and used in the genetic diversity analysis. Principal coordinate analysis confirmed the geographical distribution of the breeding lines. Cluster analysis using Rogers distance measures placed the breeding lines in several clusters and corresponded well with known pedigrees. Lines with mixed origin were classified in separate clusters and duplicate entries in the collection were identified. These mixed lines could not be placed in known heterotic groups, but could rather be used to identify new groups to be used in the breeding program. The genetic distances determined in the study can be applied to plan a more focused breeding program. Keywords: Genetic distances, maize, microsatellites. INTRODUCTION Maize is one of the most widely grown crops worldwide and certainly an important staple food in the African diet. It is used as human food, animal feed and provides an important source of income and employment for a large proportion of the population. Maize breeding is monopolized by large multi-national private companies, with little attention given to more specific needs of local markets. The public sector breeding program is essential in breeding and providing varieties for this niche market as well as providing inbred lines to local private companies. Due to ever decreasing research funds, it is however, essential to scale down the amount of testcrosses and the size of current breeding programs. Several heterotic groups are currently identified and used in the local breeding program. The locally *Corresponding author. charlotte.mienie@nwu.ac.za. Tel: is being used extensively in crosses. The I heterotic group is considered to be the most advantageous for yield adaptation in South Africa. In hybrid combination with the I-group, the US Cornbelt lines, related to the Lancaster line, Mo17, seems to be the preferred choice. Smaller heterotic groups include the K64 group. Genetic diversity among inbred lines has traditionally been based on morphological data such as endosperm type, pedigree record and amount of heterosis expressed by the hybrid. This method has several limitations as morphological characters do not always reliably portray genetic relationships due to environmental interactions. Testcross designs with numerous testers are extremely expensive, labour intensive and require large-scale field evaluations. Molecular analyses can lower the cost involved in plant breeding. It also provides a means for determining the purity of hybrids produced in the seed industry and can save seed companies and farmers money in terms of ensuring the quality of the seed. Knowledge of the relationships among lines would also

2 124 Afr. J. Biotechnol. help identify a set of inbreds that have maximal diversity for the analysis of the effects of genetic background (Liu et al., 2003). The ideal marker system is highly polymorphic, co-dominant, accurate, reproducible, high-throughput and low cost (both in terms of capitalinvestment and cost per assay). For decades, simple sequence repeats (SSR) or microsatellites, have been the genetic markers of choice, because they are economical to score, have high allelic diversity and are usually selectively neutral (Smith et al., 1997). Large numbers of single nucleotide polymorphisms (SNPs) are already available and have been used extensively in genotyping maize. However, it is still not cost effective in South Africa to genotype large numbers of inbreds for the purpose of association analysis and it is thus necessary to identify a set of lines that capture the maximum number of alleles or haplotypes and determine population structure. Hamblin et al. (2007) compared the use of SSR and SNP markers in maize. The different mutational properties of these two classes of markers resulted in differences in heterozygosities and allele frequencies, but SSRs performed better in clustering germplasm into populations. SSRs provided more resolution in measuring genetic distance based on allelesharing. Their results suggested that large numbers of SNP loci would be required to replace highly polymorphic SSRs in studies of diversity and relatedness (Hamblin et al., 2007).The relationship between mid-parentheterosis of single-cross hybrids and the genetic distance of their parental inbreds, determined with molecular markers, were investigated both in theory (Charcosset and Essioux, 1994) and numerous experiments with maize and other crops (Brummer, 1999). Xia et al. (2004) concluded from their study of CIMMYT sub-tropical maize inbreds that the SSR based genetic distances, calculated with a modified Roger s distance measure, in combination with field evaluations, provided a solid basis for the detection of heterotic groups. The objectives of our research were to: 1) fingerprint local maize genotypes according to the most appropriate and cost-effective procedure for determining genetic distances of breeding lines and 2) compile a database of local maize breeding material according to DNA data. MATERIALS AND METHODS Plant material The pedigrees of hybrids studied in this paper are commercial intellectual property. Therefore, detailed information is not allowed. Seeds from breeding lines and hybrids were obtained from plant breeders at ARC-GCI in Potchefstroom and Cedara, South Africa and planted in small pots for germination. Young leaves from approximately 20 plants from each line were harvested and two bulks made up from equal amounts of leaf material from ten plants each. We assumed that the bulk sample will capture all heterozygosity present in the genotypes. Leaf material was freeze dried and ground to a fine powder. SSR genotyping DNA was extracted from each bulk sample using a modified CTAB extraction technique (Saghai-Maroof et al., 1984) and diluted to 50 ng/µl. Microsatellite marker genotyping involved the use of 96 fluorescently labelled SSR selected from published markers ( to be evenly distributed throughout the maize genome (Figure 1). The SSR primers were labelled with four different dyes [6FAM (blue), HEX (green), NED (yellow) and PET (red)] and combined in 12 multiplex groups (Table 1) with each containing seven to ten primer pairs according to colour and size avoiding overlapping of the same colour. PCR amplifications were carried out on a Techne 384-well thermal-cycler in a final reaction volume of 5 to10 µl, containing 50 ng genomic DNA, 1X multiplex PCR master mix (Qiagen, Valencia, USA) containing HotStarTaq DNA polymerase, multiplex PCR buffer, 2 mm MgCl 2, 250 mm of each dntp (datp, dctp, dgtp, dttp), and 0.05 µm of each multiplex fluorescent primer mix. The PCR products were diluted 1:20 and 1 µl was added to HiDi-formamide (Life Technologies) containing 0.12 µl GeneScan- LIZ500 as size standard and electrophoresed on an ABI Prism3130xl sequencer. Fragments were analyzed with Gene mapper 4.0 (Applied Biosystems) software and allele sizes was verified. A list of microsatellite loci and their chromosomal locations is given in Table 1. Data analysis Genetic distances of breeding lines and hybrids tested were calculated with the Powermarkerver 3.25 (Liu and Muse, 2005) using the Rogers (1972) parameter. The polymorphic information content (PIC) for each marker was determined using Powermarkerver 3.25 software. Average linkage [unweighted paired group method using arithmetic averages (UPGMA,)] clustering was calculated based on Rogers distance (RD) estimates between pairs of inbred lines for the yellow and white lines and hybrids separately. To evaluate the robustness of the UPGMA dendrogram, the cophenetic correlation was calculated (Sneath and Sokal, 1973) utilizing NTSYS ver 2.21 software (Rohlf, 2009).The data was transformed with the dcenter module and Eigenvectors calculated. Principal coordinates analysis (PCoA) was carried out on the calculated distances usingntsys ver software (Rohlf, 2009). RESULTS A total of 96 SSR primer sets were used to fingerprint the breeding lines and hybrid samples. The SSR markers with more than 20% missing values were discarded and data from 80 markers were used in the final analysis. The PIC values of markers varied between 0.2 and 0.9. The average number of alleles per locus was 12.7 for a total of 1043 genotypes tested, with a total of 1874 alleles amplified (Table 1). The number of alleles amplified varied between 6 and 36 per locus for the total population screened. When looking at the separate populations tested, the mean number of alleles per locus was the highest for the yellow maize breeding lines, which also had the highest entries in the screening program (Table 2). In PCoA of the RD of the breeding lines, the white and

3 Mienie and Fourie 125 Figure 1. Maize genomic map indicating SSR markers used in the study (based on IBM distances). yellow groups were clearly separated in two clusters, with relatively few overlapping genotypes (Figure 2). When regarding the two localities where the breeding lines originated, it was clear that the white lines could be separated into three clearly distinguished groups, with the lines from Potchefstroom grouping in two separate clusters. The yellow breeding lines were much more heterogeneous between the two localities, with

4 Panel Marker Dye Bin Repeat Afr. J. Biotechnol. Table 1. Multiplex grouping of SSR markers. M1 M2 umc2162 PET 6.06 (CCG)5 umc FAM (CT)17 phi FAM 8.03 CCG phi046 VIC 3.08 ACGC phi VIC 5.04 AAG phi062 VIC ACG phi089 NED 6.08 ATGC phi NED 1.03 ACC umc1256 VIC 2.09 (CAT)5 phi VIC 2.10 AGAT phi NED 9.05 AGCT phi96342 NED ATCC umc FAM 1.04 (TG)6 umc1279 PET 9.00 (CCT)6 phi PET 3.00 ACCG phi96100 PET 2.00 ACCT bnlg118 6-FAM 5.07 (AG)n phi076 6-FAM 4.11 AGCGGG phi032 6-FAM 9.04 AAAG M3 phi114 VIC 7.03 GCCT umc1196 NED CACACG phi050 PET AAGC phi123 PET 6.07 AAAG M4 umc FAM 8.02 (TCGA)4 phi053 6-FAM 3.05 ATAC umc1295 VIC 7.00 (AT)6 umc1657 VIC 9.00 (GACGG(4 phi VIC 5.02 AGGCG umc1757 NED 4.01 (TCC)7 umc1109 PET 4.10 (ACG)4 phi059 PET ACC

5 Mienie and Fourie 127 M5 M6 Table 1. Contd. phi FAM 1.10 AGC umc FAM 3.06 (TTG)8 phi072 VIC 4.00 AAAC phi011 VIC 1.09 AGC phi008 NED 5.03 GGC phi079 NED 4.05 AGATG phi093 NED 4.08 AGCT bnlg391 PET phi112 PET 7.01 AG phi FAM 1.03 AGCT phi041 6-FAM (CAT)5 umc1399 VIC CTAG)5 umc1122 VIC 1.06 (CGT)7 phi034 NED 7.02 CCT phi031 NED 6.04 GTAC umc1126 PET 2.00 ACC phi127 PET 2.08 AGAC M7 umc FAM 5.06 (CT)17 umc FAM 8.06 (GCTGGG)5 bnlg FAM 1.00 (AG)17 phi083 VIC 2.04 AGCT phi070 NED 6.00 AGCTG umc1545 PET 7.00 (AAGA)4 phi029 PET 3.04 AG/AGCG phi PET 4.01 ACC M8 umc FAM (TCG)6 bnlg1792k8 6FAM 7.00 (AG)16 phi VIC 6.04 AGCC phi VIC 1.11 ACC phi NED 6.07 AGC phi073 NED 3.05 AGC phi PET 7.04 AGG phi080k15 PET 8.00 AGGAG

6 128 Afr. J. Biotechnol. Table 1. Contd. M9 umc FAM 7.00 (GGC)6 nc130 6-FAM 5.00 AGC phi FAM 3.02 ACC phi064 VIC 1.11 ATCC phi116 VIC 7.06 ACTG/ACG umc1143 NED 6.00 AAAAT umc1136 NED 3.10 (GCA)5 umc1777 PET 8.00 AGCC phi084 PET GAA M10 phi FAM 8.03 AAGC phi VIC 2.00 ACGG phi VIC 9.06 AAG bnlg2291k4 VIC 4.06 (AG)17 umc1555 NED 2.03 (TTCA)7 phi014 NED GGC bnlg2305k4 NED 5.06 (GA)6 phi065 PET 9.03 CACTT M11 bnlg161k8 VIC 6.00 (AG)n bnlg1754w3 PET 3.09 (AG)20 umc2105k3 PET 3.00 (CCAT)4 bnlg1940k9 NED 2.08 (AG)18 bnlg FAM 2.02 (AG)19 umc1932 NED 7.00 (GA)8 bnlg1953 NED 1.02 (AG)17 M12 umc1586 NED 9.03 (ATC)5 umc1955 PET umc1603 PET 1.00 (GCC)4 umc1230 PET 2.00 (TAA)8 only a few lines falling in definite clusters. The lines originating from CIMMYT, Mexico, clustered together with the Cedara material for both white and yellow lines. These lines were used in the Cedara breeding program over a long period of time and therefore the result was as expected. The white lines were compared to some anchor lines with known heterotic grouping (Figures 3 and 4). The lines with known pedigrees related to K64, clustered together (r=0.91), with only one line with larger RD from the rest of the group. A large number of white lines clustered with the Lancaster line Mo17. The rest of the groups could be separated into distinguishable clusters by the UPGMA derived dendrogram (Figure 4). PCoA and cluster

7 Mienie and Fourie 129 Table 2. Average and range of number of alleles per locus for 1043 maize genotypes. Population Number of genotype Number of allele Mean alleles / locus Range allele White breeding lines Yellow breeding lines Hybrids Total Figure 2. Principal coordinates analysis of white and yellow breeding lines from the two localities. analysis of yellow breeding lines revealed a small group of lines clustering with Mo17 (Figures 5 and 6) (r=0.72), with the rest of the lines with mixed origin. The cluster analysis (Figure 6) identified several related groups of lines indicating close genetic relationships. These results show the extent to which these lines were interbred in the breeding program over several decades. One hundred and ninety three (193) hybrids were included in the study to explore the diversity achieved and the genotypes were compared to locally cultivated maize cultivars produced by private companies. Five distinct groups of ARC hybrids could be identified. No overlap could be seen between ARC hybrids and those from the private companies tested (Figure 7). Cluster analysis (not shown) revealed several identical hybrids included in the entries (r = 0.88). DISCUSSION Maize breeding is a never-ending challenge for developing new inbred lines and cultivars with improved yield, as well as tolerance to a diverse spectrum of biotic and abiotic stress conditions, such as diseases, pests, low soil fertility and drought. New cultivars must also conform to changing industry requirements such as grain quality for milling and ethanol production. Climatic changes such as global warming pose new challenges to breeders for developing cultivars that will perform well under changed conditions. Although seed companies produce their own hybrids, they remain dependent on foundation-seed suppliers for inbred lines and even sometimes locally adapted hybrids. The ARC-GCI has a large germplasm collection of

8 130 Afr. J. Biotechnol. M37W B73 I137TNW Mo17 K64 Figure 3. Principal coordinates analysis of white breeding lines compared with anchor lines of known heterotic groups. inbred lines, containing both white and yellow lines, at its disposal. These lines needed to be characterized on a molecular level to determine close relationships or genetic distances to narrow down the possibilities for utilizing this material in testcrosses. It also served the purpose of identifying duplicate lines, as well as genetic drift within well-known breeding lines. During the past decade, the technology used in fingerprinting of field crops developed from restriction fragment length polymorphism (RFLP) to amplified fragment length polymorphism (AFLP), SSR and SNP. The RFLP technique was laborious and time consuming and only single data points could be analyzed per reaction. The AFLP technique was less time consuming and labour intensive and rendered up to 100 data points per reaction, but it was difficult to duplicate results over time and between different laboratories. The SSR technique is a simple polymerase chain reaction (PCR) technique, which lends itself to high throughput of samples when multiplex reactions are used with an automatic genetic analyzer like the ABI3130xl. The technique is also easy to duplicate among different laboratories. Although the SNP technique can analyze thousands of data points per run, one has to look at the application that you need the analysis for. The SSR technique can be utilized to give low coverage of the whole genome of maize, giving an overall indication of the genetic material present in a specific population. Knowledge of the relationships among lines can help identify a set of specific inbreds that have maximal diversity for the analysis of the effects of background (Liu et al., 2003), to be used further in experimentation and association analysis of specific traits. A similar African study was conducted by Legesse et al. (2007), to determine the diversity present in an African breeding population in Ethiopia and Zimbabwe. They found that they could distinguish between their inbred lines with as little as 27 SSR loci, which grouped their lines in groups corresponding to adaptation to different altitudes and consistent with known pedigree information. In our local line collection, the breeding program has used lines from different countries and sources and the end result is a totally different population from the original due to interbreeding between different heterotic groups. New grouping of the breeding lines became of vital importance for improvement of the crop. SSR markers were utilized in this study in the genotyping of the germplasm collection of the ARC-GCI from two different localities and were used at a density of eight markers per chromosome. The 80 markers utilized amplified a total of 1874 alleles giving an average of 23.4 alleles per locus, which is higher than observed by Van Inghelandt et al. (2010). Results were used to determine the genetic relationships among the inbred lines and

9 Mienie and Fourie 131 A1012W I137tnW I137TNW I37TNW R118W RO552W R119W B125W CB215 KPROL CB218 CED201 A1P100 P614MSV B73 P612MSV P616MSV B103W U2540W U2550W M162W U2548W M848W B141W V121W K358 K K64227 K K64229 K K K64228 K K K64224 K K K64222 K K K K64R22 P588MSV P2579W SO607W SO713W E184 VH118W CED093 L116W CED094 CED095 CED096 CED098 CED097 CED099 CED100 D940Y J80W RO421W M37W2 RO424W RO452W P590MSV SO181W P606MSV P604MSV P592MSV P594MSV BO163W T1162W KO54W RO549W R2581W R2582W R70W IMMYTJL9 K2874W U37W MMYTJL11 U2511W MMYTJL12 MMYTJL13 CB197 CB200 CB199 CB198 CB389 CB390 CB391 CB392 CB393 CB394 CB395 CB406 CB407 CB291 CB293 CB295 CB344 CB347 CB350 CB351 CB349 CB348 CB352 CB353 CB354 CB355 CB356 CB358 CB209 CB213 CB327 CB330 CB332 CB334 CB331 CB333 CB359 CB364 CB366 CB365 CB360 CB363 CB219 CB220 CB221 CB222 CB223 CB224 CB299 CB300 CB301 CB302 CB304 CB305 CB306 CB307 CB310 CB309 CB308 CB311 CB312 CB314 CB313 CB315 CB320 CB316 CB319 CB317 CB318 CB373 CB374 CB321 CB322 CB323 CB335 CB336 CB339 CB337 CB338 CB341 CB340 CB343 CB381 CB382 CB384 CB386 CB388 CB376 CB378 CB379 CB380 CB404 CB405 CB396 CB397 CB398 CB399 CB400 CB232 CB234 CB237 CB236 CB235 CB238 CB239 CB244 CB247 CB246 CB248 CB249 CB250 CB255 CB257 CB260 CB256 CB258 CB259 P598MSV CB410 Mo17 P Genetic distance Figure 4. Associations among white inbred lines revealed by average linkage (UPGMA) cluster analysis based on Rogers distance. Figure 5. Principal coordinates analysis of yellow breeding lines compared with anchor lines of known heterotic groups.

10 132 Afr. J. Biotechnol. Genetic Distance ARG CIMMYTJL2 CNO4P_686 CIMMYTJL1 CNO4P_685 CIMMYTJL15 CNO4P_699 CIMMYTJL7 CNO4P_691 CIMMYTJL8 CNO4P_692 CIMMYTJL16 CNO4P_700 CNO4P_693 CN99_1154_2 CIMMYTJL17 CNO4P_701 CIMMYTJL6 CNO4P_690 I34 I35 I37 CIMMYTJL3 CNO4P_687 CIMMYTJL4 CNO4P_688 CIMMYTJL5 CNO4P_689 CIMMYTJL14 CNO4P_ ARG CNO4P_695 CNO4P_696 CNO4P_697 A272 B312Y A281 E283S A441_5 N2575Y M708Y CN99_534_1 CNOO_253_3 CN99_116_1 CN99_81_1 CN99_80_1 JK_12 JK_10 JK_18 JK_6 JK_2 JK_3 JK_39 JK_5 JK_7 JK_40 JK_16 JK_19 JK_22 JK_24 JK_26 JK_42 JK_27 D160P R2871PE JK_4 VO481Y VO495Y VO501Y VO500Y A7082Y B2519Y R2565Y U42Y I137TN CNOO_522_RY CNOO_523_RY U666Y I137TN_P KO439Y BY230LM_1729 CNOO_106_1 CNOO_106_3 CNOO_522 CNOO_523 BY272LM_1771 BY273LM_1772 BY275LM_1774 BY285LM_1784 BY286LM_1785 BY283LM_1782 BY284LM_1783 BY276LM_1775 BY277LM_1776 BY278LM_1777 BY279LM_1778 BY282LM_1781 BY280LM_1779 BY281LM_1780 BY287LM_1786 BY289LM_1788 BY290LM_1789 BY291LM_1790 BY296LM_1795 BY297LM_1796 BY298LM_1797 BY364LM_1863 BY389LM_1888 BY393LM_1892 BY394LM_1893 BY395LM_1894 BY399LM_1898 BY400LM_1899 BY402LM_1901 BY401LM_1900 BY397LM_1896 BY398LM_1897 CN99_1016_1 CN99_1021_3 CN99_1030_1 CN99_1031_1 CN99_1036_1 CN99_1042_1 CNOO_453 BY372LM_1871 BY374LM_1873 BY375LM_1874 BY377LM_1876 BY370LM_1869 BY371LM_1870 OO_106_1_72_3 OO_106_3_72_3 OO_122_2_72_3 OO_112_1_72_3 CNOO_72_2A CNOO_72_3 CNOO_521 CNOO_521_RY CNOO_111_2 CNOO_1112_1 CNOO_1112_2 CNOO_122_2 CNOO_104_1 CNOO_104_2 O_106_3_104_1 B1138T J2678T P203Y JK_21 BY208LM_1707 BY209LM_1708 BY210LM_1709 BY180LM_1679 YC25 BY381LM_1880 A8102Y B2584Y B2585Y I42Y BY309LM_1808 B2600Y BY310LM_1809 BY311LM_1810 BY305LM_1804 BY306LM_1805 BY303LM_1802 BY304LM_1803 BY318LM_1817 BY319LM_1818 K130Y K180Y UO705Y UO718Y P362Y TO1292Y SO826Y R2582Y E30Y K559Y V2779Y KO800Y U28Y A8451Y DO620Y U12Y CN99_213_1 D940Y D940Y_1 KO326Y SO178Y SO181Y S193Y SO1224Y CN99_476_1 CN99_485_1 CN99_709_1 CN99_711_1 CN99_712_1 CN99_716_1 CN99_719_1 CN99_723_1 VO617Y CN99_723 CN01_578 CN99_459_1 CNO1_578 CN01_579 CN99_462_1 CN99_450_1 CN99_456_1 CN99_467_1 CN99_1123 CN99_1123_1 CN99_361_2 CN99_368 CN99_368_1 CN99_426_1 CN99_481_1 CN99_490_1 CN99_486_1 CN99_496_1 96Y_K169Y_S6 CN99_375_1 CN99_378_1 196Y_V613Y_S6 CN99_417_1 CN99_964_1 CN99_389 CN99_389_1 CN99_396_1 CN99_399_1 CN99_392_1 CN99_401_1 CN99_782_1 CN99_241_1 CN99_245_1 CN99_257_1 CN98_1461 U127Y CN99_281_1 CN99_284_1 CN99_290_1 Y_D940Y_1_S6 CN99_294_1 CN99_296_1 Y_D940Y_1_S7 CN99_302_1 CN99_323_1 CN99_526_1 CN99_528_1 CN99_529 CN99_529_1 B2678Y CNOO_69_1 CN99_519_1 BY320LM_1819 BY321LM_1820 BY322LM_1821 BY323LM_1822 BY325LM_1824 BY326LM_1825 BY327LM_1826 BY328LM_1827 BY329LM_1828 BY330LM_1829 BY331LM_1830 BY332LM_1831 BY335LM_1834 BY334LM_1833 BY333LM_1832 B2603Y BO461Y KO679Y BY250LM_1749 BY255LM_1754 BY253LM_1752 BY254LM_1753 BY256LM_1755 BY261LM_1760 BY262LM_1761 BY263LM_1762 BY264LM_1763 BY265LM_1764 BY258LM_1757 BY259LM_1758 CNOO_203_4 CN99_1068_1 CN99_1072_1 B2627Y BY313LM_1812 BY314LM_1813 BY299LM_1798 BY300LM_1799 BY301LM_1800 U101Y U117Y UO688Y CNOO_61_2 CNOO_62_1 B2536Y B2537Y P152Y M27Y M28Y U71Y S198Y S247Y S1247Y S297Y SO1191Y S218Y B37 B73 FR1050 BY408LM_1907 BY410LM_1909 BY411LM_1910 BY409LM_1908 VO430Y VO430Y VO434Y BY232LM_1731 BY233LM_1732 BY234LM_1733 BY235LM_1734 BY236LM_1735 BY237LM_1736 BY240LM_1739 CNOO_166_1 BY406LM_1905 BY407LM_1906 CNOO_161_2 C103 C103_Ht1A C103_Htn Mo17 Mo17Ht Mo17HT Mo17_2HtHtN Mo17HtHtN Mo17_2HTHTN P298Y TO1274Y BY346LM_1845 BY359LM_1858 BY361LM_1860 BY363LM_1862 BY362LM_1861 BY413LM_1912 BY414LM_1913 BY416LM_1915 BY415LM_1914 BY419LM_1918 BY417LM_1916 BY418LM_1917 BY420LM_1919 FR4310 BY347LM_1846 BY348LM_1847 BY352LM_1851 BY349LM_1848 BY350LM_1849 BY351LM_1850 BY353LM_1852 BY354LM_1853 BY356LM_1855 BY355LM_1854 BY357LM_1856 BY358LM_1857 BY369LM_1868 NC258 BY169LM_1668 CNOO_1_1 BY336LM_1835 BY337LM_1836 BY338LM_1837 BY339LM_1838 BY340LM_1839 BY343LM_1842 BY341LM_1840 FR4910 AP10Y AP10Y_AP6Y AP6Y_AP10Y AP10Y_AP3Y AP6Y_AP3Y AP12Y AP12Y_AP6Y AP12Y_AP3Y AP3Y_AP12Y AP6Y AP4Y AP5Y BY152LM_1651 BY005LM_1504 BY115LM_1614 BY162LM_1661 BY164LM_1663 BY116LM_1615 BY117LM_1616 BY120LM_1619 BY121LM_1620 BY163LM_1662 BY114LM_1613 BY020LM_1519 BY021LM_1520 BY022LM_1521 BY006LM_1505 BY007LM_1506 BY167LM_1666 BY168LM_1667 BY155LM_1654 BY181LM_1680 BY183LM_1682 BY182LM_1681 BY184LM_1683 BY023_LM1522 BY024LM_1523 BY025LM_1524 BY081LM_1580 BY082LM_1581 BY122LM_1621 BY078LM_1577 BY080LM_1579 BY079LM_1578 BY067LM_1566 BY068LM_1567 BY069LM_1568 BY070LM_1569 BY149LM_1648 BY150LM_1649 BY151LM_1650 BY146LM_1645 BY148LM_1647 BY143LM_1642 BY144LM_1643 BY145LM_1644 BY215LM_1714 BY216LM_1715 BY221LM_1720 BY222LM_1721 BY223LM_1722 BY219LM_1718 BY220LM_1719 BY217LM_1716 BY026LM_1525 BY028LM_1527 BY027LM_1526 BY029LM_1528 BY085LM_1584 BY087LM_1586 CN99_14_1 CNOO_472 BY099LM_1598 BY100LM_1599 BY101LM_1600 BY102LM_1601 BY104LM_1603 BY103LM_1602 BY105LM_1604 BY106LM_1605 BY108LM_1607 BY109LM_1608 BY111LM_1610 BY112LM_1611 BY110LM_1609 BY113LM_1612 BY175LM_1674 BY176LM_1675 BY177LM_1676 BY178LM_1677 BY179LM_1678 BY266LM_1765 BY268LM_1767 BY267LM_1766 BY269LM_1768 BY271LM_1770 CNOO_466 BY270LM_1769 BY241LM_1740 BY242LM_1741 BY244LM_1743 BY243LM_1742 CNOO_185_1 CNOO_185_2 BY245LM_1744 BY246LM_1745 BY247LM_1746 BY249LM_1748 BY248LM_1747 CNOO_185_3 CNOO_182_1 CNOO_182_2 CNOO_184_2 BY001LM_1500 BY004LM_1503 I16 I9 BY002LM_1501 BY003LM_1502 BY059LM_1558 BY060LM_1559 BY061LM_1560 BY062LM_1561 BY063LM_1562 BY064LM_1563 BY065LM_1564 BY066LM_1565 BY073LM_1572 BY074LM_1573 BY076LM_1575 BY075LM_1574 BY077LM_1576 BY038LM_1537 BY040LM_1539 BY045LM_1544 BY173LM_1672 BY174LM_1673 BY196LM_1695 BY197LM_1696 BY198LM_1697 BY200LM_1699 BY204LM_1703 BY205LM_1704 BY201LM_1700 BY202LM_1701 BY203LM_1702 BY206LM_1705 BY207LM_1706 BY199LM_1698 BY090LM_1589 BY091LM_1590 BY092LM_1591 BY093LM_1592 BY094LM_1593 BY095LM_1594 BY032LM_1531 BY035LM_1534 BY036LM_1535 BY037LM_1536 BY042LM_1541 BY051LM_1550 BY052LM_1551 BY044LM_1543 BY050LM_1549 BY047LM_1546 BY048LM_1547 BY043LM_1542 BY049LM_1548 BY046LM_1545 BY096LM_1595 BY097LM_1596 BY098LM_1597 BY186LM_1685 BY188LM_1687 BY187LM_1686 BY189LM_1688 BY190LM_1689 BY133LM_1632 BY134LM_1633 BY135LM_1634 BY136LM_1635 E739 BY137LM_1636 BY138LM_1637 BY140LM_1639 BY142LM_1641 BY139LM_1638 BY141LM_1640 BY053LM_1552 BY071LM_1570 BY072LM_1571 BY088LM_1587 BY089LM_1588 BY218LM_1717 BY153LM_1652 BY154LM_1653 BY156LM_1655 BY157LM_1656 BY165LM_1664 BY166LM_1665 BY211LM_1710 BY212LM_1711 BY213LM_1712 BY214LM_1713 BY041LM_1540 BY172LM_1671 BY054LM_1553 BY055LM_1554 BY056LM_1555 BY057LM_1556 BY083LM_1582 BY403LM_1902 BY404LM_1903 BY008LM_1507 BY011LM_1510 BY012LM_1511 BY013LM_1512 BY014LM_1513 BY015LM_1514 BY010LM_1509 BY009LM_1508 BY016LM_1515 BY017LM_1516 BY123LM_1622 BY124LM_1623 BY125LM_1624 BY127LM_1626 BY129LM_1628 BY131LM_1630 BY132LM_1631 BY170LM_1669 BY171LM_1670 BY030LM_1529 BY031LM_1530 BY158LM_1657 BY159LM_1658 BY161LM_1660 BY160LM_1659 BY191LM_1690 BY192LM_1691 BY193LM_1692 BY194LM_1693 BY195LM_ ARG 1251ARG 1308ARG D424ARG A413 B73VH A632 A632_Ht1 B68 B68_Ht1 B68_Htn E123Y CN99_12_1 CN99_7_1 CN99_8_1 V26 V26_Ht2 V26_Ht1 V26_Ht3 Figure 6. Associations among yellow inbred lines revealed by average linkage (UPGMA) cluster analysis based on Rogersdistance. Figure 7. Principal coordinates analysis of hybrids tested.

11 Mienie and Fourie 133 hybrids. Principal coordinates analysis clearly separated white and yellow lines as expected from the pedigree data that were available from the breeding programs. These groups were further analysed separately. In the analysis of white breeding lines based on Rogers estimates, the lines clearly were separated in two different groups with PCoA analysis. A large group of lines were grouped together with Mo17, a Lancaster line, in accordance with pedigree data. All the white lines could not be grouped into specific heterotic groups and some were of mixed origin. These lines were developed through crosses between the different groups in the breeding program and it is thus not possible to group them with currently known heterotic groups. The cluster analysis gave a more detailed grouping of lines (Figure 4): the K-group was derived from K64 crosses. K64 is a Kansas inbred known for fairly good drought tolerance. The CB group derived from inbreeding US Corn belt hybrids clustered with Mo17, a Lancaster line. A 3rd group was identified as a mixed group containing combinations of Corn Belt material with Sahara or I-group lines as well as lines with maize streak virus resistance. The M37W group was derived from the Australian inbred 21A. Lines originating from CIMMYT also clustered in this group as a sub-group. The I137TN group is the major South African group known for its superior combining ability for grain yield, especially in combination with US Corn Belt material.the group originated from the locally developed I137TN inbred. I137TN was selfed out of a yellow endosperm local variety cross between the two varieties Teko Yellow x. Rogers JS (1972).Measures of genetic similarity and genetic distance.studies in Genetics VII. University of Texas Publishing, 7213: Rohlf F (2009). NTSYS pc.numerical Taxonomy system. Exeter Publishing, Setauket. Saghai-Maroof MA, SolimanKM, Jorgenson R,Allward RW(1984). Ribosomal DNA spacer length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc. Natl. Acad. Sci. USA 81: Smith JSC, Chin ECL, Shu H, Smith OS, WalLSJ, Senior ML, Mitchell SE, Kresovich S, Ziegle J (1997). An evaluation ofthe utility of SSR loci as molecular markers in maize (Zea mays L.): comparisonswith data from RFLPs and pedigree. Theor. Appl. Genet. 95: Sneath PHA,Sokal RR(1973). Numerical taxonomy. Freeman, San Francisco. Van Inghelandt D, Melchinger AE, Lebreton C, Stich B (2010). Population structure and genetic diversity in a commercial maize breeding program assessed with SSR and SNP markers. Theor. Appl. Genet. 120: Xia XC, Reif JC, Hoisington DA, Melchinger AE, Frisch M, Warburton ML (2004). Genetic diversity among CIMMYT maize inbred lines investigated with SSR markers: I. Lowland tropical maize. Crop Science. 44: ACKNOWLEDGEMENT The authors would like to acknowledge the financial support of the Maize Trust. REFERENCES Brummer EC (1999).Capturing heterosis in forage crop cultivardevelopment. Crop Sci. 39: Charcosset A,Essioux L (1994).The effect of population structureon the relationship between heterosis and heterozygosity atmarker loci.theor. Appl. Genet. 89: Hamblin MT, Warburton ML, Buckler ES (2007).Empirical Comparison of Simple Sequence Repeats and Single Nucleotide Polymorphisms inassessment of Maize Diversity and Relatedness.PLoS ONE 2(12): e1367. doi: /journal.pone Legesse BW, Myburgh AA, Pixley KV, Botha AM. (2007) Genetic diversity of African maize inbred lines revealed by SSR markers. Hereditas 144: Liu K, Goodman M, Muse S, Smith JS, Buckler E,Doebly J (2003). Genetic structure and diversity among maize inbred lines as inferred from DNA microsatellites. Genetics 165: Melchinger AE, Messmer MM, Lee M, Woodmana WL, Lamkey KR(1991) Diversity andrelationships among U.S. maize inbredsrevealed by restriction fragment lengthpolymorphisms. Crop Science. 31: