Maize (Zea mays ssp. mays L.) is the crop with the third highest cultivation area and second highest production worldwide (FAO). After its domestication about 9,000 years ago in a restricted valley in south-central Mexico, it spread throughout the Americas over thousands of years. Following the discovery of the New World by Columbus in 1492, maize was introduced in Europe and to other parts of the world through trade and colonization (Mir et al., 2013).
Furthermore, maize was introduced in Africa nearly five centuries ago (Prasanna, 2012), and has become the most important food crop with an annual production of more than 63 million metric tons in 2010 (Westengen et al., 2012). In a study conducted for tracing the genetic footprints of global maize diffusion using SSR markers, American landraces were clustered into seven groups namely; “Northern US flints”, “Mexican highlands”, “Tropical lowlands”, “Andes”, “Middle North-American”, “Northern South-American”, and “Middle South-American” among which the Northern South-American, Tropical lowlands, and the Middle South-American clusters seemed to be the origins of the African maize landraces (Mir et al., 2013).
In Angola, maize is the staple food, but only limited breeding works have been done because of the prolonged civil war (1975-2002). The very low yield (500-700 kg/ha) achieved by small-scale farmers (70% of all maize producers), is one of the major constraints towards attaining food security in the country. The only way to overcome the constraints is to breed for more productive and well adapted hybrids and open pollinated varieties (OPVs), and to promote education to small-scale farmers to be able to use these new production technologies. The success depends on producing inbred lines from the local gemplasm and initiation of a sustainable breeding program for hybrids and OPVs production.
Maize has enormous genetic diversity that offers incredible opportunities for enhancement (Prasanna, 2012). Since 1980s, molecular markers have been widely used to assess the genetic diversity of maize (Li et al., 2002). Especially, SSR markers have been adopted as major tool for genetic diversity analysis as they are co-dominant, multi-allelic, highly polymorphic, and randomly distributed throughout the genome (Hoxha et al., 2004). For example, studies on genetic diversity of major maize inbred lines or landraces in China, Japan, Brazil, USA and central Europe using SSR markers were reported (Lu and Bernardo, 2001; Enoki et al., 2002; Li et al., 2002; Pinto et al., 2003; Reif et al., 2005). For African maize, highland and mid-altitude inbred lines from CIMMYT programs in Ethiopia and Zimbabwe were analyzed using SSR markers (Legesse et al., 2007), and Striga-resistant maize inbred lines developed at the International Institute of Tropical Agriculture (IITA) were analyzed using SSR and AFLP markers (Menkir et al., 2005).
In this study, the genetic diversity of 89 maize landraces of Angola were analyzed to get basic knowledge needed in breeding for high-yielding varieties with good adaptability to local condition.
MATERIALS AND METHODS
Plant Materials and DNA Extraction
Eighty nine (89) Angola maize landrace lines were provided by the National Gene Bank of Angola (Table 1). These maize landrace lines were collected from different parts of Angola (Fig. 1). They were planted in the field of Chianga Research Station in Huambo, Angola. A part of the harvested seeds were ground to make grain powder. The genomic DNA was extracted from the grain powder using IncloneTM plant genomic DNA extraction kit according to manufacturer’s instruction. In brief, 100 mg of maize grain powder was mixed with 600 μl of Buffer ICL (lysis buffer) in a 1.5 ml microcentrifuge tube and incubated at 65°C for 30-60 min. To remove RNA, 3 μl of RNase A (4 mg/ml) was added, and the mixture was incubated at 37°C for 15 min. The 200 μl of buffer IPS (precipitation buffer) was added, and the tubes were put on ice for 10 min and centrifuged at 12,000 rpm for 3 min. The supernatant was transferred to a new 1.5 ml microcentrifuge tube and one volume of Isopropanol was added for DNA precipitation. The DNA pellet was acquired by centrifugation and washed with 500 μl of Buffer IWS (washing buffer). After complete drying, the DNA pellet was dissolved in 100 ul of DNA hydration solution.
Five Uganda maize inbred lines and six Korean maize hybrid varieties were included in this genetic diversity analysis (Tables 2 and 3).
Genotyping Using SSR Markers
Twenty seven (27) maize SSR markers for genotyping that had been used for genetic diversity analysis of African maize inbred lines (Legesse et al., 2007) were used in this study. At first, the optimal annealing temperature of each SSR marker was decided by gradient PCR on DNA Engine TM DYAD thermal cycler (MJ Research). The PCR profile was 94°C for 3 min, followed by 35 cycles of 94°C for 40 sec, annealing temperature for 40 sec and 72°C for 1 min, followed by final extension of 72°C for 7 min. The PCR products stained with Loading Star dye (Dyne Bio) were electrophoresed on 3% (w/v) agarose gel. Also, the PCR products were electrophoresed on the QIAxcel System (QIAGEN) for more accurate PCR product size measurement. The PCR product size measured by the QIAxcel System was recorded and further processed using Allelo- Bin software (Prasanth et al., 1997) to produce discrete allele sizes. Thus processed QIAxcel allele size data were used in the following data analysis. Among the 27 maize SSR markers tested in this study, two markers showed poor PCR amplification and one marker showed too many bands. Therefore, excluding these three markers, 24 SSR markers produced genotype data to be analyzed. The primer sequences of these markers were shown in Table 4.
Data Analysis
The SSR genotype data were analyzed using Power- Marker 3.0 program (Liu and Muse, 2005) to get basic marker statistic values including allele number, maximum allele frequency (MAF), heterozygosity, gene diversity and Polymorphism information content (PIC). To analyze genetic diversity and relationship, the genotype data were inputted to DARwin5 program (Perrier and Jacquemoud- Collet, 2006) and, the genetic dissimilarity values among maize lines were acquired. Based on genetic dissimilarity data, phylogenetic tree of maize lines was constructed using MEGA4 program (Tamura et al., 2007) by Neighbor- Joining method.
RESULTS AND DISCUSSION
The 89 Angola maize land race lines were analyzed in this study. These lines originated in diverse locations of Angola (Fig. 1). Angola has relatively large land area of about 1,246,620 km2, and various agro-ecological regions including dry tropical, desert tropical, humid tropical and central plateau regions. This indicates that the Angola maize landrace lines should have very high genetic diversity.
Through genotyping using 24 SSR markers, a total of 254 alleles were detected, and the average number of alleles per marker was 10.6 (Table 5). The Umc2038 and Phi032 markers showed the lowest allele number (4), and the Nc003 marker showed the highest allele number (24). The dinucleotide repeat SSR markers (Phi037, Nc003, Phi054) showed higher allele number than tri, tetra, and pentanucleotide repeat SSR markers, which was in accordance with other studies. Maximum allele frequency ranged from 0.15 to 0.86 with average of 0.46. Gene diversity which means the probability that two randomly chosen alleles from the population are different ranged from 0.23 to 0.93 with average of 0.67. The PIC value which is the indicator of marker informativeness ranged from 0.23 to 0.92 with average of 0.64. Fourteen (14) SSR markers (Umc1153, Phi427434, Umc1568, Phi109275, Umc1677, Bnlg108, Phi115, Phi034, Phi054, Bnlg602, Phi037, Bnlg238, Nc003, Bnlg619) showed PIC values more than 0.6 indicating their potential informativeness to detect differences among the maize lines(Fig. 2).
The average of pair-wise genetic dissimilarity was 0.657. Within Angola maize landrace lines, the average of pair-wise genetic dissimilarity was 0.643. In the phylogenetic tree (Fig. 3) deduced from the pair-wise genetic dissimilarity, the Korean waxy maize hybrid varieties bred in GWARES for human consumption (Mibaek2, Heukjeom2, Miheukchal) clustered closely together which indicates the effectiveness of this analysis in revealing genetic relationship. Also, the Korean maize hybrid varieties bred at the NICS as livestock feed (Shingwangok and Gangdaok) showed close genetic relationship. But, a Korean maize hybrid variety, Gwangpyeongok showed far genetic relationship with other Korean maize varieties. Uganda maize inbred lines (WL 118-9, WL 118-16, WL 429-8, WL 429- 14) clustered together except for one line (WL 118-1-1), which also demonstrates the effectiveness of this analysis. Interestingly, Angola maize landrace accessions showed very high genetic diversity, and it is difficult to divide them into discrete groups. This might be due to the fact that the number of landrace lines tested here is small compared with large land area and very diverse agro-ecosystem of Angola.
Assessment of genetic diversity by molecular markers is useful in plant breeding by assisting in the selection of parental combinations for developing progenies with maximum genetic variability and by describing heterotic groups (Semagn et al., 2012). SSR markers are a valuable complementation to field trials for identifying heterotic groups (Reif et al., 2003). Correlations of genetic distance measured by SSR markers and heterosis were mostly positive and significant (Reif et al., 2003). The data produced in this study can be useful in making parental combinations for breeding diverse high-yielding maize varieties with good adaptability to local conditions. We are developing inbred lined with Angola maize landrace germplasm tested in this study. The present data indicates that almost all lines tested in this study can be bred to genetically distinct inbred lines and heterosis effect of their hybrids will be high.
ACKNOWLEDGEMENT
This research was supported by grants from the Korea- Africa Food and Agriculture Cooperation Initiative (KAFACI) of the Rural Development Administration (RDA), Republic of Korea, and the Instituto de Investigação Agronómica (IIA), Republica de Angola. We thank Mr. António Francisco for his technical support in field work.
적 요
앙골라에 잘 적응하는 다수성 옥수수 품종 육성을 위한 기 초지식을 얻기 위해 앙골라 옥수수 재래종 자원 89점을 대상 으로 24개의 SSR 마커를 이용하여 유전적 다양성 분석을 실 시한 결과는 다음과 같다.
총 254개의 allele가 탐지되었고 마커당 평균 allele 수는 10.6이었다.
마커의 PIC 값은 0.23 ~ 0.92 범위에 있었으며 평균값이 0.64이었다.
앙골라 옥수수 재래종 유전자원들은 매우 높은 유전적 다양 성을 보여 계통간 비유사성(dissimilarity) 평균값이 0.643 이었 고, 뚜렷하게 소수의 그룹들로 구분되지 않았다.
이러한 고도의 다양성을 가진 재래종 옥수수 유전자원을 이 용하여 앙골라에 잘 적응하는 다양한 다수성 품종을 육성할 수 있을 것으로 기대된다.
