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Combining high protein quality and hard endosperm traits through phenotypic and marker assisted selection in maize

E. Ravindra Babu, V.P. Mani and H. S. Gupta

Vivekananda Institute of Hill Agriculture, Indian Council of Agricultural Research, Almora – 263 601, Uttaranchal, India


Maize endosperm consisting of approximately 9-12 per cent protein is, however, deficient in two essential amino acids viz., lysine and tryptophan, which leads to poor net protein utilization and low biological value of traditional maize varieties. The low nutritive value of maize is genetically corrected in the biofortified form known as Quality Protein Maize (QPM), which contains twice the amount of lysine and tryptophan with high protein biological value. The opaque-2 gene along with necessary modifiers is primarily responsible for the enhanced protein quality. Four normal inbreds viz., CM212, CM141, CM145 and V25, which are parents of three single cross hybrids were targeted for conversion into high quality protein versions using three SSR markers located within opaque2. Two SSR markers viz., phi057 and umc1066 were co-dominant while phi112 was dominant in nature. Upon screening of appropriate back cross populations (BC) with phi057 and umc1066, around 50 per cent plants in each BC population were found to be heterozygous for the opaque-2 (Qq) gene. The selected BC2 progenies were advanced to selfing generation and similar molecular marker analysis was performed to identify the desirable homozygous recessive individuals (qq) in the selfed populations. The converted maize lines had twice the amount of lysine and tryptophan than the native lines and hence could be used as potential food and feed alternatives in resource-poor and marginal areas in general and hill ecosystems in particular. Based on the results obtained in our experiment, we propose that foreground selection in an early (BC1) generation combined with background selection at a later generation (BC2) along with the phenotypic selections for quantitative/continuously distributed traits would result in rapid genetic gain in a cost effective manner.

Media summary

An integrated MAS strategy with phenotypic selection aids in cost-effective conversion of normal maize lines to Quality Protein Maize, which are nutritionally superior.

Key words

Quality protein maize, SSR markers, MAS, opaque2


Maize (Zea mays L.) plays a very important role in human and animal nutrition. However, the normal maize protein is of very poor quality owing to deficiency in two essential amino acids - lysine and tryptophan, high leucine - isoleucine ratio and low biological value. A breakthrough came in the 1960s, with the discovery of the enhanced nutritional quality of the maize mutant opaque-2 (o2) (Mertz et al., 1964). The lysine value of opaque-2 maize is 3.3 to 4.0 g/100 g of protein, which is more than twice that of endosperm from the normal maize (1.3 g lysine/100 g protein). The decreased level of zein (5-27%) in opaque-2 maize in contrast to normal endosperm (54-59%) considerably improves its nutritional quality. Subsequently, plant breeders throughout the world made vigorous efforts to incorporate o2 into high yielding commercial cultivars but the numerous agronomic and processing problems associated with o2 prevented its acceptance (Glover and Mertz, 1987). The opaque-2 maize did not become popular with farmers as well as consumers mainly because of reduced grain yield, chalky and dull kernel appearance and susceptibility to ear rots and stored grain pests. The low nutritive value of normal endosperm and poor agronomic & keeping quality of opaque-2 maize is corrected in genetically improved and biofortified form known as Quality Protein Maize (QPM). QPM is a genotype in which the opaque-2 gene has been incorporated along with associated modifiers and contains twice the amount of lysine and tryptophan as compared to normal maize endosperm. Despite the nutritional superiority and improved agronomic performance, large-scale QPM cultivation and adoption is yet to gain significant momentum in many developing countries primarily due to lack of good number of commercial QPM hybrids in the market. A location specific QPM hybrid development strategy necessitates an efficient line conversion program for enhancing the protein quality of normal inbreds, which are heterotic to each other and well adapted to different region-specific intensified cropping systems.

The opaque-2 gene is recessive in nature and the modifiers behave as a multigenic trait. Although conventional breeding procedures have been used to convert commercial lines to QPM forms, the procedure is highly cumbersome and is not directly related toward improvement of grain quality. Rapid advances in genome research and molecular technology have led to the use of DNA marker assisted selection (MAS) which holds promise in enhancing selection efficiency and expediting the process of development of new varieties/hybrids with higher yield potential. (Ribaut and Hoisington, 1998). While marker assisted foreground selection (Tanksley, 1983 and Melchinger, 1990) helps in identifying the gene of interest without extensive phenotypic assays, marker assisted background selection (Hospital et al., 1992; Visscher et al., 1996; Frisch et al., 1999a,b) expedites significantly the rate of genetic gain/recovery in a backcross breeding program. Here, we report combining of high protein quality and kernel modification in early maturing normal maize inbreds through an integrated strategy of marker assisted selection for opaque2 and phenotypic selection for modifier genes. It is also demonstrated that foreground selection for opaque-2 in early (BC1) generation combined with background selection for recipient genome at later (BC2) generation results in rapid genetic gain and substantial cost savings.

Materials & Methods

The parental inbreds targeted for conversion into QPM forms viz., CM 212, CM 145, CM 141 and V 25 form three superior single cross hybrids with high grain yield potential. The donor QPM lines viz., CML 170, CML 173, CML 176, CML 180 and CML 184 were obtained from CIMMYT. A rapid and simple DNA extraction method was standardized which can be worked with or without liquid nitrogen. The SDS based procedure is inexpensive and suited to large scale MAS experiments in maize. PCR amplifications conditions were optimized according to the requirement of SSR assays. Foreground selection for opaque-2 was performed using three SSR markers viz., phi 057, phi 112 and umc 1066. Background marker analysis was conducted with 88 SSR markers spanning all the 10 chromosomes of maize genome. PCR products were separated in superfine agarose gels. Biochemical analyses were performed to estimate total protein content, tryptophan content and endosperm modification through microkjeldahl, colorimetry and light box assays respectively.

Results and Discussion

Polymorphism assay between normal and QPM inbreds

Parental polymorphism analysis was conducted between nine normal and six QPM inbreds using three SSR markers viz., phi 057, phi 112 and umc 1066 located as internal repetitive elements within the opaque2 gene. The marker, phi 112 exhibited dominant polymorphism between normal and QPM inbreds. Absence of approximately 420 bp DNA fragment in the PCR amplified product of QPM inbreds clearly distinguished them from the normal inbreds. However, such presence-absence polymorphism is only of limited use because it could not be used in discriminating homozygous (QQ) and heterozygous (Qq) backcross progenies. The presence of this particular band in one of the QPM inbred viz., CML 184 indicates possible pollen contamination from the normal maize during seed maintenance. Thus, this marker could be of use in checking the seed purity during routine field maintenance of QPM inbreds. The markers viz., phi 057 and umc 1066 exhibited co-dominant polymorphism between normal and QPM inbreds. Phi 057 amplified around 430bp fragment in normal and 440bp fragment in QPM inbreds. umc 1066 amplified 460bp fragment in normal and 480 bp fragment in QPM inbreds. Such codominant polymorphism enable their potential utility in MAS programs as they could successfully discriminate between all the three possible genotypes for the opaque-2 gene viz., dominant homozygotes (QQ) and heterozygotes (Qq) and recessive homozygotes (qq). Accordingly, the BC progenies of following crosses viz., CM 212 (QQ) X CML 176 (qq), CM 145 (QQ) X CML 173 (qq) and V 25 (QQ) X CML 176 9 (qq) were subjected to MAS and phenotypic selections.

Foreground Selection for opaque-2 in BC1 generation

Foreground selection using either phi 057 or umc 1066 could identify heterozygous (Qq) progenies that occurred with 50% frequency in a given backcross population. In the BC1population of CM 212 X CML 176, 90 plants were heterozygous for opaque-2 gene out of a total of 209 plants. Similarly, the other two crosses contained 89 and 93 heterozygotes for opaque-2 out of a total of 199 and 205 plants respectively. This confirmed with the expected frequency of heterozygotes for a particular gene in any back cross population. Since these markers are located within the opaque-2 gene, the individuals could be scored directly for the gene eliminating the probability of occurrence of false negatives and positives. Identification of heterozygotes in the seedling stage prior to pollination aided in the rejection of non-target BC progenies (dominant homozygotes) resulting in substantial saving of labor and material resources. Only these marker-identified heterozygotes were further advanced to 2nd back cross generation. Phenotypic selection for earliness, plant vigour and disease resistance reactions among marker selected BC1 progenies resulted in selection of 52, 58 and 63 ears from the three crosses viz., CM 212 X CML 176, CM 145 X CML 173 and V 25 X CML 176.

Foreground and Background selection in BC2 generation

Marker assisted background analysis of the BC/recombinant progenies is useful in determining the relative contribution of donor parent. In BC2 generation, foreground selection aided in eliminating 50% of the undesirable progenies. Out of the remaining marker selected heterozygotes, 20 plants in each cross were subjected to back-ground analysis using 88 SSR markers spanning all the 10 chromosomes of maize genome. The recipient genome content varied from 83.7 to 94.8% in the analyzed BC2 plants. Ears from the five BC2 progenies in each cross that contained maximum amount of recipient genome were chosen for raising further F2 generation.

Figure 1. Identification of homozygous recessive individuals in BC2F2 generation employing umc 1066. The first two lanes correspond to non QPM (P1) and QPM (P2) parents while rest of them are individuals in BC2F2 population. The individual plants indicated by * are homozygotes for recessive opaque2 mutant allele

Phenotypic selection for kernel modification in BC2F2 generation

The kernels from F2 plants segregated for hardness and different levels of modification. In each cross, it could be observed through light box screening that three classes of kernel modification existed viz., less than 25%, 25-50% and more than 50% opaque. The frequency of fully opaque and completely modified kernels was very low. This demonstrates that there could be several minor genes controlling kernel modification in quality protein maize. Earlier studies of Lopes and Larkins (1995) revealed existence of two additive modifier genes that significantly influence the endosperm modification in their population. The biochemical analysis of each of these classes showed that tryptophan and lysine content has recorded up to 100% increase in all the three categories. The tryptophan content varied from 0.88 to 1.10% in converted lines of all the categories of modification. The native recipient lines contained 0.38 to 0.41% tryptophan. However, it has been shown in the earlier studies that kernels with more than 50% opaqueness have poor keeping quality owing to higher grain moisture content and susceptibility to storage pests. Hence, only those kernels that displayed less than 50% opaqueness and contained significant enhancement in the lysine and tryptophan content were selected.

Table 1. Biochemical estimation of protein quality parameters in the donor, recipient and converted lines


Total Protein content (%)

Tryptophan content (%)

Endosperm modification

CML 176




CML 173




CM 212




CM 145




50%opaque progeny



Semi-opaque/semi soft

25%opaque progeny




100%opaque progeny




DNA markers allow us to identify the allelic composition of genotypes in a segregating population. As previously described by Tanksley (1989), the main advantages of using DNA markers vs. conventional selection is to accelerate the fixation of recipient alleles at non-target regions and to identify the genotypes containing crossovers close to target genes (Ribaut and Hoisington, 1998). Based on the results obtained in our experiment, we propose that foreground selection in an early (BC1) generation combined with background selection at a later generation (BC2) along with the phenotypic selections for quantitative/continuously distributed traits would result in rapid genetic gain in a cost effective manner.


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