Hereditas 151: 20–27 (2014)
Marker assisted selection of low phytic acid trait in maize (Zea mays L.) S. SURESHKUMAR1, P. TAMILKUMAR2, N. SENTHIL3, P. NAGARAJAN3, A. U. THANGAVELU3, M. RAVEENDRAN1, S. VELLAIKUMAR3, K. N. GANESAN4, R. BALAGOPAL5, G. VIJAYALAKSHMI3 and V. SHOBANA3 1
Department of Plant biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India Department of seed science and technology, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 3 Department of plant molecular biology and bioinformatics, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 4 Center for plant breeding and genetics, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 5 Department of Veterinary and Animal Sciences, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 2
Sureshkumar, S., Tamilkumar, P., Senthil, N., Nagarajan, P., Thangavelu, A.U., Raveendran, M., Vellaikumar, S., Ganesan, K.N., Balagopal, R., Vijayalakshmi, G. and Shobana, V. 2014. Marker assisted selection of low phytic acid trait in maize (Zea mays L.). – Hereditas 151: 20–27. Lund, Sweden. eISSN 1601-5223. Received 27 September 2013. Accepted 13 December 2013. Maize is the third important major food crop. Breeding for low phytate maize genotypes is an effective strategy for decreasing the content of kernel phytic acid (a chelator of cations such as Ca2⫹ and Fe3⫹) and thereby increasing the bioavailability of nutritive minerals in human diet and animal feed. Previous studies have established that a mutant plant with a lpa2-2 allele accumulates less phytic acid in seeds. Therefore, the marker assisted backcross breeding (MABB), which involves introgression of lpa2-2 recessive allele (which confer low phytate trait) from a lpa2-2 mutant line into a well-adapted line using backcrosses and selection of lines possessing lpa2-2 allele in each backcross population using molecular markers, is an effective strategy for developing low phytate maize. So far, no studies have developed any lpa2-2 allele specific molecular markers for this purpose. Here, using backcross and selfed progenies, obtained by crossing low phytate mutant line ‘EC 659418’ (i.e. donor of lpa2-2 allele) into agronomically superior line ‘UMI395’, we have validated that a SSR marker ‘umc2230’, located 0.4 cM downstream of lpa2-2, cosegregate, in a Mendelian fashion, with low phytic acid trait. Therefore umc2230 can be dependably used in MABB for the development of low phytate maize. S. Sureshkumar, Department of Plant biotechnology, Lab no. 218, CPMB & B, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu 641003, India. E-mail: [email protected]
Maize is an important animal feed and human diet. A major concern in maize is its phytic acid (PA) content, which may go up to 4.0 mg g⫺1 of seed. PA, also known as myo-inositol hexakisphosphate, Ins(1,2,3,4,5,6)P6, or InsP6, is a primary storage compound of phosphorus in seeds accounting for up to 80% of the total seed phosphorus and contributing as much as 1.5% to the seed dry weight. The negatively charged phosphate in PA strongly binds to metallic cations of Ca, Fe, K, Mg, Mn and Zn making them insoluble and thus unavailable as nutritional factors (BOHN et al. 2008). When released during food or feed processing or in the gut, PA binds minerals and makes them unavailable and hence PA is an anti-nutritional factor, which causes malnutrition in human (ZHOU and ERDMAN 1995). Also, PA reduces the phosphorous availability required for growth in monogastric animals, which digest PA poorly. Moreover, undigested PA eliminated by the monogastric animals into the environment leads to an increase in phosphorous level in the environment and contributes to water pollution © 2014 The Authors. This is an Open Access article.
through eutrophication (CROMWELL and COFFEY 1991). Supplementation of feed with inorganic phosphate or with industrially produced phytase enzyme, which breaks down PA and releases phosphorous for animal use, can address the phosphorous requirement for animal growth and reduce phosphorous pollution. However, phosphate and phytase supplementation increase the animal feeding costs. Therefore, breeding for low phytate maize genotypes is an effective strategy for decreasing the content of kernel phytic acid (a chelator of mineral cations such as K⫹, Ca2⫹, Fe3⫹, Mg2⫹ and Zn2⫹) and thereby 1) increasing the bioavailability of nutritive minerals in human diet, 2) increasing the availability of phosphorous in animal feed, 3) decreasing the environmental pollution by phosphorous released from undigested and unutilized phytic acid derived from animal feed, and 4) reducing the amount of phytase used to supplement animal feed for breaking down seed derived phytate and release phosphorous for animal growth (ERTL et al. 1998; MENDOZA et al. 1998). DOI: 10.1111/j.1601-5223.2013.00030.x
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Marker assisted selection of low phytic acid trait in maize
Many low phytic acid mutants have been generated successfully by disrupting phytic acid biosynthesis through mutagenesis breeding in maize, rice, barley, and soybean (RABOY et al. 2000) and they were used in genetic breeding for the development of low phytate lines (RABOY et al. 2001). So far, in maize, three low phytic acid (lpa) mutants have been isolated, viz. lpa1, lpa2 and lpa3. These mutant lines are important genetic resources to develop low phytic acid maize crops. The lpa1 mutation is caused by a mutation in a gene that encodes transmembrane transporter protein (ZmMRP4), which is hypothesized to load phytic acid into protein storage vacuoles of maize seed. The lpa2 mutation is caused by a mutation in inosotol phosphate kinase gene (ZmIpk4), which along with other kinases leads to phytic acid synthesis. The lpa2-1 mutation is caused by genomic sequence rearrangement in the ZmIpk. The lpa2-2 mutation, caused by a single nucleotide change (i.e. C to T at nucleotide position 158), generates a stop codon in the N-terminal region of the ZmIpk open reading frame (SHI et al. 2003). The lpa3 mutation is caused by a mutation in a gene that encodes myo-Ins kinase, which catalyzes the production of Ins(3)P1 in maize seed. Compared with wild-type kernels, the lpa 1, lpa2-1, lpa3 mutations achieved 66%, 50% and 50% reduction in phytic acid content, respectively (RABOY et al. 2000; SHI et al. 2005). The lpa2-2 mutation achieved a 30% reduction in phytic acid content and a three-fold increase in inorganic phosphate (SHI et al. 2003). The mutant lines are temperate maize lines that are not adapted to local tropical and subtropical conditions. Therefore, there is a need to have the lpa locus introgressed into locally adapted agronomically superior lines to improve their nutritional benefit. Marker assisted backcross breeding (MABB) provides a great opportunity for transfer of desirable trait of interest into the genetic background of a recipient genotype by recurrent backcrossing and also to recover the recurrent parent genome as rapidly and completely as possible. Therefore, MABB that involves introgression of lpa2-2 recessive allele for low phytate trait from the donor lpa2-2 mutant into a locally well-adapted agronomically superior line using a series of backcrosses and selection of lines possessing lpa2-2 trait from each backcross progenies, with the help of markers, is an effective strategy for developing low phytate maize. The selection of lines possessing lpa2-2 trait from each backcross progenies is a challenging task because it requires destructive sampling to measure the amount of phytic acid in maize grain. Also, the selection takes time and therefore the selection has to be deferred until when adequate seed can be produced to allow destructive sampling. Therefore, the development of a co-dominant molecular marker will enable quicker selection and make maize breeding for LPA efficient and fast, and it will enable the earlier release of lpa2-2 varieties. Recently,
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researchers (NAIDOO et al. 2012, 2013) have successfully developed and applied the lpa1-1 SNP marker for foreground selection in a backcross-breeding programme. Profiling of plants using SNP marker includes a PCR step and a post PCR step (i.e. high resolution melt analysis). However, so far no studies have developed any lpa2-2 allele specific marker that can be used in MABB for selecting plants with lpa2-2 alleles. Therefore, it is desirable to find other markers, such as a SSR (short sequence repeat) marker that is tightly associated with the lpa2-2 allele. The genetic map of chromosome 1 of maize shows that there are several SSR markers linked with lpa2-2 locus in 1.05 bin location of the short arm of chromosome one of maize genome and SSR marker “umc2230” is in proximity (0.4 cM downstream) of the lpa2-2 allele (Fig. 1). In this study, we introgressed lpa2-2 allele (low phytate trait) from a low phytate mutant line ‘EC 659418’ (donor line), developed by Victor Raboy, USDA, into a welladapted agronomically superior line ‘UMI395’ (recipient line) and raised F1 plants. Subsequently F1s are both selfed and backcrossed with ‘UMI395’ (as recurrent parent) to develop F2, BC1F1, BC2F1 and BC1F2 progenies (Fig. 2). By analyzing the above progenies, we attempted to verify whether the ‘umc2230’ marker, which is tightly linked to lpa2-2 allele, cosegregate with the low seed phytic acid
Fig. 1. Genetic map of SSR markers in chromosome 1 of maize. Positions of lpa2-2 allele (indicated by an arrow) and SSR marker ‘umc2230’ (indicated by an arrowhead) are shown.
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Hereditas 151 (2014) F1 was further used to develop ‘backcross progenies’ (i.e. BC1F1 and BC2F1) by back crossing with recurrent parent (UMI 395) and ‘selfed progenies’ (i.e. F2 and BC1F2) by selfing. The parents and 50 plants from each category of derived populations were used to analyze the association/cosegregation of SSR marker ‘umc2230’ and lpa2-2 mutant (low phytic acid) trait and verify the efficiency of umc2230 to identify the low phytate maize lines. Profiling of SSR marker ‘umc2230’ in plants
Fig. 2. General scheme for the development of backcross and selfed progenies. The line UMI 395 (well-adapted agronomically superior line) was used as female and recurrent parent. The line EC659418 (source of lpa2-2 allele that confers low phytate trait) was used as male and donor parent. The numbers 1, 2 and 3 refers to the generation in which the respective crops were raised.
trait and hence, validate that umc2230 can be utilized as an efficient marker in MABB for low phytate maize development. MATERIAL AND METHODS Plant material For this study, UMI 395 (the popular well-adapted agronomically superior and high phytate line) was used as female and recurrent parent. The temperate mutant line EC659418 (source of lpa2-2 allele that confers low phytate trait/donor for low phytate trait) was used as male and donor parent. Backcross breeding programme MABB was attempted for the introgression of the alien low phytate (lpa2-2) locus from the temperate mutant line EC 659418 (lpa2-2 donor) obtained from Victor Raboy, USDA, into UMI 395 line. UMI 395 and EC 659418 lines were raised during Kharif (i.e. cropping season encompassing July to October), 2010 in two staggered sowings at three days interval (to achieve programmed pollination) at the Wheat Research Station, IARI, Wellington, Ooty, due to temperate climatic nature mutant donor line. These two lines were hybridized to generate F1 lines.
A modified version of the CTAB Method (DELLAPORTA et al. 1983) was used for DNA extraction. PCR amplification of SSR marker ‘umc2230’, which encompasses the core repeat (AGC)5, was done using the forward primer 5′ – AACGCGACGACTTCCACAAG- 3′ and the reverse primer 5′ – ACACGTAATGTCCCTACGGTCG-3′. A modified PCR programme (touchdown PCR) was used in order to avoid the amplification of non-specific spurious PCR products by the primers. PCR was performed with the following program; first initial hold was at 95°C for 3 min. The second hold starting with denaturation step at 94°C for 30 s, annealing at 63°C up to 57°C for 30 s (12 cycles, reducing 0.5°C per cycle) and the extension step at 72°C for 45 s. The third hold starting at denaturation temperature of 94°C for 30 s, annealing at 59°C for 30 s and extension at 72°C for 45 s for 45 cycles). Eventually the final extension step was done at 72°C for 10 min. The amplified fragments were resolved in a 3% agarose gel for analyzing the amplicons. Phytic acid estimation in parents and F1 Phytic acid was estimated by the method of DAVIES and REID (1979). One gram of material was ground and extracted with HNO3 by continuous shaking, filtered and made up to suitable volume with water. To 1.4 ml of the filtrate, 1 ml of ferric ammonium sulphate (21.6 mg in 100 ml water) was added, mixed and placed in a boiling water bath for 20 min. The contents were cooled and 5 ml of isoamyl alcohol was added and mixed. To this, 0.1 ml of 25% ammonia solution was added, shaken thoroughly and centrifuged at 3000 rpm for 10 min. The alcoholic layer was separated and the color intensity was read at 465 nm against amyl alcohol blank after 15 min. Sodium phytate standards were run along with the sample. The results were expressed as milligram of phytic acid per gram of seed. Phytic acid phenotyping by high inorganic phosphorous (HIP) assay To analyze the phytic acid trait (i.e. phytic acid phenotyping), we used ‘high inorganic phosphorous’ (HIP)
Hereditas 151 (2014)
Marker assisted selection of low phytic acid trait in maize
assay. Since wild-type seeds contain low levels of inorganic phosphorus, and since reductions in seed phytic acid phosphorus in lpa mutants are usually matched by molar-equivalent increases in inorganic phosphorus, estimation of inorganic phosphorous can help us indirectly estimate the phytic acid content of maize kernels. The seeds obtained were crushed and assayed for free inorganic phosphorous using a microtiter based colorimetric assay (CHEN et al. 1956; RABOY et al. 2000). The phytate reduction was visually classified as high (dark blue color), low (light blue color), Intermediate (intermediate blue color) based on the intensity of blue color developed, which is proportional to the amount of free inorganic phosphorous content in the kernel. In order to ascertain the association between umc2230 marker and phytate trait, phytic acid phenotyping of three sets of four randomly selected seeds, obtained from the cobs of plants (in which the SSR marker had been genotyped by PCR), was done using HIP assay. Subsequently, the correlation between phytic acid trait of cobs and the alleles of the umc2230 was analyzed. For HIP assay, individual seeds were weighed (in order to select seeds of almost uniform weight to get normalized HIP assay results), crushed and extracted overnight in 1 ml of 0.4 M hydrochloric acid per mg (approximate seed weight) at 4°C. Samples (10 μl) of these extracts were mixed with 90 μl distilled water and 100 μl of colorimetric reagent (one volume of 3 M sulphuric acid, one volume 2.5% (w/v) ammonium molybdate, one volume 10% (v/v) ascorbic acid, and two volumes distilled water prepared freshly. The assays were incubated at room temperature for approximately one hour. Individual seed extracts were visually scored for the presence of HIP basing upon Prussian blue color (DOROTHY and HERRETTE 1955) development. Minimum number of seeds to be assayed per cob in a HIP assay In order to study the cosegregation of umc2230 marker trait with phytate trait (i.e. presence of 180 bp allele with low phytate trait, 175 bp allele with high phytate trait, and presence of both the alleles (i.e. 180 bp ⫹ 175 bp) with intermediate phytate trait, in the backcross and selfed progenies, umc2230 profiling was done using DNA isolated from leaves and phytate profiling was done using extracts from kernels of the cobs of the respective plants. However, due to the fact that cob is multi-seeded and the genetic makeup of all the seeds of a cob are different owing to segregation of 180 bp and 175 bp alleles (i.e. 1:2:1 and 1:1 in case of selfed and backcross progenies respectively), it is proper to analyze the phytic acid trait (i.e. whether high, intermediate or low) of individual seeds separately, and not the average phytate trait of a mixture of
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seeds of a cob, in order to know the phytic acid trait of the cob and hence ascertain the correlation between phytate and marker trait in a plant. However, since it is impossible to analyze the phytic acid trait using HIP assay in all the kernels of a cob, and since we know the theoretical outcome of the phytate trait in selfed progenies (i.e. 25% of the cobs will have high phytate seeds, which can produce only light blue color, 25% of the cobs will have low phytate seeds, which can produce only dark blue color, and 50% of the cobs will have intermediate phytate seeds, which can produce only intermediate blue color,) and in backcross progenies (i.e. 50% of the cobs will have high phytate seeds, which can produce only light blue color, and 50% of the cobs will have intermediate phytate seeds, which can produce only intermediate blue color), we used the ‘Bernoulli trial formula’ and found that the minimum number of seeds to be analyzed in a cob in order to conclude whether a cob is heterogeneous or homogenous for phytic acid trait is four. Therefore, we sampled four randomly selected seeds from a cob per replication and three replications were done to ensure the veracity of the HIP results. RESULTS Genetic map of SSR markers in chromosome 1 of maize A genetic map of SSR markers in chromosome 1 of maize was drawn using the map chart 2.2 software using SSR markers information available in MaizeGDB database. It can be seen that SSR marker ‘umc2230’ is in close proximity (0.4 cM from lpa2-2) to lpa2-2 allele (Fig. 1). The development of backcross and selfed progenies In the first generation, UMI 395 and EC659418 were crossed and F1 population was developed. In the second generation, F1 was backcrossed with UMI 395 (used as recurrent parent) for the production of backcross progenies BC1F1. Also F1 was selfed to produce F2 population. In the third generation, BC1F1 was backcrossed with UMI 395 (used as recurrent parent) for the production of backcross progenies BC2F1. Also BC1F1 was selfed to produce BC1F2 population (Fig. 2). Profiling of SSR marker ‘umc2230’ Profiling of umc2230 was done using PCR assay in UMI 395, EC659418 and F1 progenies. For this, leaves from UMI 395, EC659418 and F1 lines were analyzed by PCR. PCR amplification of SSR marker ‘umc2230’ resulted in the amplification of fragments 180 bp and 175 bp in UMI 395 and EC659418 lines, respectively and amplification of both 175 bp and 180 bp in F1 (Fig. 3). It can be seen that
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Fig. 3. Profiling of SSR marker ‘umc2230’ in F1 along with the parents. The figure shows that PCR amplification of SSR marker ‘umc2230’ resulted in the amplicons 180 bp ⫹ 175 bp, 175 bp and 180 bp in F1 (heterozygous), UMI 395 (homozygous for 175 bp) and EC659418 (homozygous for 180 bp) lines respectively, indicating that ‘UMC2230’ is polymorphic in parents and F1 and hence can differentiate all the three lines. Mr denotes marker lane loaded with 100 bp DNA ladder.
umc2230 has the ability to distinguish the parents and F1 through the polymorphism in banding pattern between lines. Further, umc2230 was used to profiling the banding pattern in F2, BC1F1, BC1F2 and BC2F1 progenies (Fig. 4, Supplementary material Appendix 1 Table A1, A2). Analysis of segregation of SSR marker in different generation The pattern of segregation of umc2230 marker in different population (F2, BC1F1, BC1F2 and BC2F1 progenies) was analyzed by χ2-test (Table 1). It revealed that the umc2230 marker alleles (i.e. 180 bp amplicon and 175 bp amplicon) segregated in Mendelian fashion, i.e. 1:2:1 in selfed progenies (F2 and BC1F2 progenies) and 1:1 in backcross (BC1F1 and BC2F1) progenies, indicating that umc2230 is a reliable marker for MABB. Profiling of phytate content Profiling of the phytic acid phenotype was done using HIP assay in UMI 395, EC659418 and F1 progenies. For this, cobs from UMI 395, EC659418 and F1 plants were taken and from each cob, four randomly selected kernels were assayed for their phytate content using HIP assay.
Fig. 4. Profiling of SSR marker ‘umc2230’ in some of the F2 progenies along with the parents EC659418 and UMI 395. Lane 1-395/418-S8-2, lane2-395/418-S8-10, lane 3-395/ 418-S8-3, lane 4-395/418-S8-4, lane 5-395/418-S8-6, lane6395/418-S8-13, lane 7-395/418-S8-6, lane 8-395/418-S8-5 and lane 9-395/418-S8-8.
The mean phytic acid content of EC659418 (which is homozygous for lpa2-2 allele), UMI 395 (homozygous for high phytate allele), and F1 (heterozygous) are 1.6 mg g⫺1, 2.6 mg g⫺1 and 2 mg g⫺1 respectively. In HIP assay, it is possible to visually differentiate kernels based on their phytate content, (i.e. intensity of blue color products (ammonium phosphomolybdate) produced is indirectly proportional to the phytate content and directly proportional to free phosphorus content). Phenotyping of phytic acid in parents and F1 (Fig. 5A) revealed that due to the presence of low phytate and hence high level of inorganic phosphorous, dark blue color was produced in HIP assay by kernels of donor parent (EC659418), which possess low phytate (lpa2-2) allele. Due to the presence of high phytate and hence low level of inorganic phosphorous, light blue color was produced by kernels of recipient parent (UMI 395), which possess wild type (high phytate) allele. However, due to the presence of intermediate level of phytic acid, intermediate intensity of blue color was produced by kernels of F1s, which possess both low and high phytate alleles. Phenotyping of phytic acid in backcross population (Fig. 5B, BC1F1 data in Supplementary material Appendix 1 Table A1, BC2F1 data in Table A2) revealed that 50% of the cobs of backcross population had kernels expressing only high phytate trait (light blue color) and
Table 1. Analysis of segregation of umc2230 marker in different populations. umc2230 marker type⫹ Generation F2 BC1F1 BC1F2 BC2F1
Total no. of plants
Expected ratio
Donor parent type (180 bp)
Heterozygous type (180/175 bp)
Recurrent parent type (175 bp)
χ2test∗
50 50 50 50
1:2:1∗ 1:1 1:2:1 1:1
11 – 9 –
26 24 27 22
13 26 14 28
0.24 0.08 1.32 0.72
∗probability value in χ2-distribution table at 0.05 significant levels is 5.991 and 3.84 for 1:2:1 ratio and 1:1 ratio, respectively. †50 seeds from every cob were raised and genotyped for this analysis. ‘S’ indicates selfing and ‘C’ denotes backcrossing.
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Fig. 5. Phenotyping of phytic acid. (A) Phenotyping of phytic acid in parents and F1. Photo of microtiter plates showing the result of HIP assay done using kernels of parents and F1 progeny is shown in panel A(i) and its pictorial representation is shown in the panel A(ii). Dark blue color is produced by kernels of donor parent (EC659418), which possesses low phytate (lpa2-2) allele. Light blue color is produced by kernels of recipient parent (UMI 395), which possesses wild type (high phytate) allele. Intermediate blue color is produced by kernels of heterozygous F1s, which possesses both low and high phytate alleles. S1, S2, S3 and S4 denote four randomly selected kernels selected for HIP assay (B) Phenotyping of phytic acid in backcross population (i.e. BC1F1, BC2F1). 50% of the cobs of backcross population are expected to have kernels expressing only high phytate trait. A sample phenotyping of phytic acid in these backcross population is shown in panel B(i). Remaining 50 % of the cobs of backcross population is expected to have a mixture of kernels (i.e. some expressing high and some intermediate phytate trait). A sample phenotyping of phytic acid in these backcross population is shown in B(ii). S1, S2, S3 and S4 denote four randomly selected kernels selected for HIP assay and R1, R2 and R3 denote three replications of HIP assay. (C) Phenotyping of phytic acid in selfed population (i.e. F2, BC1F2). 25% of cobs of selfed population are expected to have kernels expressing only high phytate trait. A sample phenotyping of phytic acid in these selfed populations is shown in C(i). 50% of the cobs of selfed population are expected to have a mixture of kernels (i.e. some expressing high, some intermediate and some low phytate trait). A sample phenotyping of phytic acid in these selfed populations is shown in C(ii). 25% of the cobs of selfed population is expected to have kernels expressing only low phytate trait. A sample phenotyping of phytic acid in these selfed populations is shown in C(iii). S1, S2, S3 and S4 denote four randomly selected kernels selected for HIP assay and R1, R2 and R3 denote three replications of HIP assay.
the remaining 50% of the cobs of backcross population had mixture of kernels (i.e. some expressing high phytate (light blue color) and some intermediate phytate trait (intermediate blue color)).
Phenotyping of the phytic acid in selfed population (Fig. 5C, F2 data in Supplementary material Appendix 1 Table A1, BC1F2 data in Table A2) revealed that 25% of cobs of selfed population had kernels expressing only
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high phytate trait (light blue color), and 25% of the cobs of selfed population had kernels expressing only low phytate trait (dark blue color). The remaining 50% of the cobs of selfed population had mixture of kernels (i.e. some expressing high, some intermediate and some low phytate trait). Importantly, phenotyping of phytic acid in backcross and selfed population (Supplementary material Appendix 1 Table A1, A2) indicated that the allele 180 bp is associated with low phytate trait and allele 175 bp is associated with high phytate trait, and alleles 180 bp ⫹ 175 bp is associated with intermediate phytate trait. DISCUSSION Breeding for maize genotypes with low kernel phytic acid content is an effective strategy for increasing the bioavailability of minerals in human diet and availability of phosphorous in animal feed, decreasing the environmental phosphorous pollution, and reducing the cost of phytase supplementation in animal feeds. The MABB that involves introgression of lpa2-2 recessive allele (which confers low phytate trait) from the donor lpa2-2 mutant into a well-adapted agronomically superior line with the help of a series of backcrosses and selection of lines possessing lpa2-2 allele in each backcross population with the help of markers is an effective strategy for developing low phytate maize. So far, no studies have developed any lpa2-2 allele specific marker that can be used in MABB for selecting plants with lpa2-2 alleles. Therefore, it is desirable to find markers, such as SSR marker, that is tightly associated and hence cosegregating with lpa2-2 allele. Therefore this study was taken up with the objective of finding a SSR marker that co-segregate with lpa2-2 allele. From the genetic map of SSR marker, it was found that SSR marker ‘umc2230’ is in proximity (0.4 cM downstream) with lpa2-2. Therefore, we aimed to verify whether umc2230 can co-segregate in a Mendelian fashion with low phytic acid trait and hence linked to lpa2-2 allele. In this regard, we developed F1, F2, BC1F1, BC2F1 and BC1F2 progenies by crossing lpa2-2 mutant line ‘EC 659418’ (which showed low phytate trait) into a well-adapted agronomically superior line ‘UMI395’ (which had high phytate trait). We confirmed, by PCR profiling of the umc2230 marker, that umc2230 could easily differentiate the parents and F1 by showing polymorphism in the amplicons in PCR. Next, we profiled the phytic acid trait (using HIP assay) and umc2230 marker trait (by PCR) in F2, BC1F1, BC2F1 and BC1F2 progenies. Our results showed that the umc2230 alleles (180 bp and 175 bp fragments) segregated in Mendelian fashion, i.e. 1:2:1 in selfed progenies (F2 and BC1F2 progenies) and 1:1 in backcross (BC1F1 and BC2F1) progenies. The results
Hereditas 151 (2014) also showed that the plant with 180 bp amplicon expressed low phytate trait, plant with 175 bp amplicon expressed high phytate trait and the plant had both the amplicons expressed intermediate phytate trait in HIP assay, indicating that allele 180 bp is associated with low phytate trait and allele 175 bp is associated with high phytate trait. In short, this study has validated that SSR marker umc2230 cosegregates with phytate trait conferred by lpa2-2 alleles in a mendelian fashion in both selfed and backcross progenies, and hence umc2230 marker is linked with lpa2-2 allele and therefore, umc2230 can be dependably used in a MABB for the efficient selection and transfer of low phytate trait to any well adapted plant for the development of low phytate nutritive maize lines. Acknowledgements – The financial support by Dept of Biotechnology, Ministry of Science and Technology, Government of India, sponsored project on ‘The program support for research and development in agricultural biotechnology-Phase-II at TNAU, Coimbatore’ (Project sanction no. No.BT/PR5095/ AGR/2/847/2012, Date 16/05/2012) is kindly acknowledged. The lpa2-2 mutant line supplied by Dr. Victor Raboy, USDA, is kindly acknowledged.
REFERENCES Bohn, L., Meyer, A. S. and Rasmussen, S. K. 2008. Phytate: impact on environment and human nutrition. A challenge for molecular breeding. – J. Zhejiang Univ. Sci. B 9: 165–191. Chen, P. S., Toribara, T. Y. and Warner, H. 1956. Microdetermination of phosphorus. – Anal. Chem. 28: 1756–1758. Cromwell, G. L. and Coffey, R. D. 1991. Phosphorus: a key essential nutrient, yet a possible major pollutant. Its central role in animal nutrition. – In: Lyons, T. P. (ed.), Biotechnology in the feed industry. Alltech Tech Publishers, Nicholasville, KY, pp. 133–145. Davies, N. and Reid, H. 1979. An evaluation of the phytate, zinc, copper, iron and manganese contents of, and Zn availability from, soya-based textured-vegetable-protein meat-substitutes or meat-extenders. – Br. J. Nutr. 41: 579–589. Dellaporta, S. L., Wood, J. and Hicks, J. B. 1983. A plant DNA Mini preparation ver. 2. – Plant Mol. Biol. Rep. 1: 19–22. Dorothy, R. and Harrette, B. 1955. The application of the Prussian blue stain to previously stained films of blood and bone marrow. – Blood J. 10: 160–166. Ertl, D. S., Young, K. A. and Raboy, V. 1998. Plant genetic approaches to phosphorus management in agricultural production. – J. Environ. Qual. 27: 299–304. Mendoza, C., Viteri, F. E., Lonnerdal, B. et al. 1998. Effect of genetically modified, low-phytic acid maize on absorption of iron from tortillas. – Am. J. Clin. Nutr. 68: 1123–1128. Naidoo, R., Watson, G. M. F., Derera, J. et al. 2012. Markerassisted selection for low phytic acid (lpa1-1) with single nucleotide polymorphism marker and amplified fragment length polymorphisms for background selection in a maize backcross breeding programme. – Mol. Breeding 30: 1207–1217. Naidoo, R., Watson, G. M. F., Tongoona, P. et al. 2013. Development of a single nucleotide polymorphism (SNP)
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marker for detection of the low phytic acid (lpa1-1) gene used during maize breeding. – Afr. J. Biotechnol. 12: 892–900. Raboy, V., Gerbasi, P. F., Young, K. A. et al. 2000. Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1. – Plant Physiol. 124: 355–368. Raboy, V., Young, K. A., Dorsch, J. A. et al. 2001. Genetics and breeding of seed phosphorus and phytic acid. – J. Plant Physiol. 158: 489–497.
Supplementary material (available as Appendix HRD-00030 at ⬍www.oikosoffice.lu.se/appendix⬎). Appendix 1.
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Shi, J., Wang, H. Y., Wu, J. et al. 2003. The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene. – Plant Physiol. 131: 507–515. Shi, J., Wang, H. and Hazebroek, J. 2005. The maizelowphyticacid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds. – Plant J. 42: 708–719. Zhou, J. R. and Erdman, J. W. Jr. 1995. Phytic acid in health and disease. – Crit. Rev. Food Sci. Nutr. 35: 495–508.
Marker assisted selection of low phytic acid trait in maize (Zea mays L.) S. SURESHKUMAR1, P. TAMILKUMAR2, N. SENTHIL3, P. NAGARAJAN3, A. U. THANGAVELU3, M. RAVEENDRAN1, S. VELLAIKUMAR3, K. N. GANESAN4, R. BALAGOPAL5, G. VIJAYALAKSHMI3 and V. SHOBANA3 1
Department of Plant biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India Department of seed science and technology, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 3 Department of plant molecular biology and bioinformatics, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 4 Center for plant breeding and genetics, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 5 Department of Veterinary and Animal Sciences, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu, India 2
Sureshkumar, S., Tamilkumar, P., Senthil, N., Nagarajan, P., Thangavelu, A.U., Raveendran, M., Vellaikumar, S., Ganesan, K.N., Balagopal, R., Vijayalakshmi, G. and Shobana, V. 2014. Marker assisted selection of low phytic acid trait in maize (Zea mays L.). – Hereditas 151: 20–27. Lund, Sweden. eISSN 1601-5223. Received 27 September 2013. Accepted 13 December 2013. Maize is the third important major food crop. Breeding for low phytate maize genotypes is an effective strategy for decreasing the content of kernel phytic acid (a chelator of cations such as Ca2⫹ and Fe3⫹) and thereby increasing the bioavailability of nutritive minerals in human diet and animal feed. Previous studies have established that a mutant plant with a lpa2-2 allele accumulates less phytic acid in seeds. Therefore, the marker assisted backcross breeding (MABB), which involves introgression of lpa2-2 recessive allele (which confer low phytate trait) from a lpa2-2 mutant line into a well-adapted line using backcrosses and selection of lines possessing lpa2-2 allele in each backcross population using molecular markers, is an effective strategy for developing low phytate maize. So far, no studies have developed any lpa2-2 allele specific molecular markers for this purpose. Here, using backcross and selfed progenies, obtained by crossing low phytate mutant line ‘EC 659418’ (i.e. donor of lpa2-2 allele) into agronomically superior line ‘UMI395’, we have validated that a SSR marker ‘umc2230’, located 0.4 cM downstream of lpa2-2, cosegregate, in a Mendelian fashion, with low phytic acid trait. Therefore umc2230 can be dependably used in MABB for the development of low phytate maize. S. Sureshkumar, Department of Plant biotechnology, Lab no. 218, CPMB & B, Tamil Nadu Agricultural University, Coimbatore, Tamilnadu 641003, India. E-mail: [email protected]
Maize is an important animal feed and human diet. A major concern in maize is its phytic acid (PA) content, which may go up to 4.0 mg g⫺1 of seed. PA, also known as myo-inositol hexakisphosphate, Ins(1,2,3,4,5,6)P6, or InsP6, is a primary storage compound of phosphorus in seeds accounting for up to 80% of the total seed phosphorus and contributing as much as 1.5% to the seed dry weight. The negatively charged phosphate in PA strongly binds to metallic cations of Ca, Fe, K, Mg, Mn and Zn making them insoluble and thus unavailable as nutritional factors (BOHN et al. 2008). When released during food or feed processing or in the gut, PA binds minerals and makes them unavailable and hence PA is an anti-nutritional factor, which causes malnutrition in human (ZHOU and ERDMAN 1995). Also, PA reduces the phosphorous availability required for growth in monogastric animals, which digest PA poorly. Moreover, undigested PA eliminated by the monogastric animals into the environment leads to an increase in phosphorous level in the environment and contributes to water pollution © 2014 The Authors. This is an Open Access article.
through eutrophication (CROMWELL and COFFEY 1991). Supplementation of feed with inorganic phosphate or with industrially produced phytase enzyme, which breaks down PA and releases phosphorous for animal use, can address the phosphorous requirement for animal growth and reduce phosphorous pollution. However, phosphate and phytase supplementation increase the animal feeding costs. Therefore, breeding for low phytate maize genotypes is an effective strategy for decreasing the content of kernel phytic acid (a chelator of mineral cations such as K⫹, Ca2⫹, Fe3⫹, Mg2⫹ and Zn2⫹) and thereby 1) increasing the bioavailability of nutritive minerals in human diet, 2) increasing the availability of phosphorous in animal feed, 3) decreasing the environmental pollution by phosphorous released from undigested and unutilized phytic acid derived from animal feed, and 4) reducing the amount of phytase used to supplement animal feed for breaking down seed derived phytate and release phosphorous for animal growth (ERTL et al. 1998; MENDOZA et al. 1998). DOI: 10.1111/j.1601-5223.2013.00030.x
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Many low phytic acid mutants have been generated successfully by disrupting phytic acid biosynthesis through mutagenesis breeding in maize, rice, barley, and soybean (RABOY et al. 2000) and they were used in genetic breeding for the development of low phytate lines (RABOY et al. 2001). So far, in maize, three low phytic acid (lpa) mutants have been isolated, viz. lpa1, lpa2 and lpa3. These mutant lines are important genetic resources to develop low phytic acid maize crops. The lpa1 mutation is caused by a mutation in a gene that encodes transmembrane transporter protein (ZmMRP4), which is hypothesized to load phytic acid into protein storage vacuoles of maize seed. The lpa2 mutation is caused by a mutation in inosotol phosphate kinase gene (ZmIpk4), which along with other kinases leads to phytic acid synthesis. The lpa2-1 mutation is caused by genomic sequence rearrangement in the ZmIpk. The lpa2-2 mutation, caused by a single nucleotide change (i.e. C to T at nucleotide position 158), generates a stop codon in the N-terminal region of the ZmIpk open reading frame (SHI et al. 2003). The lpa3 mutation is caused by a mutation in a gene that encodes myo-Ins kinase, which catalyzes the production of Ins(3)P1 in maize seed. Compared with wild-type kernels, the lpa 1, lpa2-1, lpa3 mutations achieved 66%, 50% and 50% reduction in phytic acid content, respectively (RABOY et al. 2000; SHI et al. 2005). The lpa2-2 mutation achieved a 30% reduction in phytic acid content and a three-fold increase in inorganic phosphate (SHI et al. 2003). The mutant lines are temperate maize lines that are not adapted to local tropical and subtropical conditions. Therefore, there is a need to have the lpa locus introgressed into locally adapted agronomically superior lines to improve their nutritional benefit. Marker assisted backcross breeding (MABB) provides a great opportunity for transfer of desirable trait of interest into the genetic background of a recipient genotype by recurrent backcrossing and also to recover the recurrent parent genome as rapidly and completely as possible. Therefore, MABB that involves introgression of lpa2-2 recessive allele for low phytate trait from the donor lpa2-2 mutant into a locally well-adapted agronomically superior line using a series of backcrosses and selection of lines possessing lpa2-2 trait from each backcross progenies, with the help of markers, is an effective strategy for developing low phytate maize. The selection of lines possessing lpa2-2 trait from each backcross progenies is a challenging task because it requires destructive sampling to measure the amount of phytic acid in maize grain. Also, the selection takes time and therefore the selection has to be deferred until when adequate seed can be produced to allow destructive sampling. Therefore, the development of a co-dominant molecular marker will enable quicker selection and make maize breeding for LPA efficient and fast, and it will enable the earlier release of lpa2-2 varieties. Recently,
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researchers (NAIDOO et al. 2012, 2013) have successfully developed and applied the lpa1-1 SNP marker for foreground selection in a backcross-breeding programme. Profiling of plants using SNP marker includes a PCR step and a post PCR step (i.e. high resolution melt analysis). However, so far no studies have developed any lpa2-2 allele specific marker that can be used in MABB for selecting plants with lpa2-2 alleles. Therefore, it is desirable to find other markers, such as a SSR (short sequence repeat) marker that is tightly associated with the lpa2-2 allele. The genetic map of chromosome 1 of maize shows that there are several SSR markers linked with lpa2-2 locus in 1.05 bin location of the short arm of chromosome one of maize genome and SSR marker “umc2230” is in proximity (0.4 cM downstream) of the lpa2-2 allele (Fig. 1). In this study, we introgressed lpa2-2 allele (low phytate trait) from a low phytate mutant line ‘EC 659418’ (donor line), developed by Victor Raboy, USDA, into a welladapted agronomically superior line ‘UMI395’ (recipient line) and raised F1 plants. Subsequently F1s are both selfed and backcrossed with ‘UMI395’ (as recurrent parent) to develop F2, BC1F1, BC2F1 and BC1F2 progenies (Fig. 2). By analyzing the above progenies, we attempted to verify whether the ‘umc2230’ marker, which is tightly linked to lpa2-2 allele, cosegregate with the low seed phytic acid
Fig. 1. Genetic map of SSR markers in chromosome 1 of maize. Positions of lpa2-2 allele (indicated by an arrow) and SSR marker ‘umc2230’ (indicated by an arrowhead) are shown.
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Hereditas 151 (2014) F1 was further used to develop ‘backcross progenies’ (i.e. BC1F1 and BC2F1) by back crossing with recurrent parent (UMI 395) and ‘selfed progenies’ (i.e. F2 and BC1F2) by selfing. The parents and 50 plants from each category of derived populations were used to analyze the association/cosegregation of SSR marker ‘umc2230’ and lpa2-2 mutant (low phytic acid) trait and verify the efficiency of umc2230 to identify the low phytate maize lines. Profiling of SSR marker ‘umc2230’ in plants
Fig. 2. General scheme for the development of backcross and selfed progenies. The line UMI 395 (well-adapted agronomically superior line) was used as female and recurrent parent. The line EC659418 (source of lpa2-2 allele that confers low phytate trait) was used as male and donor parent. The numbers 1, 2 and 3 refers to the generation in which the respective crops were raised.
trait and hence, validate that umc2230 can be utilized as an efficient marker in MABB for low phytate maize development. MATERIAL AND METHODS Plant material For this study, UMI 395 (the popular well-adapted agronomically superior and high phytate line) was used as female and recurrent parent. The temperate mutant line EC659418 (source of lpa2-2 allele that confers low phytate trait/donor for low phytate trait) was used as male and donor parent. Backcross breeding programme MABB was attempted for the introgression of the alien low phytate (lpa2-2) locus from the temperate mutant line EC 659418 (lpa2-2 donor) obtained from Victor Raboy, USDA, into UMI 395 line. UMI 395 and EC 659418 lines were raised during Kharif (i.e. cropping season encompassing July to October), 2010 in two staggered sowings at three days interval (to achieve programmed pollination) at the Wheat Research Station, IARI, Wellington, Ooty, due to temperate climatic nature mutant donor line. These two lines were hybridized to generate F1 lines.
A modified version of the CTAB Method (DELLAPORTA et al. 1983) was used for DNA extraction. PCR amplification of SSR marker ‘umc2230’, which encompasses the core repeat (AGC)5, was done using the forward primer 5′ – AACGCGACGACTTCCACAAG- 3′ and the reverse primer 5′ – ACACGTAATGTCCCTACGGTCG-3′. A modified PCR programme (touchdown PCR) was used in order to avoid the amplification of non-specific spurious PCR products by the primers. PCR was performed with the following program; first initial hold was at 95°C for 3 min. The second hold starting with denaturation step at 94°C for 30 s, annealing at 63°C up to 57°C for 30 s (12 cycles, reducing 0.5°C per cycle) and the extension step at 72°C for 45 s. The third hold starting at denaturation temperature of 94°C for 30 s, annealing at 59°C for 30 s and extension at 72°C for 45 s for 45 cycles). Eventually the final extension step was done at 72°C for 10 min. The amplified fragments were resolved in a 3% agarose gel for analyzing the amplicons. Phytic acid estimation in parents and F1 Phytic acid was estimated by the method of DAVIES and REID (1979). One gram of material was ground and extracted with HNO3 by continuous shaking, filtered and made up to suitable volume with water. To 1.4 ml of the filtrate, 1 ml of ferric ammonium sulphate (21.6 mg in 100 ml water) was added, mixed and placed in a boiling water bath for 20 min. The contents were cooled and 5 ml of isoamyl alcohol was added and mixed. To this, 0.1 ml of 25% ammonia solution was added, shaken thoroughly and centrifuged at 3000 rpm for 10 min. The alcoholic layer was separated and the color intensity was read at 465 nm against amyl alcohol blank after 15 min. Sodium phytate standards were run along with the sample. The results were expressed as milligram of phytic acid per gram of seed. Phytic acid phenotyping by high inorganic phosphorous (HIP) assay To analyze the phytic acid trait (i.e. phytic acid phenotyping), we used ‘high inorganic phosphorous’ (HIP)
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assay. Since wild-type seeds contain low levels of inorganic phosphorus, and since reductions in seed phytic acid phosphorus in lpa mutants are usually matched by molar-equivalent increases in inorganic phosphorus, estimation of inorganic phosphorous can help us indirectly estimate the phytic acid content of maize kernels. The seeds obtained were crushed and assayed for free inorganic phosphorous using a microtiter based colorimetric assay (CHEN et al. 1956; RABOY et al. 2000). The phytate reduction was visually classified as high (dark blue color), low (light blue color), Intermediate (intermediate blue color) based on the intensity of blue color developed, which is proportional to the amount of free inorganic phosphorous content in the kernel. In order to ascertain the association between umc2230 marker and phytate trait, phytic acid phenotyping of three sets of four randomly selected seeds, obtained from the cobs of plants (in which the SSR marker had been genotyped by PCR), was done using HIP assay. Subsequently, the correlation between phytic acid trait of cobs and the alleles of the umc2230 was analyzed. For HIP assay, individual seeds were weighed (in order to select seeds of almost uniform weight to get normalized HIP assay results), crushed and extracted overnight in 1 ml of 0.4 M hydrochloric acid per mg (approximate seed weight) at 4°C. Samples (10 μl) of these extracts were mixed with 90 μl distilled water and 100 μl of colorimetric reagent (one volume of 3 M sulphuric acid, one volume 2.5% (w/v) ammonium molybdate, one volume 10% (v/v) ascorbic acid, and two volumes distilled water prepared freshly. The assays were incubated at room temperature for approximately one hour. Individual seed extracts were visually scored for the presence of HIP basing upon Prussian blue color (DOROTHY and HERRETTE 1955) development. Minimum number of seeds to be assayed per cob in a HIP assay In order to study the cosegregation of umc2230 marker trait with phytate trait (i.e. presence of 180 bp allele with low phytate trait, 175 bp allele with high phytate trait, and presence of both the alleles (i.e. 180 bp ⫹ 175 bp) with intermediate phytate trait, in the backcross and selfed progenies, umc2230 profiling was done using DNA isolated from leaves and phytate profiling was done using extracts from kernels of the cobs of the respective plants. However, due to the fact that cob is multi-seeded and the genetic makeup of all the seeds of a cob are different owing to segregation of 180 bp and 175 bp alleles (i.e. 1:2:1 and 1:1 in case of selfed and backcross progenies respectively), it is proper to analyze the phytic acid trait (i.e. whether high, intermediate or low) of individual seeds separately, and not the average phytate trait of a mixture of
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seeds of a cob, in order to know the phytic acid trait of the cob and hence ascertain the correlation between phytate and marker trait in a plant. However, since it is impossible to analyze the phytic acid trait using HIP assay in all the kernels of a cob, and since we know the theoretical outcome of the phytate trait in selfed progenies (i.e. 25% of the cobs will have high phytate seeds, which can produce only light blue color, 25% of the cobs will have low phytate seeds, which can produce only dark blue color, and 50% of the cobs will have intermediate phytate seeds, which can produce only intermediate blue color,) and in backcross progenies (i.e. 50% of the cobs will have high phytate seeds, which can produce only light blue color, and 50% of the cobs will have intermediate phytate seeds, which can produce only intermediate blue color), we used the ‘Bernoulli trial formula’ and found that the minimum number of seeds to be analyzed in a cob in order to conclude whether a cob is heterogeneous or homogenous for phytic acid trait is four. Therefore, we sampled four randomly selected seeds from a cob per replication and three replications were done to ensure the veracity of the HIP results. RESULTS Genetic map of SSR markers in chromosome 1 of maize A genetic map of SSR markers in chromosome 1 of maize was drawn using the map chart 2.2 software using SSR markers information available in MaizeGDB database. It can be seen that SSR marker ‘umc2230’ is in close proximity (0.4 cM from lpa2-2) to lpa2-2 allele (Fig. 1). The development of backcross and selfed progenies In the first generation, UMI 395 and EC659418 were crossed and F1 population was developed. In the second generation, F1 was backcrossed with UMI 395 (used as recurrent parent) for the production of backcross progenies BC1F1. Also F1 was selfed to produce F2 population. In the third generation, BC1F1 was backcrossed with UMI 395 (used as recurrent parent) for the production of backcross progenies BC2F1. Also BC1F1 was selfed to produce BC1F2 population (Fig. 2). Profiling of SSR marker ‘umc2230’ Profiling of umc2230 was done using PCR assay in UMI 395, EC659418 and F1 progenies. For this, leaves from UMI 395, EC659418 and F1 lines were analyzed by PCR. PCR amplification of SSR marker ‘umc2230’ resulted in the amplification of fragments 180 bp and 175 bp in UMI 395 and EC659418 lines, respectively and amplification of both 175 bp and 180 bp in F1 (Fig. 3). It can be seen that
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Fig. 3. Profiling of SSR marker ‘umc2230’ in F1 along with the parents. The figure shows that PCR amplification of SSR marker ‘umc2230’ resulted in the amplicons 180 bp ⫹ 175 bp, 175 bp and 180 bp in F1 (heterozygous), UMI 395 (homozygous for 175 bp) and EC659418 (homozygous for 180 bp) lines respectively, indicating that ‘UMC2230’ is polymorphic in parents and F1 and hence can differentiate all the three lines. Mr denotes marker lane loaded with 100 bp DNA ladder.
umc2230 has the ability to distinguish the parents and F1 through the polymorphism in banding pattern between lines. Further, umc2230 was used to profiling the banding pattern in F2, BC1F1, BC1F2 and BC2F1 progenies (Fig. 4, Supplementary material Appendix 1 Table A1, A2). Analysis of segregation of SSR marker in different generation The pattern of segregation of umc2230 marker in different population (F2, BC1F1, BC1F2 and BC2F1 progenies) was analyzed by χ2-test (Table 1). It revealed that the umc2230 marker alleles (i.e. 180 bp amplicon and 175 bp amplicon) segregated in Mendelian fashion, i.e. 1:2:1 in selfed progenies (F2 and BC1F2 progenies) and 1:1 in backcross (BC1F1 and BC2F1) progenies, indicating that umc2230 is a reliable marker for MABB. Profiling of phytate content Profiling of the phytic acid phenotype was done using HIP assay in UMI 395, EC659418 and F1 progenies. For this, cobs from UMI 395, EC659418 and F1 plants were taken and from each cob, four randomly selected kernels were assayed for their phytate content using HIP assay.
Fig. 4. Profiling of SSR marker ‘umc2230’ in some of the F2 progenies along with the parents EC659418 and UMI 395. Lane 1-395/418-S8-2, lane2-395/418-S8-10, lane 3-395/ 418-S8-3, lane 4-395/418-S8-4, lane 5-395/418-S8-6, lane6395/418-S8-13, lane 7-395/418-S8-6, lane 8-395/418-S8-5 and lane 9-395/418-S8-8.
The mean phytic acid content of EC659418 (which is homozygous for lpa2-2 allele), UMI 395 (homozygous for high phytate allele), and F1 (heterozygous) are 1.6 mg g⫺1, 2.6 mg g⫺1 and 2 mg g⫺1 respectively. In HIP assay, it is possible to visually differentiate kernels based on their phytate content, (i.e. intensity of blue color products (ammonium phosphomolybdate) produced is indirectly proportional to the phytate content and directly proportional to free phosphorus content). Phenotyping of phytic acid in parents and F1 (Fig. 5A) revealed that due to the presence of low phytate and hence high level of inorganic phosphorous, dark blue color was produced in HIP assay by kernels of donor parent (EC659418), which possess low phytate (lpa2-2) allele. Due to the presence of high phytate and hence low level of inorganic phosphorous, light blue color was produced by kernels of recipient parent (UMI 395), which possess wild type (high phytate) allele. However, due to the presence of intermediate level of phytic acid, intermediate intensity of blue color was produced by kernels of F1s, which possess both low and high phytate alleles. Phenotyping of phytic acid in backcross population (Fig. 5B, BC1F1 data in Supplementary material Appendix 1 Table A1, BC2F1 data in Table A2) revealed that 50% of the cobs of backcross population had kernels expressing only high phytate trait (light blue color) and
Table 1. Analysis of segregation of umc2230 marker in different populations. umc2230 marker type⫹ Generation F2 BC1F1 BC1F2 BC2F1
Total no. of plants
Expected ratio
Donor parent type (180 bp)
Heterozygous type (180/175 bp)
Recurrent parent type (175 bp)
χ2test∗
50 50 50 50
1:2:1∗ 1:1 1:2:1 1:1
11 – 9 –
26 24 27 22
13 26 14 28
0.24 0.08 1.32 0.72
∗probability value in χ2-distribution table at 0.05 significant levels is 5.991 and 3.84 for 1:2:1 ratio and 1:1 ratio, respectively. †50 seeds from every cob were raised and genotyped for this analysis. ‘S’ indicates selfing and ‘C’ denotes backcrossing.
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Fig. 5. Phenotyping of phytic acid. (A) Phenotyping of phytic acid in parents and F1. Photo of microtiter plates showing the result of HIP assay done using kernels of parents and F1 progeny is shown in panel A(i) and its pictorial representation is shown in the panel A(ii). Dark blue color is produced by kernels of donor parent (EC659418), which possesses low phytate (lpa2-2) allele. Light blue color is produced by kernels of recipient parent (UMI 395), which possesses wild type (high phytate) allele. Intermediate blue color is produced by kernels of heterozygous F1s, which possesses both low and high phytate alleles. S1, S2, S3 and S4 denote four randomly selected kernels selected for HIP assay (B) Phenotyping of phytic acid in backcross population (i.e. BC1F1, BC2F1). 50% of the cobs of backcross population are expected to have kernels expressing only high phytate trait. A sample phenotyping of phytic acid in these backcross population is shown in panel B(i). Remaining 50 % of the cobs of backcross population is expected to have a mixture of kernels (i.e. some expressing high and some intermediate phytate trait). A sample phenotyping of phytic acid in these backcross population is shown in B(ii). S1, S2, S3 and S4 denote four randomly selected kernels selected for HIP assay and R1, R2 and R3 denote three replications of HIP assay. (C) Phenotyping of phytic acid in selfed population (i.e. F2, BC1F2). 25% of cobs of selfed population are expected to have kernels expressing only high phytate trait. A sample phenotyping of phytic acid in these selfed populations is shown in C(i). 50% of the cobs of selfed population are expected to have a mixture of kernels (i.e. some expressing high, some intermediate and some low phytate trait). A sample phenotyping of phytic acid in these selfed populations is shown in C(ii). 25% of the cobs of selfed population is expected to have kernels expressing only low phytate trait. A sample phenotyping of phytic acid in these selfed populations is shown in C(iii). S1, S2, S3 and S4 denote four randomly selected kernels selected for HIP assay and R1, R2 and R3 denote three replications of HIP assay.
the remaining 50% of the cobs of backcross population had mixture of kernels (i.e. some expressing high phytate (light blue color) and some intermediate phytate trait (intermediate blue color)).
Phenotyping of the phytic acid in selfed population (Fig. 5C, F2 data in Supplementary material Appendix 1 Table A1, BC1F2 data in Table A2) revealed that 25% of cobs of selfed population had kernels expressing only
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high phytate trait (light blue color), and 25% of the cobs of selfed population had kernels expressing only low phytate trait (dark blue color). The remaining 50% of the cobs of selfed population had mixture of kernels (i.e. some expressing high, some intermediate and some low phytate trait). Importantly, phenotyping of phytic acid in backcross and selfed population (Supplementary material Appendix 1 Table A1, A2) indicated that the allele 180 bp is associated with low phytate trait and allele 175 bp is associated with high phytate trait, and alleles 180 bp ⫹ 175 bp is associated with intermediate phytate trait. DISCUSSION Breeding for maize genotypes with low kernel phytic acid content is an effective strategy for increasing the bioavailability of minerals in human diet and availability of phosphorous in animal feed, decreasing the environmental phosphorous pollution, and reducing the cost of phytase supplementation in animal feeds. The MABB that involves introgression of lpa2-2 recessive allele (which confers low phytate trait) from the donor lpa2-2 mutant into a well-adapted agronomically superior line with the help of a series of backcrosses and selection of lines possessing lpa2-2 allele in each backcross population with the help of markers is an effective strategy for developing low phytate maize. So far, no studies have developed any lpa2-2 allele specific marker that can be used in MABB for selecting plants with lpa2-2 alleles. Therefore, it is desirable to find markers, such as SSR marker, that is tightly associated and hence cosegregating with lpa2-2 allele. Therefore this study was taken up with the objective of finding a SSR marker that co-segregate with lpa2-2 allele. From the genetic map of SSR marker, it was found that SSR marker ‘umc2230’ is in proximity (0.4 cM downstream) with lpa2-2. Therefore, we aimed to verify whether umc2230 can co-segregate in a Mendelian fashion with low phytic acid trait and hence linked to lpa2-2 allele. In this regard, we developed F1, F2, BC1F1, BC2F1 and BC1F2 progenies by crossing lpa2-2 mutant line ‘EC 659418’ (which showed low phytate trait) into a well-adapted agronomically superior line ‘UMI395’ (which had high phytate trait). We confirmed, by PCR profiling of the umc2230 marker, that umc2230 could easily differentiate the parents and F1 by showing polymorphism in the amplicons in PCR. Next, we profiled the phytic acid trait (using HIP assay) and umc2230 marker trait (by PCR) in F2, BC1F1, BC2F1 and BC1F2 progenies. Our results showed that the umc2230 alleles (180 bp and 175 bp fragments) segregated in Mendelian fashion, i.e. 1:2:1 in selfed progenies (F2 and BC1F2 progenies) and 1:1 in backcross (BC1F1 and BC2F1) progenies. The results
Hereditas 151 (2014) also showed that the plant with 180 bp amplicon expressed low phytate trait, plant with 175 bp amplicon expressed high phytate trait and the plant had both the amplicons expressed intermediate phytate trait in HIP assay, indicating that allele 180 bp is associated with low phytate trait and allele 175 bp is associated with high phytate trait. In short, this study has validated that SSR marker umc2230 cosegregates with phytate trait conferred by lpa2-2 alleles in a mendelian fashion in both selfed and backcross progenies, and hence umc2230 marker is linked with lpa2-2 allele and therefore, umc2230 can be dependably used in a MABB for the efficient selection and transfer of low phytate trait to any well adapted plant for the development of low phytate nutritive maize lines. Acknowledgements – The financial support by Dept of Biotechnology, Ministry of Science and Technology, Government of India, sponsored project on ‘The program support for research and development in agricultural biotechnology-Phase-II at TNAU, Coimbatore’ (Project sanction no. No.BT/PR5095/ AGR/2/847/2012, Date 16/05/2012) is kindly acknowledged. The lpa2-2 mutant line supplied by Dr. Victor Raboy, USDA, is kindly acknowledged.
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Supplementary material (available as Appendix HRD-00030 at ⬍www.oikosoffice.lu.se/appendix⬎). Appendix 1.
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