Marker-assisted introgression of lpa2 locus responsible for low-phytic acid trait into an elite...

Marker-assisted introgression of lpa2 locus responsible for low-phytic acid traitinto an elite tropical maize inbred (Zea mays L.)S . S U R E S H K U M A R

1, P A R A M A S I V A M T A M I L K U M A R2, A R U M U G A M U . T H A N G A V E L U

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N A T E S A N S E N T H I L1,5, P O T H I R A J N A G A R A J A N

1, S A M P T H R A J A N V E L L A I K U M A R1, K A L I P A T T Y N . G A N E S A N

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R A M A C H A N D R A N B A L A G O P A L4 and M U T H U R A J A N R A V E E N D R A N

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1Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India;2Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore 641 003, India; 3Department ofMillets, Tamil Nadu Agricultural University, Coimbatore 641 003, India; 4Department of Veterinary and Animal Sciences, TamilNadu Agricultural University, Coimbatore 641 003, India; 5Corresponding author, E-mail: [email protected]

With 7 figures and 3 tables

Received October 21, 2013/Accepted March 4, 2014Communicated by T. L€ubberstedt

AbstractMaize is an important food and feed crop worldwide. Phytic acid (PA), inmaize kernel, is an antinutritional factor. PA chelates mineral cations andcauses mineral deficiency in humans and phosphorous deficiency in ani-mals. The undigested PA excreted by monogastric animals causes phos-phorous eutrophication. Therefore, development of low-phytate maize isindispensable. The low-phytate locus (lpa2 allele) has been transferredfrom low-phytate mutant line ‘EC 659418’ into an elite inbred UMI 395through marker-assisted backcross breeding (MABB). The MABBinvolved three backcrosses followed by two selfing steps, including‘foreground selection’, that is, selecting lines with lpa2 allele with the helpof a codominant SSR marker ‘umc2230’ and ‘background selection’, thatis, selecting plants having genetic background similar to that of the recur-rent parent using 50 codominant SSR markers. Two low-phytate lpa2 lineswith genome similar (>90% similarity) to that of recurrent parent havebeen identified. These lines can be used as parent in future hybridizationprogrammes for obtaining low-phytate high-yielding maize hybrids.

Key words: Marker-assisted selection — phytic acid — maize— lpa2

Maize (Zea mays L.) has multiple facets of usage such as humanconsumption (12%), livestock feed (58%) and industrial purposes(30%). Maize is a staple food for 310 million people over 20countries, contributing 20–30% of total calories in human diets.Phytic acid (PA) {myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate[InsP(6)]} is ubiquitous in eukaryotic cells and constitutes themajor storage form of phosphate in plant seeds (from 60% to80%) (Raboy 2007). During maturation, PA is accumulated inthe protein storage vacuole in inclusions called globoids; thephosphate groups present in PA are able to form phytate saltsbinding important mineral cations such as calcium, magnesium,potassium, iron and zinc. In mature maize kernels, 80% of PA islocalized in the scutellum and the remaining 20% in the aleuronelayer (O’Dell et al. 1972).Major concern in maize is its PA content, which may go up

to 4 mg/g of seed. PA is an antinutritional factor, for it chelatesmineral nutrients (calcium, manganese, iron, magnesium, zinc,etc.) and renders minerals in phytate non-bioavailable to humansand renders phosphorus in phytate non-bioavailable to mono-gastric animals such as swine, poultry and fish (Brinch-Pedersenet al. 2002). Moreover, PA is an environmental pollutant; thephosphorus released from undigested PA excreted by monogas-tric animals causes phosphorous pollution.

Therefore, development of new maize cultivars with ‘low-PA/high available Phosphorus’ characteristics could increase the bio-availability of minerals in humans and phosphorus in monogas-tric animals and reduce phosphorus pollution to environment andreduce amount of phosphorus supplementation required in ani-mal feeds and hence the associated economic loss to feed indus-try (Ertl et al. 1998). Such grain would also offer more availableFe and Zn for human nutrition (Mendoza et al. 1998).Several low-PA mutants have been generated successfully in

maize, rice, barley and soybean by disrupting PA biosynthesisthrough mutagenesis breeding (Larson et al. 1998, Raboy et al.2000, Wilcox et al. 2000), and they were used in genetic breedingas donors for the development of low-phytate lines (Raboy 2001).So far, in maize, three low-PA (lpa) mutants have been isolated,viz., lpa1, lpa2 and lpa3. These mutant lines are used as importantdonors for the development of low-phytate maize. The lpa2-1mutation is caused by genomic sequence rearrangement in theZmIpk, and lpa2-2 is caused by a nucleotide mutation (i.e. C to Tat nucleotide position 158) that generated a stop codon in theN-terminal region of ZmIpk open reading frame (Shi et al., 2003).The lpa3 mutation is caused by a mutation in a gene that encodesmyo-Ins kinase, which catalyses the production of Ins(3)P1 inmaize seed. Compared with wild-type kernels, the lpa1, lpa2-1and lpa3 mutations achieved 66%, 50% and 50% reduction in phy-tic acid content, respectively (Raboy 2002, Shi et al. 2005). Thelpa2-2 mutation achieved a 30% reduction in phytic acid contentand a threefold increase in inorganic phosphate (Shi et al., 2003).The mutant lines are temperate maize lines that are not adapted tolocal tropical and subtropical conditions. Therefore, there is a needto have the lpa locus introgressed into locally adapted agronomi-cally superior lines to improve their nutritional benefit.There are two strategies to reduce the seed PA content in the

major crops. One is by the breeding approach (which involvesintrogression of a locus responsible for lowering seed phytatecontent), and another is the transgenic approach (which involvestransfer of an alien gene responsible for lowering seed phytatecontent). In this study, we have used marker-assisted backcrossbreeding (MABB) approach towards this end.MABB that entails the use of molecular markers in the selec-

tion process has been shown to be cost-effective (Abalo et al.2009) and is most useful for the introgression of recessive traits.The main aim of MABB is to introgress a trait of interest intothe genetic background of a recipient genotype by recurrentbackcrossing and also to recover the recurrent parent genome as

Plant Breeding, 133, 566–578 (2014) doi:10.1111/pbr.12185© 2014 Blackwell Verlag GmbH

rapidly and completely as possible. In each backcross generation,there is variation around the mean of a trait, which can beexploited to gain advantage in each generation.In our previous study (under review), we have validated that a

SSR marker ‘umc2230’, which is in proximity (0.4 cM or1479 kbp downstream) with lpa2, cosegregate, in a Mendelianfashion, with low-PA trait. Therefore, umc2230 can be depend-ably used in MABB for the efficient selection and transfer oflow-phytate (lpa2) trait and hence development of low-phytatemaize lines. Therefore, we utilized MABB, involving marker‘umc2230’, to facilitate introgression of lpa2 trait (conferred bylpa2-2 allele) from the lpa2 mutant germplasm EC 659418,developed by Victor Raboy, USDA, into our regionally adaptedand productive elite inbred UMI 395. Due to the facts that UMI395 is one of the parental line of popular maize hybrid COH(M)5 released from our Tamil Nadu Agricultural University,Coimbatore, and that it exhibits high yield performance alongwith combining ability, we chose UMI 395 as an ideal recipientline for transferring of the low-phytate trait.

Material and MethodsPCR-based foreground and background screening: DNA extraction:The leaves of 12- to 15-day-old seedlings were collected from the field.Modified CTAB method (Dellaporta et al. 1983) was followed for DNAextraction. The leaves were ground with 500 ll of CTAB buffer, and theextract was transferred into 1.5-ml tube and incubated for 30 min at 65°Cwith occasional mixing after adding PVP and ß–mercaptoethanol. Thetubes were removed from the water bath and allowed to cool at roomtemperature. Equal volume of chloroform/isoamyl alcohol mixture(24 : 1) was added and mixed by inversion for 15 min. The sample wascentrifuged at 4000 rpm for 20 min, and the clear aqueous phase wastransferred to a new sterile tube. Equal volume of phenol/chloroform(1 : 1) was added. The sample was centrifuged at 1680 g for 20 min, andthe clear aqueous phase was transferred to a new sterile tube. One-thirdvolume of ice-cold isopropanol and 3 M sodium acetate were added andmixed gently by inversion. Then, the sample was centrifuged at 4000 rpmfor 20 min to pellet the DNA, and the supernatant was discarded. TheDNA pellet was washed with 70% alcohol. After washing, the DNApellet was air-dried. Depending upon the size of the pellet, DNA wasdissolved in 100–200 ll of 19 TE (pH 8.0) and stored at 4°C.

Foreground screening: Foreground screening (i.e. selection of a donorparent allele linked to a marker) was performed to confirm the introgres-sion of lpa2 locus conferring the low-phytate trait in recombinant lines.Here, foreground screening was performed by profiling of codominantalleles of SSR marker ‘umc2230’ linked to lpa2 locus. For profiling,PCR amplification of umc2230, which encompasses the core repeat(AGC)5, was performed using the forward primer 50-AACGCGAC-GACTTCCACAAG-30 and the reverse primer 50-ACACGTAATGTCCCTACGGTCG-30. A modified PCR program (touchdown PCR)was used to avoid the amplification of non-specific spurious PCR prod-ucts by the primers. PCR was performed with the following program:First, initial hold was at 95°C for 3 min; the second hold starting withdenaturation step at 94°C for 30 s, annealing at 63°C up to 57°C for30 s (12 cycles, reducing 0.5°C per cycle) and the extension step at72°C for 45 s; and the third hold starting at denaturation temperature of94°C for 30 s, annealing at 59°C for 30 s and extension at 72°C for45 s for 45 cycles. Eventually, the final extension step was performed at72°C for 10 min. The amplified fragments were resolved in a 3% aga-rose gel for analysing the amplicons. PCR amplification of 175-bp/175-bp, 175-bp/180-bp and 180-bp/180-bp fragments was expected in UMI395, F1 (UMI 395 9 EC 659418) and EC 659418, respectively.

Background screening: Background screening (i.e. selection of recurrentparent alleles using several molecular markers) was performed to confirmthe recovery of recurrent parent genome in the recombinant lines. Here,

for the background analysis, we used 50 codominant polymorphic mark-ers to analyse 10 chromosomes in maize genome (i.e. approximately fivemarkers per chromosome). The list of background markers used is shownin Table S1. The background screening data for all the 10 chromosomesshowing intervals in cM were graphically represented using the software,Graphical Genotypes (GGT V2.0; www.plantbreeding.wur.nl).

Phytic acid phenotyping: The analysis of PA trait in kernels (i.e. PAphenotyping) was performed to confirm the introgression of lpa2 locusin recombinant lines using high inorganic phosphorous (HIP) assay(Chen et al. 1956) and direct PA assay (Davies and Reid 1979).

Single-seed High Inorganic Phosphorous (HIP) assay: To analyse thePA trait of individual seeds, we used HIP assay. Usually, the higher thefree inorganic phosphorous in kernels, the lower the phytate content andvice versa. Therefore, estimation of inorganic phosphorous can help usindirectly estimate the PA content of maize kernels. Here, the seedsobtained were assayed for free inorganic phosphorous using a microtiter-based colorimetric assay (as described by Chen et al. 1956). In HIPassay, based on the intensity of blue colour developed, which is propor-tional to the amount of free inorganic phosphorous content in the kernel,the phytate content of the individual seeds can be classified as high PA(i.e. low free inorganic phosphorus, which produces light blue colour),low PA (i.e. high free inorganic phosphorus, which produces dark bluecolour) and intermediate PA (i.e. almost near high PA, which producesintermediate blue colour) (Figure S1). To ascertain the associationbetween umc2230 marker and phytate trait, PA phenotyping of 12 ran-domly selected seeds, obtained from the cobs of plants selected by fore-ground screening, was performed using HIP assay. For HIP assay,individual seeds were weighed (in order to select seeds of almost uni-form weight to get normalized HIP assay results), crushed and extractedovernight in 1 ml of 0.4M hydrochloric acid per mg (approximate seedweight) at 4°C. Samples (10 ll) of these extracts were mixed with 90 lldistilled water and 100 ll of colorimetric reagent (one volume of 3 M

sulphuric acid, one volume 2.5% (w/v) ammonium molybdate, one vol-ume 10% (v/v) ascorbic acid and two volumes of distilled water preparedfreshly). The assays were incubated at room temperature for approxi-mately 1 h. Individual seed extracts were visually scored for the presenceof HIP based upon Prussian blue colour (Dorothy and Harrette 1955)development.

Direct PA assay: Phytic acid was estimated by the method of Daviesand Reid (1979). For the estimation of PA in plants, a few seeds wereharvested from the cob of the plant selected and ground into powder.One gram of seed powder was extracted with HNO3 by continuous shak-ing, filtered and made up to suitable volume with water. To 1.4 ml ofthe 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. Thecontents were cooled, and 5 ml of isoamyl alcohol was added andmixed. To this, 0.1 ml ammonia solution was added, shaken thoroughlyand centrifuged at 940 g for 10 min. The alcoholic layer was separated,and the colour intensity was read at 465 nm against amyl alcohol blankafter 15 min. Sodium phytate standards were run along with the sample.The results were expressed as mg PA g per dry wt.

Marker-assisted backcross breeding: Marker-assisted backcrossbreeding was attempted for the introgression of lpa2 locus from thetemperate lpa2 mutant line EC 659418 (source of lpa2 allele that conferslow-phytate trait) obtained from Victor Raboy, USDA, into UMI 395 line(a popular well-adapted agronomically superior and high-phytate line).

Plant materials: UMI 395 was used as female and recurrent parent. TheEC 659418 was used as male and donor parent. As the donor parent istemperate, the donor parent multiplication and development of F1s werecarried out in Wheat Research Station, IARI, Wellington, Ooty (a coolerand higher altitude region in Tamilnadu). The other backcross lines weredeveloped in the Tamil Nadu Agricultural University, Coimbatore(a tropical zone in Tamilnadu).

Molecular breeding and development of low-phytic acid maize 567

Development of F1 and other backcross lines: Parent 1 (P1) ‘UMI 395’and parent 2 (P2) ‘EC 659418’ were raised in two staggered sowings at3-day interval at the Wheat Research Station, IARI, Wellington, Ooty, toachieve programmed pollination. These two lines were hybridized togenerate F1 (UMI 395 9 EC 659418) lines.

Development of BC1F1 lines: The F1 plants were raised along with therecurrent parent (UMI 395). The F1 plants were genotypically confirmed(using foreground selection as described above) to be heterozygous forumc2230 alleles ‘180-bp/175-bp’ lines. The F1 plants (female parent)were backcrossed to the recurrent parent UMI 395 (male parent) to pro-duce seeds of BC1F1 generation.

Development of BC2F1 and BC3F1 lines: BC1F1 plants (female parent)were raised and backcrossed with recurrent parent (UMI 395) (male parent).Introgressed plants with 180-bp/175-bp genotype and expressing mixed-PAphenotype [i.e. a cob with both high-PA kernels (containing PA contentcomparable to that of recurrent parent) and intermediate-PA kernels (con-taining PA content comparable to that of F1)] were selected and forwardedfor growing in the next breeding step, and non-introgressed lines (i.e. plantswith 175-bp/175-bp genotype and expressing only high-PA-trait kernels)were excluded. Similarly, BC3F1 lines were developed using BC2F1 plants.

Development of BC3F2 lines: BC2F1 plants were raised and selfed toproduce BC3F2 generation. The plants with 180-bp/175-bp genotype andexpressing mixed-PA phenotype and plants with 175-bp/175-bp genotypeand expressing only high-PA-trait kernels were excluded. Introgressedlines, with 180-bp/180-bp genotype and expressing only low-PA-traitkernels, were selected, and they were subjected to background screening(described above) to verify whether its genome is identical to recurrentparent. Thus, low-phytate lines with agronomical traits almost identicalto UMI 395 were developed.

ResultsForeground screening of SSR marker linked to lpa2 locus inparents, backcross and selfed progeny

In our previous study, we obtained the sequence of SSR marker‘umc2230’ from the maize database (www.maizegdb.org) andstudied the polymorphism between the EC 659418 (lpa2 mutant/low-phytate donor) and UMI 395 (high-phytate genotype). Wehave found that codominant umc2230 is located just 0.4 cMaway from the lpa2 region on the short arm of chromosome 1 inmaize genome, which is polymorphic for parents and hybrid, thatis, PCR amplification of the marker resulted in 175-bp/175-bp,175-bp/180-bp and 180-bp/180-bp fragments in UMI 395, F1(UMI 395 9 EC 659418) and EC 659418, respectively (Fig. 1).The plants were scored based on the umc2230 alleles present[i.e. ‘175 bp/175 bp’ or ‘175 bp only’ if they contain only UMI395 parent allele; ‘180 bp/180 bp’ or ‘180 bp only’ if it containsonly EC 659418 parent allele; and ‘180 bp/175 bp’ if they con-tain both UMI 395 and EC 659418 alleles (Fig. 2)]. Therefore,at each breeding step, we used this polymorphic marker linked tolpa2 locus for our foreground screening for identification of lpa2introgressed low-phytate lines [lpa2 locus in homozygous condi-tion (indicated by 180-bp/180-bp marker allele banding pattern)or in heterozygous condition (indicated by 180-bp/175-bp pat-tern)] from wild-type high-phytate lines [LPA2 locus in homozy-gous condition (indicated by 175-bp/175-bp pattern)].In our study, first, the F1s of the cross UMI 395 9 EC

659418 were raised in the field, and using foreground screening,they were confirmed as heterozygous (Figs 1 and 2). Thehybrids thus confirmed were used as female parents to backcrosswith recurrent male parent UMI 395, and backcross generationBC1F1 was developed (Fig. 2).

The seeds harvested from hybrid plant #8 (i.e. ‘395/418-c8’)were used for raising BC1F1 progeny (Table 1). Of 50 BC1F1progeny analysed by foreground screening, 22 progeny showedthe presence of alleles from both the parents (180-bp/175-bpbanding pattern) indicating that plants possess the lpa2 locus inheterozygous condition and therefore produce intermediate-phy-tate kernels and high-phytate kernels when backcrossed withrecurrent parent with 175-bp/175-bp banding pattern, and remain-ing 28 progeny showed only alleles of recurrent parent (175-bp/175-bp banding pattern) indicating that plants possess the LPA2locus in homozygous condition and therefore produce only high-phytate kernels when backcrossed with recurrent parent with 175-bp/175-bp banding pattern. We backcrossed 10 heterozygouslines (female parent) with UMI 395 (male parent) and used PAphenotyping for selecting cobs of BC2F1 generation (Table 2).The seeds harvested from BC1F1 plant #40 (i.e. ‘395/418-c8-

c40’) were used for raising BC2F1 progeny. Of 50 BC2F1 prog-eny analysed by foreground screening, 18 progeny showed 180-bp/175-bp banding pattern and 32 progeny showed ‘175-bp/175-bp’ banding pattern. We backcrossed 10 heterozygous lines(female parent) with UMI 395 (male parent) to develop BC3F1generation.The seeds harvested from BC2F1 plant #44 (i.e. ‘395/418-c8-

c40-c44’) were used for raising BC3F1 progeny. Of 50 BC3F1progeny analysed by foreground screening, 35 progeny showed180-bp/175-bp banding pattern and 15 progeny showed ‘175-bp/175-bp’ banding pattern. We selfed 10 heterozygous lines todevelop BC3F2 generation.

Fig. 1: Profiling of alleles of SSR marker ‘umc2230’ in F1 along withthe parents. The figure shows that PCR amplification of a codominantSSR marker ‘umc2230’, which is linked to lpa2 locus, resulted in theamplicons 180 bp/175 bp (i.e. heterozygous) in F1, 175 bp/175 bp (i.e.homozygous for 175 bp) in high-phytate parent UMI 395 and 180 bp/180 bp (i.e. homozygous for 180 bp) in low-phytate EC 659418 line,indicating that ‘umc2230’ is polymorphic in parents and F1 and hencecan differentiate all the three lines. As the locus that governs the PA con-tent is linked to ‘umc2230’, the 175-bp/175-bp allele pattern of umc2230marker indicates the presence of LPA2/LPA2 (wild-type alleles in homo-zygous condition, which confers high-phytate trait), the 180-bp/175-bpallele pattern of umc2230 marker indicates the presence of lpa2/LPA2(alleles in heterozygous condition, which confers intermediate-phytatetrait, and the 180-bp/180-bp allele pattern of umc2230 marker indicatesthe presence of lpa2/lpa2 (mutant alleles in homozygous condition,which confers low-phytate trait)

568 S. SURESH KUMAR , P . TAM ILKUMAR , A. U. THANGAVELU e t a l .

The seeds harvested from BC3F1 plant #21 (i.e. ‘395/418-c8-c40-c44-s21’) and #50 (i.e. ‘395/418-c8-c40-c44-s50’) wereraised as BC3F2 progeny, and the progeny were selfed. Of 100BC3F1 progeny of ‘395/418-c8-c40-c44-s21’ analysed by fore-ground screening, 15 progeny showed ‘180-bp/180-bp’ bandingpattern, 57 progeny showed 180-bp/175-bp banding pattern, and28 progeny showed 175-bp/175-bp banding pattern. The line‘395/418-c8-c40-c44-s21-s36’ which showed 180-bp/180-bpbanding pattern was selected as low-phytate line.Of 100 BC3F1 progeny of ‘395/418-c8-c40-c44-s50’ analysed

by foreground screening, 25 progeny showed ‘180-bp/180-bp’banding pattern, 49 progeny showed 180-bp/175-bp banding pat-tern, and 26 progeny showed 175-bp/175-bp banding pattern.The line ‘395/418-c8-c40-c44-s50-s31’ which showed 180-bp/180-bp banding pattern was selected as low-phytate line.In short, foreground selection helped the selection of interme-

diate-phytate lines from high-phytate lines in segregatingbackcross populations (i.e. ‘395/418-c8’ from BC1F1, ‘395/418-c8-c40’ from BC2F1, ‘395/418-c8-c40-c44’ from BC3F1) andtwo low-phytate lines homozygous for lpa2 alleles (‘395/418-c8-c40-c44-s21-s36’ and ‘395/418-c8-c40-c44-s50-s31’) from high-and intermediate-phytate lines in segregating selfed BC3F2populations (Table 1).

Background screening of polymorphic SSR markers foridentification of plants with recurrent parent genome

Background screening (i.e. profiling the banding pattern ofalleles of background polymorphic markers) was performed inthe selected two low-phytate lines to estimate the recovery of thegenome of recurrent parent. For this background survey, the

polymorphic SSR marker survey was carried out using 234 SSRmarker sequences obtained from MaizeGDB database (www.maizegdb.org). These markers were located among the ten chro-mosomes of maize genome. Of the 234 SSR markers used toscreen the parents UMI 395 and EC 659418, 112 markers werefound to be polymorphic. The physical position of the polymor-phic markers was obtained from the MaizeGDB database foreach chromosome separately.The 50 SSR markers, which showed distinct alleles between

the parents, were selected for the genome-wide backgroundanalysis. The polymorphic SSR markers were distributed inchromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. These markerswere used to survey the two BC3F2 plants (i.e. 395/418-c8-c40-c44-s21-s36 and ‘395/418-c8-c40-c44-s50-s31’) for estimatingthe recovery of the background UMI 395 genotype. The PCRproducts were screened at 3.5% agarose gel and scored for eitherrecovery of UMI 395 allele (AA), introgression of EC 659418(BB) allele or heterozygous (AB) for both the parents. The plantwas scored as AA if it contains UMI 395 allele and scored asBB if it contains EC 659418 allele, and scored as AB if it con-tains both UMI 395 and EC 659418 alleles (Fig. 3).The percentage of the markers alleles that shows similarity

with that of recurrent parent is used for calculating the recoveryof recurrent parent genome. The genomes of the first (395/418-c8-c40-c44-s21-s36) and the second (‘395/418-c8-c40-c44-s50-s31’) low-phytate BC3F2 lines are 91% and 93% identical to therecurrent parent (Figs 4 and 5) (Table S2). The selected lineswere further maintained by selfing and will be used in futurehybridization programme. In short, background screening ofselected BC3F2 lines was performed using polymorphic SSRmarkers to identify the introgressed line which not only

Table 1: Pedigree of the two low phytate lines developed in this MABB study

Season Population grown Breeding steps taken Population developed Plant selected from the population developed for next generation1

First P1 and P2 Crossing of parents F1 395/418Second F1 Backcrossing with recurrent BC1F1 395/418-c8Third BC1F1 Backcrossing with recurrent BC2F1 395/418-c8-c40Fourth BC2F1 Backcrossing with recurrent BC3F1 395/418-c8-c40-c44Fifth BC3F1 Selfing BC3F2 395/418-c8-c40-c44-s21

395/418-c8-c40-c44-s50Sixth BC3F2 Selfing and selection of

low phytate lines2BC3F3 395/418-c8-c40-c44-s21-s36 and 395/418-c8-c40-c44-s50-s31

lines that are homozygous for lpa2 alleles and genotypicallyidentical to recurrent parent

1C denotes backcross line, S denotes selfed line.2In case of selfed population, the plants, which resemble the recurrent parent in almost all of agronomical traits, are screened/selected at 4 levels forselecting the low phytate lines homozygous for lpa2 alleles; (i) foreground selection based on the presence or absence of alleles of SSR marker‘umc2280’ [i.e., plants showing the banding patterns 180 bp/175 bp (similar to heterozygous F1 plant) and 175 bp/175 bp (similar to homozygousrecurrent plant) were excluded from breeding operations and only plants showing pattern 180 bp/180 bp (similar to homozygous donor plant) wasselected as low phytate line (ii) HIP assay was done to confirm whether the plants withlpa2 allele linked to180 bp allele indeed confers the low PAtrait phenotypically [i.e., to test whether the plant that is genotypically homozygous for lpa2 allele (180 bp/180 bp) produce only low phytate kernels,the plant that is genotypically homozygous for LPA2 allele (175 bp/175 bp) produce only high phytate kernels, and the heterozygous plants (180 bp/175 bp pattern) produce cob with a mixture of high and intermediate phytate kernels)] (iii) PA was directly estimated in cobs selected by foregroundand HIP analysis to confirm whether the PA content of introgressed lines (180 bp/180 bp) is lower (≤2 mg/g) than that of recurrent parent (2.66 mg/g) and F1 (2.16 mg/g), and other lines (180 bp/175 bp and 175 bp/175 bp) have high PA (2.16 mg/g and 2.66 mg/g, respectively) (iv) backgroundscreening was done only in low phytate introgressed lines (180 bp/180 bp) to confirm whether these lines have recovered almost all of the genome ofthe recurrent parent.

Fig. 2: General schema of MABB for the development of low-phytate lines. The line UMI 395 (well-adapted agronomically superior line) was usedas female and recurrent parent. The line EC 659418 (source of lpa2 allele that confers low-phytate trait) was used as donor and male parent. The F1,backcross lines (BC1F1, BC2F1, BC3F1) and selfed (BC3F2) lines were developed and screened to develop agronomically superior low-phytate lines.The numbers 1 to 6 refer to the generation in which the respective crops were raised. The selection of seeds for each generation was performed basedon foreground screening (using umc2230) and phytate estimation (using HIP and direct phytate assays) to confirm the introgression of lpa2 locus inrecombinant lines. In HIP assay, kernels with high-phytate trait (or rather low free inorganic phosphorous) produce light blue colour products, kernelswith intermediate-phytate trait (or rather intermediate free inorganic phosphorous) produce medium blue colour products, and kernels with low-phytatetrait (or rather high free inorganic phosphorous) produce dark blue colour products


possesses low-phytate 180-bp/180-bp genotype, but also hasrecovered almost all of the genome of recurrent parent.

Screening of plants for phytate trait and content

At each breeding step, the PA phenotyping using direct phytaseassay and HIP assay was performed in the segregating popula-tions to confirm whether the alleles of umc2230 indeed matchwith kernel PA phenotypes [i.e. plants with banding pattern180 bp/180 bp (or rather lpa2/ lpa2) produces only low-phytatekernels, that with 180-bp/175-bp (lpa2/LPA2) pattern producesmixture of high- and intermediate-phytate kernels and that with175-bp/175-bp (LPA2/LPA2) pattern produces only high-phytatekernels, respectively] (Fig. 1).

Quantitative estimation of PA

Phytate estimation was performed in EC 659418 (lpa2 donor),UMI 395 (high-phytate line), F1 (UMI 395 9 EC 659418) andthe developed low-phytate BC3F2 lines (Table 3). The analysisrevealed PA levels varied among the lines. EC 659418 line whichis homozygous for the mutant lpa2 allele was found to containthe minimum level of PA (1.62 mg/g), the UMI 395 line whichis homozygous for the wild-type LPA2 allele was found to con-tain the maximum level of PA (2.66 mg/g), and the F1 which isheterozygous possessing lpa2/LPA2 alleles was found to containthe intermediate level of PA (2.16 mg/g). Importantly, the newlydeveloped low-phytate BC3F3 lines which are homozygous forthe mutant lpa2 allele were also found to contain the lower levelof PA (i.e. 1.78 mg/g for the first line and 1.73 mg/g for the sec-ond line) almost similar to the donor genotype, indicating that the

MABB has successfully introgressed the low-phytate trait fromthe donor line into agronomically superior UMI 395 line.

Single-seed HIP assay

As the wild-type (i.e. high phytate) kernels have relatively lowlevels of inorganic phosphorous and low-phytate kernels havevery high levels of inorganic phosphorous, and as testing forinorganic phosphorous is quick, easy, accurate and inexpensive,the HIP phenotyping using HIP assay provides a high-through-put single-seed assay for homozygosity or heterozygosity forlpa2 alleles and homozygosity for wild-type LPA2 alleles. InHIP assay, kernels with high-phytate trait produce light blue col-our products, kernels with intermediate-phytate trait producemedium blue colour products, and kernels with low-phytate traitproduce dark blue colour products. In our MABB, the HIP assayhelped to speed up the confirmation of the lpa2 introgressionphenotypically and selection of lines expressing low-phytate ker-nels for forwarding to next generation.The facts that F1 kernels have intermediate-PA content

(Table 3) and produce medium blue colour products in HIPassay (Fig. 2, Figure S1) may be due to the near-recessivenessof the mutant lpa 2 allele, reported by Raboy et al. (2000).

Comparison of agronomical traits in parents and low-phytatelines

The mean values of agronomical traits [i.e. plant height, days to50% tasselling, days to 50% silking (days), E=ear height (cm),100 grain weight (g), yield per plant (g), PA content in kernels(mg/g)] observed in EC 659418 (lpa2 donor), UMI 395 (high-

Table 2: Criteria for selecting plants from BC1F1 population for raising BC2F1 generation

BC1F1 lines Plant ID

umc2230 markerscreening1

(Level I)

HIP assay2 (Level II) PA estimation4

(mg/g)(Level III)

Plant selected forBC2F1 populationdevelopment5Light-blue Intermediate-blue Interpretation

395/418-c8 4 180 bp/175 bp 8 4 Mixed3 –

395/418-c8 9 180 bp/175 bp 7 5 Mixed –

395/418-c8 16 180 bp/175 bp 4 8 Mixed –

395/418-c8 21 180 bp/175 bp 6 6 Mixed 1.8 Selected but notused further

395/418-c8 25 180 bp/175 bp 5 7 Mixed –

395/418-c8 37 180 bp/175 bp 4 8 Mixed –

395/418-c8 38 180 bp/175 bp 8 4 Mixed –

395/418-c8 40 180 bp/175 bp 5 7 Mixed 1.79 395/418-c8-c40selected

395/418-c8 42 180 bp/175 bp 8 4 Mixed 2.19 Not selected395/418-c8 50 180 bp/175 bp 4 8 Mixed –

‘–’ denotes that plants are excluded based on cob and seed qualities.1PCR profiling of umc2230 alleles was done using the DNA extracted from leaves of selected maize plants (first level selection by marker umc2230).2For HIP assay, we selected 12 seeds randomly from the cob of plant selected and did HIP assay for each seed separately. In HIP assay, kernels withhigh-, intermediate- and low-phytate traits produce light-, medium- and dark-blue colour products respectively. By foreground selection, it can be seenthat the plants identified as 180 bp/175 bp genotype has produced a cob that contains mixture of high- and intermediate-phytate kernels, and the plantsidentified as 175 bp/175 bp genotype has produced a cob that contains only high-phytate kernels, confirming that lpa2 allele linked to the 180 bp mar-ker allele confers the low-PA trait phenotypically (second level selection by free phosphorous content).3A cob with both high-PA kernels (containing PA content comparable to that of recurrent parent) and intermediate-PA kernels (containing PA contentcomparable to that of F1) were selected for forwarding generations.4PA estimation was done in selected cobs with good cob and seed qualities (third level selection by direct estimation of phytic acid).5In case of backcross population, the plants are screened/selected at three levels for forwarding to next backcross step; (i) foreground selection based onthe presence or absence of alleles of SSR marker ‘umc2280’ [i.e., plants which shows the banding pattern similar to heterozygous F1 plant (180 bp/175 bp) were selected and plants which shows the banding pattern similar to homozygous recurrent plant (175 bp/175 bp) were excluded from breedingoperations] (Table 1 and Fig. 1), (ii) HIP assay is done to confirm whether the plants with lpa2 allele linked to 180 bp allele indeed confers the low-PAtrait phenotypically (iii) Direct estimation of PA in kernels of plants selected by foreground and HIP analysis. Plants with low PA (≤ 2 mg/g) (i.e., lowerthan that of recurrent parent and/or equal that of F1) are selected and that with high PA (> 2 mg/g) are excluded.


phytate line), F1 (UMI 395 9 EC 659418) and the low-phytateBC3F2 lines are shown in Table 3.

Analysis of agronomical traits in BC3F2 selfed progeny

The agronomical traits of 395/418-c8-c40-c44-s50 and 395/418-c8-c40-c44-s21 (BC3F2) progeny were analysed to know whetherthe selfed population behaves agronomically similar to the recur-rent parent. The mean values for the agronomical traits of theBC3F2 progeny [i.e. plant height, days to 50% tasselling, days to50% silking (days), ear height (cm), 100-grain weight (g), yieldper plant (g), PA content in kernels (mg/g)] were almost similarto that of the recurrent parent, indicating that the BC3F2 selfedprogeny have recovered most of the genome of the recurrent par-ent (Table 3, Figs 6 and 7).

DiscussionMaize holds the unique position in world agriculture as it contrib-utes not only for food but also for feed and fodder. Together withrice and wheat, maize provides at least 30% of the food calories tomore than 4.5 billion people in 94 developing countries (VonBraun et al. 2010). In India, maize is emerging as third mostimportant crop after rice and wheat. Maize is one of the six keystaple food crops that are the focus of an international agronomiceffort to combat micronutrient malnutrition in the developingcountries through biofortification. An increment in the micronutri-ent contents in the maize kernel is expected to have a substantialhealth impact in poor families, including children and women whoare most vulnerable to the micronutrient deficiencies.

Foreground screening of parents and backcross progenyusing SSR marker umc2230 linked to lpa2 locus

DNA-based molecular markers have been developed and usedextensively in marker-assisted breeding, gene tagging, construct-ing genetic and physical linkage maps and marker-assisted selec-tion (MAS) in crops (McCouch et al. 1997). The availability anddevelopment of molecular markers are a prerequisite for identifi-cation of the gene of interest or QTLs without phenotypic selec-tion in MAS and hence great reduction in population size to behandled in MAS. The main advantage of MAS is that selectionis based on specific genes and not on phenotype.One main objective of plant breeding is the introgression of

one or more genes from a donor into the background of an elite

variety (recurrent or recipient parent) and to recover the recurrentparent genome as rapidly as possible. The availability of largenumber of SSR markers in maize, which is distributed through-out the maize genome, makes the selection more efficienttowards selection of desirable traits. Breeders transfer targetalleles from a donor variety to a popular cultivar by a repetitivebackcrossing which is slow, uncertain and often retain undesir-able chromosome segments from the donor. Marker-assistedbreeding tackles these problems by allowing breeders to identifyplants with the desired trait at early vegetative state and its selec-tion and advancement to further generations (marker-assistedforeground selection). Maize breeders try to combine the nutri-tional quality traits with yield to produce better nutrition in theanimal feed. MABB is one of the most anticipated and fre-quently cited benefits of molecular markers as indirect selectiontools in breeding programmes (Semagan et al. 2006). Markersare effective aids to selection in backcrossing in three ways.First, markers can aid selection on target alleles whose effectsare difficult to observe phenotypically. Second, markers can beused to select for rare progeny, which contain the target genewith less agronomic introgression from the donor parent. Third,markers can be used to select rare progeny that are the result ofrecombination near the target gene, thus minimizing the effectsof linkage drag (Joshi and Nayak 2010). The advantages ofusing MAS in maize improvement have been well documented(Babu et al. 2005, Naidoo et al. 2012, Zhang et al. 2012).MABB combines ‘foreground’ selection of donor alleles linkedto markers and ‘background’ selection of recurrent parent allelesin the later generation for efficient and feasible breedingapproach for speedy introgression of lpa2 locus.

Parental polymorphism survey in the lpa2 region using SSRmarker

Considerable interest has centred on the development of low-phytate grain as a solution to dietary micronutrient deficiencyand environmental phosphorous pollution. Therefore, we aimedto develop low-phytate lines using MABB. The DNA markersflanking the low-phytate locus lpa2 could facilitate efficientselection of the plants with low-phytate trait in molecular breed-ing programmes. The microsatellite DNA, also called simplesequence repeats (SSRs), has been widely used for screening theparents and to identify the polymorphism between the genotypesin maize (Chakraborti et al. 2011). However, to improve the effi-ciency of marker-assisted selection (MAS), it is important that

Fig. 3: Background screening of the developed low-phytate lines. The banding pattern produced by the amplification of polymorphic SSR markers‘umc1737’, ‘umc2226’, ‘umc1708’, ‘umc2324’, ‘umc1717’, in lines UMI 395 (P1), EC 659418 (P2) and the two low-phytate lines developed (D1 andD2) in this study are shown. The pedigrees of D1 and D2 are ‘395/418-c8-c40-c44-s21-s36’ and ‘395/418-c8-c40-c44-s50-s31’, respectively. The plantwas scored as AA if it contains UMI 395 allele and scored as BB if it contains EC 659418 allele, and scored as AB if it contains both UMI 395 andEC 659418 alleles. Lane M denotes Marker lane loaded with 100-bp DNA ladder


umc1

552

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umc1

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chromosome1

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chromosome5

chromosome6

chromosome7

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508.

18

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70

75.9

3

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80

Fig.

4:Graphical

representatio

nof

background

screeningin

developedlow-phytate

line‘395/418-c8-c40-c44-s21-s36’.The

profiling

ofallelesof

allthe50

polymorphic

SSR

markers

locatedacross

allthe10

chromosom

esin

low-phy

tate

BC3F

2lin

e‘395/418-c8-c40-c44-s21-s36’

alongwith

theparentswas

graphically

represented.

The

map,which

provides

aview

ofthepercentage

ofrecurrentparent

genomerecovery

both

atthelevelof

individual

chromosom

eandgenome,

confirmstherecovery

ofrecurrentparent

genomein

thelow-phytate

recombinant

line.

The

distance

betweenthemarkers

inchromosom

esisexpressedin

cMintervals


UM

I 395

EC 6

5941

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eter

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ous

regi

on

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686.

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umc1

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683.

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806.

90

umc1

737

1029

.96

chromosome1

chromosome2

chromosome3

chromosome4

chromosome5

chromosome6

chromosome7

chromosome8

chromosome9

chromosome10

Fig.

5:Graphical

representatio

nof

background

screeningin

developedlow-phytate

line‘418

-c8-c40-c44-s50-s31’.The

profiling

ofallelesof

allthe50

polymorphic

SSR

markers

locatedacross

allthe10

chro-

mosom

esin

low-phytate

BC3F

2lin

e‘395/418-c8-c40-c44-s50-s31’

alongwith

theparentswas

graphically

represented.

The

map,which

provides

aview

ofthepercentage

ofrecurrentparent

genomerecovery

both

atthelevelof

individual

chromosom

eandgenome,

confirmstherecovery

ofrecurrentparent

genomein

thelow-phytate

recombinant

line.

The

distance

betweenthemarkers

inchromosom

esis

expressedin

cMintervals


the recombination frequency between the target gene and themarker should be as low as possible, and this could be achievedby identifying markers as close as possible to the gene. With thedevelopment of saturated molecular marker map in maize (Chinet al. 1996, Taramino and Tingey 1996), it is now possible toidentify new markers closely linked to the gene of interest andalso the flanking markers.In our previous study, we have found that SSR marker

umc2230 which is closely linked to lpa2 locus was found tobe polymorphic between parents, and hence, we used it forforeground screening of segregating populations for selectionof plants with lpa2 trait. Elilis and Pantalone (2009) also car-ried out similar study in soybean for the introgression of twolow-phytate mutant genes into a locally adapted soybean culti-var. Naidoo et al. (2012) studied marker-assisted selection forlow PA (lpa1-1) with single nucleotide polymorphism markerand amplified fragment length polymorphisms in a maize back-cross breeding programme. They reported that as the level ofphytic acid is difficult to assess in a maize breedingprogramme, they developed a codominant single nucleotidepolymorphism (SNP) marker for detecting the single recessivelow-phytic acid (lpa1-1) gene in a BC2F1 population developedfrom a locally adapted tropical normal inbred line (P 16) andCM 32 (lpa1-1 donor). They demonstrated that the use of mar-ker-assisted selection for foreground and background selectiongreatly increased the efficiency of detection of the homozygousrecessive (99.58%) and heterozygous (99.59%) genotypes aswell as improving the recovery of the recurrent parent(92.15%) in the BC2F1 generation of the maize backcrossbreeding programme.

Background SSR marker screening for identification ofrecurrent parent genome

Marker-assisted background selection for the identification ofbackcrossed plants that have recovered the maximum amount ofgenome of recurrent parent with good agronomical type was pro-posed by Young and Tanskley and later termed as backgroundselection (Hospital and Charcosset 1997). This background

screening will aid in reducing the number of generations to pro-duce individuals with desired gene combinations but remainsisogenic to recurrent parent. The background recovery percent-age depends on the number of markers applied in screening,their equal distribution over the chromosomes, the backcrossgenerations and number of individual plants in the population.In our study, background screening was performed in the

selected two low-phytate lines to estimate the recovery of thegenome of recurrent parent. We used 50 SSR markers distributedamong 10 maize chromosomes to find that the genome of thefirst (395/418-c8-c40-c44-s21-s36) and the second (‘395/418-c8-c40-c44-s50-s31’) low-phytate BC3F2 lines are 91% and 93%identical to the recurrent parent. Using the maize genome as amodel, Jorboe et al. (1994) have reported that three backcrossgenerations and 80 markers were needed to recover 99% of therecurrent parent genotype. Naidoo et al. (2012) achieved 92.15%recovery of the recurrent parent in the BC2F1 generation in amaize backcross breeding programme for low PA (lpa1-1) usingcodominant single nucleotide polymorphism (SNP) marker-assisted selection for foreground and background selection.

Analysis of agronomical and phytate traits in the developedlines

The newly developed two low-phytate lines had lower level of PAalmost similar to that of donor genotype and had genetic back-ground and agronomical traits almost identical to that of recurrentparent, indicating that the MABB has successfully introgressed thelow-phytate trait from the donor line into UMI 395 line.In short, in the present investigation, we have developed two

low-phytate agronomically superior maize lines by introgressingthe alien low-phytate locus ‘lpa2’, which confers the low-phy-tate trait, from the temperate lpa2 mutant donor line EC659418 (Raboy et al. 2000) into the elite and locally well-adapted high-yielding inbred line UMI 395 through marker-assisted backcross breeding. Three backcross populations(BC1F1, BC2F1, BC3F1) were developed using UMI 395 asrecurrent parent to reduce step by step the donor backgroundand increase the recurrent background, which confers superior

Table 3: Mean values of agronomically important traits in parental, F1, and low-phytate genotypes

Populationstatistics1

Plant height(cm)

50% tasseling(days)

50% silking(days)

Ear height(cm)

100-grainweight (g)

Yield perplant (g)

PA content(mg/g)2

UMI 395 (Recurrent parent)Mean 119.48 60.99 64.04 59.99 27.4709 66.7344 2.66SD 3.10256 0.81023 0.80302 0.77192 2.3822 1.31595 0.06728SEM 0.31026 0.08102 0.0803 0.07719 0.23822 0.13159 0.01504

EC 659418 (Donor parent)Mean 110.34 59.89 63.2 33.61 19.7309 26.3815 1.62SD 3.02254 0.79003 0.7785 1.14499 2.34185 1.29577 0.05777SEM 0.30225 0.079 0.07785 0.1145 0.23419 0.12958 0.01292

F1Mean 170.43 45.96 48.95 68.24 33.416 80.9467 2.16SD 3.26368 0.81551 0.82112 0.7929 2.6231 1.25716 0.06415SEM 0.32637 0.08155 0.08211 0.07929 0.26231 0.12572 0.01435

BC3F2Mean 122.31 59.54 62.34 60.62 26.0215 65.4326 1.74SD 3.0441 1.20956 1.13902 1.12618 2.65056 1.43617 0.06759SEM 0.30441 0.12096 0.1139 0.11262 0.26506 0.14362 0.01511

1Agronomically important, easily observable, and comparable traits are selected. Mean values for plant height, 50% tasseling, 50% silking, ear height,100-grain weight and yield per plant are based on the observation made on 100 plants. Mean value for PA content is based on the observation madeon 20 plants.2Mean value of phytate content under each population category.


Fig. 6: Analysis of the agronomical traits of 395/418-c8-c40-c44-s21 (BC3F2) progeny. The mean values for the agronomical traits of the BC3F2 prog-eny [i.e. plant height, days to 50% tasselling, days to 50% silking (days), ear height (cm), 100-grain weight (g), yield per plant (g), PA content in ker-nels (mg/g)] are almost similar to that of the recurrent parent


Fig. 7: Analysis of the agronomical traits of 395/418-c8-c40-c44-s50 (BC3F2) progeny. The mean values for the agronomical traits of the BC3F2 prog-eny [i.e. plant height, days to 50% tasselling, days to 50% silking (days), ear height (cm), 100-grain weight (g), yield per plant (g), PA content in ker-nels (mg/g)] are almost similar to that of the recurrent parent


agronomical performance. Finally, selfing of the selected BC3F1lines was performed to get a low-phytate line that is character-istic of and identical to recurrent parent and also homozygousfor the lpa2 allele. It can be seen (Table 3) that the developedlow-phytate BC3F2 lines have recovered the agronomical traitsof the recurrent parent and phytate trait of the donor parent,indicating that MABB is efficient in introgression of interestedtrait. The two novel low-phytate lines that are similar to UMI395 but are homozygous for recessive lpa2 can be used inhybridization programmes for the production of low-phytatehigh-yielding maize hybrids.

Acknowledgements

The financial support by Department of Biotechnology, Ministry ofScience and Technology, Government of India, sponsored project on‘The program support for research and development in agricultural bio-technology-Phase-II at TNAU, Coimbatore’ (Project sanction number:No.BT/PR5095/AGR/2/847/2012, Date 16/05/2012) is kindly acknowl-edged. The lpa2-2 mutant line supplied by Dr. Victor Raboy, USDA, iskindly acknowledged.

ReferencesAbalo, G., P. Tongoona, J. Derera, and R. Edema, 2009: A comparativeanalysis of conventional and marker-assisted selection methodsin breeding maize streak virus resistance in maize. Crop Sci. 49,509—520.

Babu, R., S. K. Nair, A. Kumar, S. Venkatesh, J. C. Sekhar, N. N.Singh, G. Srinivasan, and H. S. Gupta, 2005: Two-generation marker-aided backcrossing for rapid conversion of normal maize lines toquality protein maize (QPM). Theor. Appl. Genet. 111, 888—897.

Brinch-Pedersen, H., L. D. Sorensen, and P. B. Holm, 2002: Engineeringcrop plants: getting a handle on phosphate. Trends Plant Sci. 7, 118—125.

Chakraborti, M., B. M. Prasanna, F. Hossain, S. Mazumdar, A. M.Singh, S. Guleria, and H. S. Gupta, 2011: Identification of kernel iron-and zinc-rich maize inbreds and analysis of genetic diversity using mi-crosatellite markers. J. Plant Biochem. Biotechnol. 20, 224—233.

Chen, P. S., T. Y. Toribara, and H. Warner, 1956: Microdeterminationof phosphorus. Anal. Chem. 28, 1756—1758.

Chin, E., L. Senior, H. Shu, and J. S. C. Smith, 1996: Maize sim-plerepetitive DNA sequences: abundance and allele variation. Genome39, 866—873.

Davies, N., and H. Reid, 1979: An evaluation of the phytate, zinc, cop-per, 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., J. Wood, and J. B. Hicks, 1983: A plant DNA Minipreparation version 2. Plant Mol. Biol. Rep. 1, 19—22.

Dorothy, R., and B. Harrette, 1955: The application of the Prussian bluestain to previously stained films of blood and bone marrow. Blood J.10, 160—166.

Elilis, D. L., and V. R. Pantalone, 2009: Induced plant mutations in thegenomic era. FAO 34, 316—318.

Ertl, D. S., K. A. Young, and V. Raboy, 1998: Plant genetic approachesto phosphorus management in agricultural production. J. Environ.Qual. 27, 299—304.

Hospital, F., and A. Charcosset, 1997: Marker assisted introgression ofquantitative trait loci. Genet. 147, 1469—1485.

Jorboe, S. G., W. D. Beavis, and S. J. Openshaw. 1994: Prediction ofresponses to selection in marker-assisted backcross programs by com-

puter simulation. Abstracts of the Second International Conference onPlant Genome, Scherago International Inc., 38.

Joshi, R. K., and S. Nayak, 2010: Gene pyramiding-A broad spectrumtechnique for developing durable stress resistance in crops. Biotechnol.Mol. Biol. Rev. 5, 51—60.

Larson, S. R., K. A. Young, A. Cook, T. K. Blake, and V. Raboy, 1998:Linkage mapping two mutations that reduce phytic acid content of bar-ley grain. Theor. Appl. Genet. 97, 141—146.

McCouch, S. R., X. Chen, O. Panaud, S. Temnykh, Y. Xu, Y. G. Cho,N. Huang, T. Ishii, and M. Blair, 1997: Microsatellite marker develop-ment, mapping and applications in rice genetics and breeding. PlantMol. Biol. 35, 89—99.

Mendoza, C., F. Viteri, L. B. �Ennerdal, K. A. Young, V. Raboy, and K. H.Brown, 1998: Effect of genetically modified low phytic acid maize onabsorption of iron from tortillas. Am. J. Clin. Nutr. 68, 1123—1127.

Naidoo, R., G. M. F. Watson, J. Derera, P. Tongoona, and M. D. Laing,2012: Marker assisted selection for low phytic acid (lpa1-1) with sin-gle nucleotide polymorphism marker and amplified fragment lengthpolymorphisms for background selection in a maize backcross breed-ing programme. Mol. Breeding 30, 1207—1217.

O’Dell, B. L., A. R. Deboland, and S. R. Koirtyohann, 1972: Distribu-tion of phytate and nutritionally important elements among morpholog-ical components of cereal grains. J. Agric. Food Chem. 20, 718—721.

Raboy, V., 2001: Seeds for a better future: ‘Low phytate’ grains help toovercome malnutrition and reduce pollution. Trends Plant Sci. 6,458—462.

Raboy, V., 2002: Progress in breeding low phytate crops. J. Nutr. 132,503S—505S.

Raboy, V., 2007: The ABCs of low-phytate crops. Nat. Biotechnol. 25,874—875.

Raboy, V., P. F. Gerbasi, K. A. Young, S. D. Stoneberg, S. G. Pickett,A. T. Bauman, P. P. Murthy, W. F. Sheridan, and D. S. Ertl, 2000:Origin and seed phenotype of maize low phytic acid 1-1 and low phy-tic acid 2-1. J. Plant Physiol. 124, 355—368.

Semagan, K., A. Bjornstad, and M. N. Ndjiondjop, 2006: Progress andprospects of marker assisted backcrossing as a tool in crop breedingprograms. Afr. J. Biotechnol. 5, 2588—2603.

Shi, J., H. Wang, Y. Wu, J. Hazebroek, R. B. Meeley, and D. S. Ertl,2003: The maize low-phytic acid mutant lpa2 is caused by mutation inan inositol phosphate kinase gene. Plant Physiol. 131, 507–515.

Shi, J., H. Wang, J. Hazebroek, D. S. Ertl, and T. Harp, 2005: The maizelow-phytic acid 3 encodes a myo- inositol kinase that plays a role inphytic acid biosynthesis in developing seeds. Plant J. 42, 708—719.

Taramino, G., and S. Tingey, 1996: Simple sequence repeats for germ-plasm analysis and mapping in maize. Genome 39, 277—287.

Von Braun, J., B. Derek, C. Colin, L. Tom, and W. Jeff, 2010. A draftstrategy and results framework for the CGIAR. 20 March 2010.CGIAR, The World Bank, Washington, DC

Wilcox, J. R., G. S. Premachandra, K. A. Young, and V. Raboy, 2000:Isolation of high seed inorganic P, low-phytate soybean mutants. CropSci. 40, 1601—1605.

Zhang, X., W. H. Pfeiffer, N. Palacios-Rojas, R. Babu, H. Bouis, and J.Wang, 2012: Probability of success of breeding strategies for improvingpro-vitamin A content in maize. Theor. Appl. Genet. 125, 235—246.

Supporting InformationAdditional Supporting Information may be found in the online version ofthis article:Figure S1. HIP assay for parents and F1s.Table S1. List of background SSR markers used in this study.Table S2. Profiling banding pattern of background marker alleles in thetwo low-phytate lines developed in this MABB study.


Marker-assisted introgression of lpa2 locus responsible for low-phytic acid trait into an elite...

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