Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of crtRB1 and lcyE governing higher accumulation of provitamin A in maize

Zunjare, Rajkumar U.; Chhabra, Rashmi; Hossain, Firoz; Muthusamy, Vignesh; Baveja, Aanchal; Gupta, H. S.

doi:10.1007/s13562-017-0432-8

Cited by 12 publications

(12 citation statements)

References 23 publications

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“…Gene-based InDel marker present in 3'TE and 5'TE region of crtRB1 and lcyE were used for foreground selection (Harjes et al, 2008 ; Yan et al, 2010 ; Figure S1 ; Table 2 ). PCR mediated amplification of crtRB1 and lcyE was performed using protocol standardized at Maize Genetics Unit, IARI (Zunjare et al, 2017a ). Agarose of 1.5% concentration (Lonza, Rockland, ME USA) was used for separating the amplicon at 120 V for 2–3 h along with 100 bp DNA ladder (MBA-Fermentas).…”

Section: Methodsmentioning

confidence: 99%

Development of Biofortified Maize Hybrids through Marker-Assisted Stacking of β-Carotene Hydroxylase, Lycopene-ε-Cyclase and Opaque2 Genes

et al. 2018

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Traditional yellow maize though contains high kernel carotenoids, the concentration of provitamin A (proA) is quite low (<2 μg/g), compared to recommended level (15 μg/g). It also possesses poor endosperm protein quality due to low concentration of lysine and tryptophan. Natural variant of crtRB1 (β-carotene hydroxylase) and lcyE (lycopene-ε-cyclase) cause significant enhancement of proA concentration, while recessive allele, opaque2 (o2) enhances the level of these amino acids. Development of biofortified maize enriched in proA, lysine and tryptophan thus holds significance in alleviation of micronutrient malnutrition. In the present study, marker-assisted stacking of crtRB1, lcyE and o2 was undertaken in the genetic background of four maize hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) popularly grown in India. HP704-22 and HP704-23 were used as donors, while four elite QPM parents viz., HKI161, HKI163, HKI193-1, and HKI193-2 were used as recipients. CrtRB1 showed severe segregation distortion, while lcyE segregated as per the expectation. Recovery of recurrent parent genome (RPG) among selected backcross progenies ranged from 89 to 93%. Introgressed progenies possessed high concentration of proA (7.38–13.59 μg/g), compared to 1.65–2.04 μg/g in the recurrent parents. The reconstituted hybrids showed an average of 4.5-fold increase in proA with a range of 9.25–12.88 μg/g, compared to original hybrids (2.14–2.48 μg/g). Similar plant-, ear-, and grain- characteristics of improved versions of both inbreds and hybrids were observed when evaluated with their respective original versions. Mean lysine (0.334%) and tryptophan (0.080%) of the improved hybrids were at par with the original versions (lysine: 0.340%, tryptophan: 0.083%). Improved hybrids also possessed similar grain yield potential (6,301–8,545 kg/ha) with their original versions (6,135–8,479 kg/ha) evaluated at two locations. This is the first study of staking crtRB1-, lcyE-, and o2-, favorable alleles in single genetic background. The improved inbreds can be effectively used as potential donor for independent and/or simultaneous introgression of crtRB1, lcyE, and o2 in the future breeding programme. These biofortified maize hybrids, rich in proA, lysine and tryptophan will hold great promise for nutritional security.

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Section: Methodsmentioning

confidence: 99%

Development of Biofortified Maize Hybrids through Marker-Assisted Stacking of β-Carotene Hydroxylase, Lycopene-ε-Cyclase and Opaque2 Genes

et al. 2018

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“…Regular consumption of quality protein maize (QPM) varieties with high lysine and tryptophan has benefitted millions of peoples suffering from PEM worldwide (Tandzi et al, ; Tessema et al, ). Similarly, maize varieties with high proA conditioned by β‐carotene hydroxylase1 ( crtRB1 ) gene have been recently deployed (Muthusamy et al, ; Simpungwe et al, ; Yan et al, ; Zunjare, Hossain, et al, ; Zunjare, Chhabra, Hossain, Muthusamy, et al, ; Zunjare, Chhabra, Hossain, Baveja, et al, ). Reports suggest potential impact of proA‐rich maize in alleviating VAD (Palmer et al, ).…”

Section: Introductionmentioning

confidence: 99%

Marker‐assisted introgression of rare allele of β‐carotene hydroxylase (crtRB1) gene into elite quality protein maize inbred for combining high lysine, tryptophan and provitamin A in maize

et al. 2019

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Vitamin A deficiency in humans is a widespread health problem. Quality protein maize (QPM) is a popular food rich in lysine and tryptophan, but poor in provitamin A (proA). Here, we report the improvement of an elite QPM inbred, HKI1128Q for proA using marker-assisted introgression of crtRB1-favourable allele. HKI1128 was one of the parental lines of three popular hybrids in India and was converted to QPM in our earlier programme. Severe segregation distortion for crtRB1 was observed in BC 1 F 1 , BC 2 F 1 and BC 2 F 2 . Background selection by 100 SSRs revealed mean recovery of 91.07% recurrent parent genome varying from 88.78% to 93.88%. Across years, introgressed progenies possessed higher mean β-carotene (BC: 9.22 µg/g), β-cryptoxanthin (BCX: 3.05 µg/g) and provitamin A (proA: 10.75 µg/ g) compared to HKI1128Q (BC: 2.26 µg/g, BCX: 2.26 µg/g and proA: 3.38 µg/g).High concentration of essential amino acids, viz. lysine (mean: 0.303%) and tryptophan (0.080%) in endosperm, was also retained. Multi-year evaluation showed that introgressed progenies possessed similar grain yield (1,759-1,879 kg/ha) with HKI1128Q (1,778 kg/ha). Introgressed progenies with higher lysine, tryptophan and proA hold immense potential as donors and parents in developing biofortified hybrids. K E Y W O R D Sbiofortification, lysine, provitamin A, quality protein maize, tryptophan, β-carotene hydroxylase

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“…The favourable allele had 543 bp allele and the unfavourable produced 296 bp amplicon. The new PCR assay took 1.25 h in place of 2.50 h as compared to protocol by Zunjare et al (2017a). It also gave reproducible and clear amplicons as compared to earlier protocols (Yan et al 2010;Zunjare et al 2017a).…”

Section: Uniplex-pcr Assay For Crtrb1mentioning

confidence: 88%

“…The PCR protocols for sh2 (Hossain et al 2013), o2 (Sarika et al 2018), crtRB1 (Zunjare et al 2017a) and lcyE (Zunjare et al 2018b) genes standardized at Maize Genetics Unit, ICAR-IARI, New Delhi were modified for further reducing the time of assay. Ready-to-use master mix was used which included all the reagents viz., Taq buffer, MgCl 2 , dNTPs and Taq Polymerase.…”

Section: Uniplex-pcr Assaymentioning

confidence: 99%

“…Uniplex-polymerase chain reaction-based (Uniplex-PCR) strategy is routinely used in the MAS programme. However, multiplex-PCR (Mx-PCR) assay provides significant advantage to save time and resources in comparison to uniplex-PCR assays (Markoulatos et al 2002;Frey et al 2004;Zunjare et al 2017a). Mx-PCR assay is rapid and convenient to simultaneously identify allelic combinations in the segregating generations.…”

Section: Introductionmentioning

confidence: 99%

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Development and validation of multiplex-PCR assay to simultaneously detect favourable alleles of shrunken2, opaque2, crtRB1 and lcyE genes in marker-assisted selection for maize biofortification

Baveja

Chhabra

Panda

et al. 2020

J. Plant Biochem. Biotechnol.

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Sweet corn has emerged as a popular vegetable worldwide. Commercial shrunken2 (sh2)-based sweet corn lacks lysine, tryptophan and provitamin-A, deficiency of which cause various health problems. To overcome these problems, markerassisted stacking of sh2, opaque2 (o2), b-carotene hydroxylase (crtRB1) and lycopene epsilon cyclase (lcyE) was attempted to combine nutritional quality traits in maize endosperm. Gene-based simple sequence marker (SSR) specific to o2, InDel-based marker for crtRB1 and lcyE, and SSR linked to sh2 were used in marker-assisted selection. Four backcross populations segregating for the alleles of crtRB1, lcyE, o2 and sh2 were developed. The favourable alleles of sh2 and o2 produced 103 and 150 bp amplicons, while the unfavourable alleles were 96 and 160 bp, respectively. CrtRB1 produced 543 bp favourable and 296 bp unfavourable allele, while the same for lcyE was 650 and 300 bp, respectively. To detect desirable alleles of the target genes simultaneously, multiplex-PCR (Mx-PCR) assay was developed and validated in BC 2 F 2 populations. Mx-PCR assay successfully identified the favourable alleles of four genes simultaneously, and validated the results of individual uniplex-PCR assays. Mx-PCR assay saved 68% of cost and approximately 63% of time. This is the first report of multiplexing sh2, o2, crtRB1 and lcyE genes in maize. The information generated here would be immensely useful in molecular breeding programme.

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Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of crtRB1 and lcyE governing higher accumulation of provitamin A in maize

Cited by 12 publications

References 23 publications

Development of Biofortified Maize Hybrids through Marker-Assisted Stacking of β-Carotene Hydroxylase, Lycopene-ε-Cyclase and Opaque2 Genes

Development of Biofortified Maize Hybrids through Marker-Assisted Stacking of β-Carotene Hydroxylase, Lycopene-ε-Cyclase and Opaque2 Genes

Marker‐assisted introgression of rare allele of β‐carotene hydroxylase (crtRB1) gene into elite quality protein maize inbred for combining high lysine, tryptophan and provitamin A in maize

Development and validation of multiplex-PCR assay to simultaneously detect favourable alleles of shrunken2, opaque2, crtRB1 and lcyE genes in marker-assisted selection for maize biofortification

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