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HAPLOID INDUCTION OF ALLELIC DIVERSITY POPULATIONS IN MAIZE(Zea mays L.)

Submitted byNAVEEN JAKHAR

Submitted toDepartment of Genetics & Plant Breeding

Allahabad School of AgricultureSam Higginbottom Institute of Agriculture, Technology & Sciences

(Formerly Allahabad Agricultural Institute)Deemed University, Allahabad

2015 ID 14MSCGPB029

Outline • Introduction to Haploid technology

• Objectives • Methods

• Methodology

• Outcome

INTRODUCTION Induction of maternal haploids occurs when a female plant is

pollinated by a haploid inducer line, resulting in a small amount of haploid progeny in addition to normal F1 progeny.

There are two hypotheses on haploid embryo formation. First hypothesis involves two sperm cells, one of which is non-

functional. The non-functional sperm cell fuses with the egg cell and the normal sperm cell fuses with the central cell to create a normal triploid endosperm. Subsequently, the non-functional sperm cell’s chromosomes are purged from the embryo through multiple cell divisions .

Second hypothesis involves two normal sperm cells, but one cannot fuse with the egg cell resulting in haploid embryogenesis, while fertilization by the other sperm cell creates the triploid endosperm .

OBJECTIVES• Haploid induction of allelic diversity population in

maize• Identification of superior inbred line

Methods for Producing haploids

DH-Donor/ Source germplasm (Female)

Inducer (Pollinator)

Haploid seedlings

Two doubled haploid

(DH) plants

Two new DH lines

1

2

3

4

1. Induction of haploid

2. Identification of haploids

3. Genome Doubling of Haploids

4. Self-pollination for seed multiplication

METHODS OF HAPLOID INDUCTION • Use transgenic inducers resistant to an herbicide.• Use a high oil inducer and sorting by seed oil content. With use of a transgenic herbicide-resistant inducer,

seedlings are treated with herbicide on a small area of a leaf; leaf tissue injury results on haploid seedlings (Geiger, 2009).

The high oil inducer line produces diploid seed with larger

embryo size and higher oil content than haploid seed and progeny can be identified using nuclear magnetic resonance (Chang and Coe, 2009).

METHODOLOGY

Selected races from the AD project were assigned to three elevation groups.

• The high elevation group consisted of races above 2,500 meters, • Mid elevation races between 1,500-2,499 meters, • Low elevation races originating from elevations below 1,500

meters. Six races were randomly selected from each of the three elevation

groups for a total of 18 races. These 18 races were represented in both Ex-PVP backgrounds, resulting in 36 populations. For the growing seasons of 2011 and 2012, ten replications of each population were planted in a complete randomized block design, providing 360 experimental units each year. Each replication consisted of one row containing at most ten plants.

At least three pollinations were attempted using the maternal haploid inducer RWSxRWK-76, to obtain seed for haploid screening. Kernels were sorted during the winters of 2012 and 2013 based on a color marker to differentiate between hybrids, contaminates (any seed not containing the R1-nj marker) and haploids.

Any broken, germless, or diseased kernels were discarded and not utilized in

the calculation of HIR. If the kernel expressed the purple Navajo marker (R1-nj) on the cap of the kernel, it was assumed that it resulted from pollination with the haploid inducer, and would either be scored as haploid or a hybrid. Kernels without purple caps were considered to be considered to be a hybrid and discarded.

If a kernel lacked a purple scutellum and had the R1-nj marker on the kernel

cap, the kernel was considered to be a putative haploid. The number of haploid, hybrid, and contaminate seeds were recorded to obtain haploid induction rates. The HIR was calculated by dividing the number of haploid seeds by the total number of seeds in the experimental unit.

Using a 95% confidence interval, the following model was used to analyze the results:

Yijkl = µ + Bi + Ej + BEij + R(j)k +BRi(j)k εijkl Where : B = the effect of the ith background, E = the effect of the jth elevation group, BE = the interaction of background and race to the ijth level. R = the effect of the kth race nested in the jth elevation group. BR = the interaction of background and race nested within

elevation to the ijkth level. ɛ = the residual error, and a normal distribution was assumed. Using SAS 9.2, probabilities and Tukey-Kramer comparison lines

for least squares means were calculated.

OUTCOME Across both the 2011 and 2012 growing seasons, populations with low elevation

races had significantly higher HIRs than those with high or mid elevation groups (Pr = 0.0003).

The low elevation race populations averaged a HIR of 7.13% and ranged from

6.31% for the Puya Grande race to 8.54% for the TuxpeñoNorteño race. The average HIR of the high elevation race populations averaged 6.24% with a

range of 4.47% to 7.62% from Conico and Cuzco races, respectively). Middle elevation race populations averaged 5.62% for HIR and had rates that

ranged from 4.16% HIR for the Amagaceño race to 6.41% HIR for the San Marceño.

Recurrent Ex-PVP parent effects were significant (Pr = 0.0026), with PHB47

having a higher HIR, 6.67%, than PHZ51, 5.90% over the two years). When the recurrent parent was PHB47, the HIR ranged from 4.34% for the Amagaceño race to 10.30% for the TuxpeñoNorteño race).

Finally, differences between races were significant as well (Pr = 0.0001), with TuxpeñoNorteño having the highest and Conico the lowest HIRs when means were adjusted for missing data and analyzed using the Tukey-Kramer comparison lines for least squares means .

HIR for all races averaged 6.37%, and ranged from 4.16% - 8.54%

for Amagaceño and TuxpeñoNorteño, respectively. The highest HIR was 10.30% for TuxpeñoNorteño backcrossed to PHB47, and the lowest HIR was 3.93% from Amagaceño backcrossed to PHZ5.

The interaction of background and elevation was non-significant

(Pr = 0.1639), however, the three way interaction of background and race nested in elevation was significant (Pr = 0.0066).

 

REFERENCE Geiger  HH  (2009)  Doubled  haploids.  In:JL  Bennetzen,  S  Hake  (eds.)

 Maize handbook  volume  II:  genetics  and  genomics.Springer  Science  and Business  Media,  New  York,  pp.  641–657.

  Geiger  HH,  Gordillo  GA  (2009)  Doubled  haploids  in  hybrid  maize

 breeding.  Maydica  54:  485–499.  Chang  MT,  Coe  EH  (2009)  Doubled  haploids.  In:  AL  Kriz,  BA

 Larkins  (eds)  Biotechnology  in  Agriculture  and  Forestry.  Vol.  63.  Molecular  Genetic  Approaches  to  Maize  Improvement.  Springer  Verlag,  Berlin,  Heidelberg,  pp.127–142.

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THANKS