Molecular Plant Breeding 2016, Vol.7, No.33, 1
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constant imaging which gives on-line detection of separation at a resolution of 2 bp. The experiment was performed
using manufacturer’s instructions.
4.2.4 Background analysis
To check for background genome recovery of the 11 selected ABLs, SNP genotyping was carried out along with
RP and DP using high-density genome-wide genotyping chip. The Illumina 6K Infinium SNP Chip was designed
at Cornell University from which SNPs were selected to be informative within and in between
O. sativa
subgroups and between
O. sativa
and
O. rufipogon
(Thomson, 2014). Infinium platform detects SNP alleles by
adding a fluorescence-labeled allele-specific nucleotide via single-base extension and subsequent detection of the
fluorescence color (Steemers et al., 2006). SNP genotyping was carried out following the manufacturer’s protocol
and chips were scanned in BeadArray reader (Illumina Inc., USA). SNP genotyping data were analyzed using
GenomeStudio V2011.1 (Illumina Inc., USA). Graphical genotype of ABLs was generated using Phenogram
(
).
4.3 Evaluation for blast resistance
4.3.1 Blast nursery test
Screening for blast disease resistance/susceptibility of selected ABLs was conducted at IRRI blast nursery under
UBN test following the method of Suh et al. (2009). Forty to fifty seedlings of test materials were planted in two
replications. LTH and CO39 were used as susceptible (S) checks while IR72 and IR65482-4-136-2-2 (
Pi40
) were
used as resistant (R) checks during the course of evaluation. Spreader rows with a mixture of cultivars IR72, IR36,
CO39, IR50 and IR42 were set up around each replicate to maintain diverse pathogen population. Scoring of the test
materials was carried out 14 days after infection. Disease reaction was scored using the 0-9 scale of the standard
evaluation system (SES) of IRRI (IRRI, 1996) and percent DLA.
4.3.2 Blast spray inoculation
Artificial spray inoculation of selected ABLs was carried out at the glasshouse facility of IRRI following the method
described by Jeung et al. (2007) and using 21 Philippine virulent blast strains namely: M36-1-3-10-10, JMB840610,
Ca23-49, V850256, V86010, M39-1-3-B-1, M39-1-2-21-2, Ca89, BN209, BN111, 43, M101-1-2-9-1, 9482-01-03,
IK81-3, JMB8401, Ca41, IK81-25, P06-06, B90002, M64-1-3-9-1 and Pi9-G7-2K-1. In brief, 21-day-old
seedlings were inoculated with 1.5 ×10
5
spores/ml blast isolate suspension. Seedlings were spray inoculated with
20 mL spore suspension and were kept in a greenhouse with 12/12 h (day/night) photoperiod at 90% relative
humidity for seven days. Scoring was carried out following the method described by Japan International Research
Center for Agricultural Sciences (Hayashi and Fukuta, 2009).
4.4 Agronomic and grain quality evaluation of selected ABLs
Materials were seeded in a seed box and twenty one day old seedlings were transplanted at the Experiment Station
of IRRI. Recurrent and donor parents were planted along with ABLs in 5-rows with 20cm ×20cm spacing using a
complete randomized design during 2014 wet season (WS) and 2015 dry season (DS). Major agronomic traits
such as PH, TN, days to heading (DTH), FLL, 100 GW, PL, GN, F during 2014WS and 2015DS were collected
following standard evaluation system of IRRI. Grain quality was estimated for AC, GT, GC and PV. AC is
determined by colorimetric assay of the amylase-iodine complex (Juliano, 1971). GT category was estimated by
determining the alkali spreading value. The degree of spreading of 6 head rice soaked in 10 mL of 1.7% KOH
after 23 hours at room temperature (25 ± 5
o
C) was measured and a score of 1-7 is given, depending on the
intensity of swelling and spreading, 1 being no reaction and 7 being mostly dissolved. Alkali spreading value of
1-2 corresponds to high GT, 3 for intermediate high, 4-5 for intermediate, and 6-7 for low GT (Little et al., 1958).
Gel consistency was measured following the protocol of Cagampang (1971). In brief, the consistency of a cold 4.4%
milled rice paste in 0.2 M KOH was measured by the length of the cold gel in the culture tube held horizontally
for 30 min to 1 hour. PV was measured by quantification of total nitogren content by Kjeldahl digestion method.
The ammonia produced in the reaction was determined colorimetrically as indo-phenol blue (IRRI, 2005,
.
