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IDENTIFICATION OF MOLECULAR MARKERS ASSOCIATED WITH CULM STRENGTH IN RICE (Oryza sativa L.) USING SELECTIVE LINE GENOTYPING

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Title IDENTIFICATION OF MOLECULAR MARKERS ASSOCIATED WITH CULM STRENGTH IN RICE (Oryza sativa L.) USING SELECTIVE LINE GENOTYPING
 
Creator ANITHA SHALINI, S
 
Contributor SRIVIDHYA, A
 
Subject genotypes, rice, biological phenomena, planting, alleles, genes, phenotypes, yields, chromosomes, polymorphism
 
Description Culm mechanical strength is an important agronomic trait to be focussed in current day’s
crop breeding programme, to further enhance yield levels offered by bending type lodging tolerant
semi-dwarf rice, which produce low amounts of biomass. Compared to bending type resistance,
the plants with high breaking culm strength can produce more biomass and yield, yet remain nonlodging.
Hence, the present investigation was aimed at identification of marker/s that associated
with culm strength and related traits with main emphasis given for the identification of regions
governing breaking type lodging resistance, through selective line genotyping (SLG) approach.
A total of 45 rice germplasm accessions, which includes strong culm bearing varieties/lines
from IARI, PUSA; CRRI, Cuttack, and other popular varieties and landraces (Assam rice
collection) employed in current study were phenotyped for eight culm strength (CS) related and
seven yield related traits, under field conditions. The performance of genotypes for CS and related
traits revealed that the genotype MTU 3626 was recorded lowest values for plant height (PH,
55.70cm), culm height (CH, 33.40cm), inter-nodal length (INL, 8.86 cm) and number of
internodes (NIN, 3.70); whereas Pusa 44 recorded highest phenotypic values for culm diameter
(CD, 5.81 mm), pith diameter (PD, 2.26 mm) and culm strength (CS, 34.00 g/culm). For the yield
and related traits, different genotypes recorded highest for different traits viz. BM 71 for number of
panicles/plant (NPP, 19.15), Ravi for number of filled grains/panicle (NFGP, 161.2), CN 384 for
spikelet fertility (SF, 94.8 %), and Pusa 44 for grain yield/plant (GYP, 29.31 g), wherein along
with yield traits Pusa 44 and BM 71(recorded second highest GYP) showed first (34.00 g/culm)
and third highest (28.90 g/culm) score for CS. When considered both CS and yield traits, Pusa 44
was recorded highest values for culm strength (34.00 g/culm) and GYP (29.31 g). Hence molecular
tagging of genotypes that exhibited ~100 cm plant height (non-semidwarf type) with larger CD,
CWT (culm wall thickness) and CS (pushing resistance) is boon to culm strength breeding there by
development of new plant types suitable for super rice breeding.
Correlation analysis employing culm strength (CS) and related traits revealed that,
selection for the traits PH (0.309), INL (0.237), CD (0.373) and CWT (0.540) can increase lodging
resistance in rice. Here, the low correlation values can be explained either by non linearity of the
variables or may be due to presence of one or more outlier genotypes for the trait pairs measured.
Further based on correlations conducted among culm strength and yield related traits, it can be
concluded that selection for PH, CH, INL, CD, CWT and CS affect one or more yield traits viz.
number of panicles/plant (NPP, 0.321), panicle length (PL, 0.318), thousand seed weight (TSW,
0.513) and GYP (0.380), positively. Hence selection for these CS related traits will improve yield
levels also.
Frequency distributions for majority of culm strength and yield related traits did not fit well
the normal distribution, wherein some were close to normal distribution as majority of genotypes
fall into one or two class intervals (for instance, a high number of genotypes for CS (g/culm) fall
into two class intervals i.e. 15-20 and 21-25) or may be entirely towards either left (NIN and
CWT) or right side (PL and TSW). This kind of deviation from the normal distribution could be
result of the influence of very few (one or more) major genes. Hence these genotypes are suitable
for identification of chromosomal regions (markers) associated with culm strength and related
traits.
Phenotypic ranking for each genotype was assigned based on their performance for culm
strength, as CS is an important measure in analysing genotype efficiency for lodging tolerance (as
CS is a sign of both visible (morphological) and invisible (anatomical and biochemical traits) traits
governing lodging tolerance of a plant). The genotypes Pusa 44 (34.00 g/culm) CR 401 (31.70
g/culm), and BM 71 (28.90 g/culm) stood in first, second and third ranks, respectively.
Molecular analysis to tag chromosomal regions that are associated with culm strength
related traits was carried, employing selective line genotyping (SLG) approach. Of 45 genotypes
used for phenotyping, 16 were selected (8 each from two phenotypic extremes of culm strength i.e.
high-type and low-type) based on their phenotypic ranking. Polymorphism survey was conducted
between two core DNA samples prepared from 2 extreme high-types (Pusa 44 and CR 401) and 2
extreme low-type (JGL 3855 and Chittimuthyalu) genotypes employing a total of 463 SSR
markers, covering all rice chromosomes uniformly. The screening revealed that, only 15 markers
showed polymorphic alleles that spanned on 7 chromosomes viz. four markers on Chr 1
(RM11069, RM297, RM302, RM9), three on Chr 2 (RM12690, RM324, RM555), only one on Chr
4 (RM261), four on Chr 6 (RM20037, RM238, RM527, RM586), one each on Chr 7 (RM21906),
Chr 8 (RM310) and Chr 9 (RM242); which accounts for 3.24 percentage. The percent
polymorphism observed was extremely low compared to selective recombinant genotyping (bulk
segregant analysis, BSA) which employed widely in many QTL mapping studies. Hence,
ultimately it reduces the number of markers that needs to be used for independent genotyping with
extreme phenotypes.
Genotyping of 16 selected genotypes using 15 polymorphic markers generated an allele
size range from 90 bp to 350 bp at loci RM527 and RM586, respectively and an allele number of
3-6. Allele code was assigned among 16 genotypes, for each different sized allele of a locus
separately in the descending order of their size viz. A-F (for 6 alleles of a locus). Similarity
analysis of allele code generated by seven other high-type genotypes to Pusa 44 (1st rank at
phenotypic level for CS) revealed that, among all genotypes tested, CR 401(2nd rank at phenotypic
level for CS) showed majority of alleles i.e. 13 out of 15, similar to Pusa 44, which is in
accordance with phenotypic ranking. Hence, these markers are assumed to be well associated with
the regions that govern culm strength.
Based on allele similarity generated by each genotype for 15 polymorphic loci, genotypic
rank was assigned. When compared the phenotypic and genotypic ranks of 8 high-type genotypes,
except the landrace INRC 10192, remaining all genotypes showed consensus from phenotypic
ranking to genotypic ranking (with very negligible deviations for the genotypes BM 71, MTU
1064, Annada and SV3 i.e. change in one rank position either side). Pearson correlation study
between phenotypic and genotypic rankings of high-type genotypes, excluding the landrace INRC
10192 found highly significant (p = 0.009 and R2 = 0.774), and when considered landrace with
remaining high-types, it resulted in moderately significant association (p = 0.052 and R2 = 0.495).
Hence, the markers and the genotypes are valuable sources in tagging culm strength related trait/s
closely and thereby to develop potent donor sources, respectively.
Among all polymorphic markers used, the markers RM20037, RM11069 and RM9 were
generated common allele pattern in all high-types except for MTU1064 and Annada. Hence the
degree of allelic association among high-types (generation of similar allele) is more for RM20037
(100%), followed by RM11069, RM9, RM555, RM586, RM21906 and RM242, which showed the
allelic association range of 50 - 87.5 %. Thus these markers/regions are assumed to have strong
association with lodging resistance. Hence, validatition of these markers by analyzing their
association in a segregating population derived from the respective high-type(s), will help to select
precise markers for use in a marker assisted breeding programme.
The identified markers on four chromosomes are very closely located to brittle culm/strong
culm genes, which have been reported earlier. They includes, two genes that increases cellulose
deposit in culm cell walls (secondary cell wall formation) viz. Brittle culm 7(t) [Bc7(t),
Os01g0750300] has close location with our markers RM297 (only 100 Kb) and RM302 (203 Kb)
on chromosome 1, whereas BC15/OsCTL1 (Os09g32080, chitinase-like1 protein) located nearer to
RM242 (338 Kb) on chromosome 9; and a gene that increases lignin concentration of cell walls,
OsCAD2 (Os02g0187800, cinnamyl-alcohol dehydrogenase) was located close to RM555 on
chromosome two (564 Kb). Further STRONG CULM2 (SCM2, Os06g0665400), that imparts CS
through increase in culm diameter is located nearer to our polymorphic marker RM20037 (1200
Kb) on chromosome 6 was noticed. The positions of RM261 on chromosome 4 and RM21906 on
chromosome 7 are novel regions, since there are no previous reports on mapping of these
regions/closely located cloned genes for CS related traits. These markers (regions) and genotypes
are valuable in developing alternative donor sources for these Japanese rice (japonica) and Chinese
rice (indica) based genes, where they originally identified which cannot be used directly to
improve our varieties. These regions can be rapidly delineate for their variation at closely located
reported candidate genes or of novel regions employing advanced tools like NGS based targeted
re-sequencing using the high-type genotypes, which in-turn will help to develop trait associated
molecular/genic tags or may be of functional tags.
Put together the involvement of small set of phenotyping panel and use of extremely core
set of genotyping panel for SLG, that generated most relevant association of many markers with
reported bending strength genes and as well identified novel regions as comparable to QTL
mapping, allele pattern exhibited by genotypes alike large association panel, additional knowhow
of the degree of association of a locus makes the SLG technique to be challenged as greatly
powerful.
 
Date 2017-01-03T10:14:59Z
2017-01-03T10:14:59Z
2015
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/94058
 
Language en
 
Relation D9877;
 
Format application/pdf
 
Publisher PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD