Indian Journal of Biotechnology

Vol. 19, July 2020, pp 159-168

Variability and molecular diversity of wild sugarcane germplasm collected from

low temperature regions Lohit and Changlang of Arunachal Pradesh
C. Appunu1,*, J. Ashwin Narayan1, H. K. Mahadevaswamy1, S. Karthigeyan1, R. Valarmathi1, C. Mahadevaiah1,
Ravinder Kumar2, Mintu Ram Meena2, Bakshi Ram1
1
ICAR-Sugarcane Breeding Institute, Coimbatore 641007, Tamil Nadu, India
2
ICAR-Sugarcane Breeding Institute Regional Centre, Karnal Haryana, India

Received 28 September 2019; revised & accepted 20 April 2020

Saccharum spontaneum L. is a perennial grass representing the most genetically diversified species in Saccharum
genus. It has the potential to withstand severe biotic/abiotic stresses and frequently used as donor of stress tolerant genes in
sugarcane improvement program through gene introgression. In this study, the phenotypic variation and molecular diversity
of forty nine S. spontaneum accessions collected from Lohit and Changlang regions of Arunachal Pradesh, North Eastern
India were investigated for morphometric traits and polymorphic STMS marker. The phenotypic coefficient of variation
showed ample variability for the traits viz., plant height (27.19%), stalk diameter (28.21%), single cane weight (48.97%),
internode number (22.60%) and internode length (29.15%). Further, twenty nine sequence-tagged microsatellite site (STMS)
markers generated 495 bands with an average of 14.06 polymorphic bands. The accessions specific bands in respect to
specific marker combinations were identified. The Jaccard’s similarity coefficients among these accessions ranged from
0.42 to 0.78 with an average of 0.58 and clustering using unweighted pair group method of arithmetic-average (UPGMA)
showed two major clusters with subclusters. Similarly polulation structure analysis based Bayesian approach grouped the
individuals into two subpopulations, with alpha value of 0.112. The study shows that S. spontaneum accessions collected
from Arunachal Pradesh is highly diverse, most of them will be harbouring the genes for cold tolerance and biomass. The set
of markers which produced specific bands for the specific accessions identified in the study will help in identification of the
particular accessions. The accessions studied are potential source for cold tolerance and high biomass, the results obtained in
the present study will definitely help in planning and utilising them in sugarcane improvement programme.

Keywords: Molecular diversity; STMS marker; Saccharum spontaneum; germplasm; sugarcane

Introduction cultivars are complex hybrids derived mainly from the

Saccharum genus belongs to family Poaceae which interspecific crosses made between S. officinarum and S.
has six species, viz. S. officinarum, S. barberi, S. edule, spontaneum. The significant contribution of this species
S. robustum, S. spontaneum and S. sinense. The is imparting high productivity and resistance to diseases
S. spontaneum L. is a tall (1 to 7 m in height) perennial and pests in the modern sugarcane cultivars6-7. Realising
C4 grass with deep roots and rhizomes and has diverse the importance of the species in the sugarcane varietal
genetic composition1. Almost, thirty cytotypes of S. improvement programmes, there had been concerted
spontaneum with chromosome number ranging from 2n efforts in the past to collect and conserve the S.
= 40 to 128 has been reported2-3. The species has wide spontaneum diversity. Large S. spontaneum accessions
adaptability and extensively distributed throughout representing different geographical groups are available
India. It is usually found in banks of water bodies (river, in the Indian Sugarcane Germplasm Collection, which
lakes, and ponds), alongside the road and railway tracks, have been characterized for quantitative and
alluvial plains, damp depressions, swamps, sandy soils, morphological attributes8-9. Through regular
etc4. It occurs at an altitude ranging from lowland eco- explorations, collections were made and are conserved at
region or sea-level to more than 1,800 m5. The species the field gene bank of ICAR-Sugarcane Breeding
has played a significant role in the development of Institute (SBI) Coimbatore, India. One such explorations
improved sugarcane varieties, as the modern sugarcane was aimed to collect accssions which are likely to
harbour the genes for cold tolerance from Lohit and
—————— Changlang regions of Arunachal Pradesh, a northeastern
*Author for correspondence
Tel: +914222472621; Fax: +914222472923
state which is most important region in terms of both
[email protected] abundance and diversity of Saccharum germplasm in
160 INDIAN J BIOTECHNOL, JULY 2020

India5. The tall, thick forms of S. spontaneum found

exclusively in some part of Arunachal Pradesh are
considered being a vital source of gene for vigour and
high productivity in sugarcane breeding5. Molecular
markers have been used for the genetic characterization
of germplasm in a variety of crops including sugarcane.
Various types of markers like RFLP, 5S rRNA ITS
marker, RAPD, ISSR, AFLP, SRAP, TRAP, SNP,
STMS, genomic simple sequence repeat (gSSR), EST-
derived simple sequence repeats (EST-SSRs), SCoT
have been used for the analysis of phylogeny, clone or
cultivar identification, parent evaluation, genetic
mapping, inter-species relationships and genetic
diversity among the Saccharum species, related genera
and their hybrids10-11. In the present study, we analyzed
the phenotypic variation, genetic diversity and
relationship of 49 accessions of S. spontaneum collected
from Lohit and Changlang regions of Arunachal
Pradesh, to provide a basis for utilization of the wild Fig. 1 — Gel electrophoresis of the amplified products obtained
with STMS primers NKS52 (A) and NKS53 (B). Lanes; M,
germplasm in sugarcane improvement.
Marker; 1, S. spontaneum IND 00-997; 2, IND 00-998; 3, IND
00-1001; 4, IND 00-1002; 5,IND 00-1004; 6, IND 00-1011; 7,
Materials and Methods IND 00-1012; 8, IND 00-1015; 9, IND 00-1018; 10, IND 00-
1019; 11, IND 00-1020.
Plant Material and Assessment of Trait Phenotypic Variation
Forty-nine accessions of S. spontaneum collected
Twenty nine STMS markers (Supplementary
from Lohit and Changlang regions of Arunachal
Table 2) were used for molecular diversity analysis of
Pradesh, India were used in this study
49 S. spontaneum accessions. PCR reaction was
(Supplementary Table 1 and Supplementary Fig. 1).
performed by following the procedure mentioned by
These accessions are being maintained at World Saravanakumar et al14. A total of 10 μl reaction
Germplasm Collection, Indian Council of mixture contained 10X PCR buffer with 15 mM
Agricultural Research (ICAR)-Sugarcane Breeding MgCl2, 0.5 U of Taq DNA polymerase, 100 μM of
Institute (SBI), Coimbatore, Tamil Nadu, India. To each dNTPs, 20 pmol/μl of each primer and 20 ng/μl
assess the phenotypic variation, each accession was of genomic DNA. PCR reactions were performed in
planted and replicated thrice in a randomized Thermal cycler (Eppendorf Master Cycler ProS,
replicated blocks with plot size of 3 m length and 1.2 Germany) using a single primer pair in each reaction.
m spacing between rows. The regular field The PCR cycle conditions were: initial denaturation at
management was carried out as like for commercial 94°C for 2 minute followed by 35 cycles consisting of
sugarcane production12. Observations were recorded denaturation at 94°C for 1 minute, primer annealing
for five quantitative traits viz. plant height (cm), temperature varied depends on primers for 40 second,
stalk diameter (cm), single cane weight (kg), and extension at 72°C for 40 second followed by a
internode number and internode length (cm). final extension at 72°C for 7 minute. The PCR
products obtained were resolved in 8% native PAGE
Genomic DNA Isolation and PCR Amplification
and documented using Geldoc System (Alpha Imager
Genomic DNA from a young leaf of each plant was
version 4).
isolated by following the procedure of Doyle and
Doyle13. After RNAs treatment DNA quality was Data Analysis and Phylogeny Construction
checked by 0.8% agarose and quantified using Only clearly visible and reproducible amplicons
NanoDrop spectrophotometer (ND-1000, version from the sequence tagged microsatellite site (STMS)
3.1.1, United States of America). Polymerase chain primers were considered for further analysis15. The
reaction (PCR) performed with a DNA concentration markers were scored as absent (0) or present (1),
of 20 ng/µl. genetic similarity relationship analysis between
 APPUNU et al.: VARIABILITY AND MOLECULAR DIVERSITY OF WILD SUGARCANE 161

the accessions was estimated by calculating Lohit and four were from Changlang. The altitude of
Jaccard’s similarity coefficients. Cluster analysis was the areas explored were ranged from 140 m to 1650
performed with these coefficient values and a m. Maximum number of accessions recorded the
dendrogram was constructed by the unweighted pair chromosome number of 64 (2n=64) while, one
group method of arithmetic-average (UPGMA) using accession IND 00-1064 had chromosome number of
NTSYS program (NTSYS-pc2.11)16. The quantitative 60 (2n=60). The moropological traits, like plant
traits viz. plant height, stalk diameter, single cane height, stalk diameter, single cane weight, internode
weight, internode number and internode length were number and internode length showed higly significant
used to estimate the phenotypic coefficient of variation among the accessions (Table 1). The
variation (PCV), genotypic coefficient of variation existence of high variation has been reported among
(GCV) and environmental coefficient of variation S. spontaneum clones collected in arid and semi-arid
(ECV) using SAS (SAS version 9.2 software package; zones of North Western India21. The plant height
SAS Institute, Inc.; Cary, NC) software. ranged from 89.6 to 293.3 cm with mean of 214.3 cm.
Stalk diameter ranged between 0.36 and 1.86 cm with
Population Structure mean of 1.19 cm. The single cane weight ranged from
Analysis of the population structure of 49 S. 0.018 to 0.508 kg with mean of 0.277 kg. The number
spontaneum genotypes was carried out using of internodes ranged from 8.7 to 25.3 with the average
STRUCTURE v.2.3.4, for which a Bayesian approach of 15.1 and the internode length ranged between 4.5
is used to infer the population structure17. Gene flow and 31.9 cm with mean of 18.2 cm. The phenotypic
(Nm), or the number of migrants entering a coefficient of variation (PCV), was highest for single
population in each generation, was estimated using cane weight (48.97%) followed by internode length
the formula Nm=0.25×(1−Fst)/Fst (five independent (29.15%), stalk diameter (28.21%), plant height
runs with K value ranging from 2 to 8 and five (27.19%) and number of internodes (22.60%; Table
iterations for each value of K was set)18. To determine 1). The geneotypic coefficient of variation (GCV) and
the actual K value, the following structure parameter environmental coefficient of variation (ECV) were
was set with the possibility of admixture and allele estimated (Table 1), GCV was highest for single cane
frequency: the length of the burning period was weight (47%) and lowest for plant height (18.95%)
50,000 iterations followed by 200,000 Monte Carlo while ECV was highest for plant height (19.51%) and
Markov Chain replicates. To obtain the optimal K lowest for stalk diameter (6.29%). PCV and GCV
value, it was plotted against the mean estimate of log ranged between 0-10%, 10-20%, and over 20%
probability of the data L(K). The actual number of respectively are considered as lower, medium and
sub-populations was identified using the maximum high variation levels22-23. The GCV and PCV provide
L(K) value. The final population structure was a measure to compare the variability present among
calculated with ΔK, based on the second-order rate of the traits. Values of PCV and GCV were close for
change of likelihood distribution mean L′′(K) and most of the traits except for plant height, indicating
with respect to K estimated using STRUCTURE that other traits are less influenced by environment
HARVESTER and optimal K value was obtained19-20. and variation observed was due to their genetic
Analysis of molecular variance (AMOVA) was done constitution. These germplasm may harbour different
to detect the genetic variation within and among the combination of genes which may be highly helpful in
population. genetic improvement of sugarcane for cold tolerance
and biomass.
Result and Discussion
Polymorphism Detected Using STMS Markers
Phenotypic Variation Analysis Twenty nine selected STMS markers which
A total of 49 S. spontaneum accessions collected produced scorable, reproducible and well-resolved
from Lohit and Changlang regions of Arunachal banding patterns were used for genetic diversity
Pradesh, India was analyzed for phenotypic variation studies (Supplementary Table 2). The number of
and their genetic diversity. These accessions were amplified DNA fragments by each primer ranged
collected from twenty-five locations in two major from 2 (NKS 53) to 31 (NKS 6, NKS 12, NKS 16
districts of Arunachal Pradesh (Supplementary Table and NKS 21) with an average of 17.68 fragments per
1; Supplementary Fig. 1), of which 45 were from primer (Fig. 1, Table 2). Usually, high ploidy plants
162 INDIAN J BIOTECHNOL, JULY 2020

Table 1 — Morphological diversity in agronomic traits of S. spontaneum L. clones from different areas of Arunachal Pradesh, India
Clone nos. Plant height (cm) Stalk diameter (cm) Single cane weight (kg) Internode number (no) Internode length (cm)
IND00-997 241.7 1.33 0.233 17.9 19.1
IND00-998 233.3 1.26 0.262 15.7 30.3
IND00-1001 153.3 1.37 0.297 11.7 14.9
IND00-1002 196.7 1.29 0.305 11.7 21.8
IND00-1004 235.0 1.35 0.440 12.3 16.5
IND00-1011 246.7 0.85 0.072 15.0 20.7
IND00-1012 181.7 1.66 0.200 20.3 15.6
IND00-1015 215.0 0.81 0.196 19.3 14.2
IND00-1018 251.7 1.70 0.470 11.0 21.5
IND00-1019 216.7 1.28 0.402 12.3 16.8
IND00-1020 293.3 1.34 0.403 25.3 15.8
IND00-1022 201.7 1.72 0.538 12.7 15.3
IND00-1024 225.0 1.52 0.488 13.0 14.2
IND00-1025 228.3 1.52 0.460 14.3 16.1
IND00-1026 180.0 1.10 0.235 13.7 14.9
IND00-1027 153.3 1.44 0.292 10.0 20.3
IND00-1030 193.3 1.09 0.180 14.7 17.2
IND00-1032 125.0 1.07 0.140 8.7 11.8
IND00-1034 216.7 1.38 0.355 15.7 22.4
IND00-1035 206.7 0.96 0.115 15.7 15.2
IND00-1036 176.7 1.24 0.310 18.3 12.5
IND00-1037 218.3 0.95 0.148 18.0 9.4
IND00-1038 231.7 1.37 0.337 17.0 22.8
IND00-1039 218.3 1.26 0.293 14.3 24.2
IND00-1040 241.7 1.22 0.313 16.0 21.2
IND00-1041 235.0 1.09 0.300 14.7 22.1
IND00-1042 285.0 1.47 0.413 15.7 31.9
IND00-1043 256.7 1.35 0.365 19.0 18.1
IND00-1044 221.7 1.06 0.212 17.7 15.7
IND00-1045 251.7 0.67 0.128 13.3 25.3
IND00-1046 138.3 0.65 0.062 19.3 11.1
IND00-1047 90.0 0.36 0.018 13.7 4.5
IND00-1048 110.0 0.93 0.091 9.7 9.9
IND00-1051 205.0 1.96 0.378 13.0 21.6
IND00-1054 215.0 1.38 0.286 13.3 18.1
IND00-1056 220.0 1.37 0.300 13.7 24.2
IND00-1057 236.7 1.45 0.395 16.7 22.9
IND00-1058 236.7 1.05 0.292 16.7 19.6
IND00-1059 261.7 1.23 0.363 20.7 22.2
IND00-1060 236.7 0.99 0.170 17.0 17.1
IND00-1061 253.3 1.29 0.294 15.7 21.4
IND00-1062 288.3 1.47 0.508 16.7 23.2
IND00-1063 183.7 1.51 0.436 11.3 18.9
IND00-1064 105.0 0.54 0.035 11.3 12.1
IND00-1067 176.7 1.25 0.317 11.7 15.1
IND00-1068 288.3 0.74 0.111 15.7 16.3
IND00-1069 208.3 1.28 0.421 15.7 12.4
IND00-1070 246.7 0.63 0.102 13.7 18.1
IND00-1071 268.3 0.60 0.105 13.3 16.9
Mean 214.3 1.19 0.277 15.1 18.2
General Mean 214.29 1.19 0.28 15.05 18.16
MST 6693.51** 0.32** 0.055** 30.99** 80.77**
(<.0001) (<.0001) (<.0001) (<.0001) (<.0001)
MSe 1747.36 0.0056 0.0008 1.86 1.64
General Mean 214.29 1.19 0.28 15.05 18.16
Minimum 89.6 0.36 0.018 8.7 4.5
Maximum 293.3 1.86 0.508 25.3 31.9
(Contd.)
 APPUNU et al.: VARIABILITY AND MOLECULAR DIVERSITY OF WILD SUGARCANE 163

Table 1 — Morphological diversity in agronomic traits of S. spontaneum L. clones from different areas of
Arunachal Pradesh, India (Contd.)
Clone nos. Plant height (cm) Stalk diameter (cm) Single cane weight (kg) Internode number (no) Internode length (cm)
LSD at 5% 67.75 0.12 0.046 2.21 2.08
CV(%) 19.51 6.27 10.23 9.06 7.06
PCV 27.19 28.21 48.97 22.60 29.15
GCV 18.95 27.51 47.92 20.70 28.28
ECV 19.51 6.29 10.10 9.06 7.06
** Highly significant, Values in the parenthesis indicates p-value (0.05)
Table 2 — Genetic diversity among 49 S. spontaneum accessions revealed by STMS primers
Marker Number of Number of Percentage of Size range of bands Unique fragment amplified
name bands amplified polymorphic bands polymorphic bands (%) Min Max
NKS1 22 18 81.82 216 1090 IND00-998
NKS2 24 19 79.17 180 915 -
NKS3 14 13 92.86 183 977 IND00-1024, IND00-1069
NKS6 26 24 92.31 147 1060 -
NKS7 23 19 82.61 174 490 -
NKS8 9 8 88.89 194 527 IND00-1059
NKS9 25 20 80.00 148 1613 IND00-1057
NKS12 26 21 80.77 147 2080 -
NKS16 26 19 73.08 157 944 IND00-1061
NKS21 26 22 84.62 165 693 IND00-1046
NKS24 11 11 100.00 136 993 -
NKS25 17 13 76.47 153 884 IND00-1068
NKS28 25 17 68.00 119 696 -
NKS29 12 9 75.00 145 425 IND00-1062
NKS30 16 11 68.75 129 696 IND00-1062
NKS31 13 8 61.54 101 582 -
NKS38 13 9 69.23 128 590 IND00-1036
NKS42 16 12 75.00 182 1375 -
NKS45 19 18 94.74 104 772 IND00-1039
NKS46 4 3 75.00 116 554 IND00-1047
NKS48 17 15 88.24 103 554 IND00-1047
NKS49 23 22 95.65 100 1035 -
NKS50 19 18 94.74 112 806 IND00-1041
NKS51 23 19 82.61 130 645 IND00-1041
NKS52 3 3 100.00 110 540 IND00-1043
NKS53 2 2 100.00 102 339 -
NKS56 14 13 92.86 129 407 -
NKS57 13 10 76.92 101 482 IND00-1036
NKS61 13 12 92.31 128 356 -

with high heterozygosity show multiple bands per range of 300-800 bp. The total number of bands
marker per clone. The high number of DNA scored was 495, of which 408 were polymorphic
fragments obtained in S. spontaneum is possibly due (82.40%) with an average of 14.06 per primer (Table
to the ploidy level and as well as hetrozygosity. High 2). The polymorphism ranged from 61.54 (NKS 31)
number of amplicons were recorded in Saccharum to 100% (NKS 24, NKS 52 and NKS 53), revealing
interspecific and intergeneric hybrids using STMS a high degree of polymorphism among these clones.
markers24 than in commercial cultivars, this could be The different group of markers revealed a high
possibly due to the high similarity between the degree of polymorphism in S. spontaneum26-33.
commercial varieties. The chromosome number of S.
spontaneum accessions used in this study were of Germplasm Accession Specific Markers
2n=60 (IND 00-1064) and 2n=64 (all other Out of 29 primers tested, the combined profile
accessions)25 indicating that their ploidy level is of polymorphic bands provided S. spontaneum
high. The size of the amplified fragment varied from accession specific markers that could distinguish from
100 to 2080 bp, but most of the bands obtained in the each other (Table 2). Unique markers were
164 INDIAN J BIOTECHNOL, JULY 2020

identified either by their presence or absence in a difference obtained with commercial sugarcane
particular accession. Unique DNA fragment amplified varieties based on STMS markers42-43. Lower
by some of the markers will be useful for variation in commercial varieties could be attributed
identification/differentiation of clones and germplasm to the utilization of only few germplasm accessions in
verification (Table 2). Different group of markers varietal development process and also due to varital
including STMS have been reported to be improvement is concentrated on few important traits.
successfully used for identification or differentiation Lu et al44 reported relatively higher genetic diversity
of individuals in Saccharum species clones and of 69% in S. spontaneum from five different countries
Saccharum hybrids34-37. These unique markers could based on RFLP markers. However the present
also be used for identification of specific varieties in a estimate of genetic diversity is higher than that has
mislabeled field and selfed progeny in conventional been reported for the Chinese S. Spontaneum
sugarcane breeding38 and marker-assisted selection in (Hui et al. 2001, Fan et al. 2001). Arunachal
varietal development programme39. STMS markers Pradesh clones showed the maximum intragroup
have been successfully used to identify the related diversity consistent with its phenotypic diversity as
species, landraces, new plant lines and varieties of represented by tall, medium and dwarf clones. The
many other crops of Poaceae family40-41. These clone relatively high difference in the Arunachal Pradesh
specific markers identified in the study would also be group is expected since this is the most variable group
helpful in monitoring the transmission of genomic among the Indian S. spontaneum in terms of
regions introgressed from specific accession of S. morphological variation45. Manechini et al46 reported
spontaneum clones in the progeny generations. that based on microsatellite marker analysis highest
putative exclusive alleles (39%) of the basic
Similarity Coefficient Analysis germplasm group with S. spontaneum were not found
Pairwise genetic similarity coefficient among the in the Brazilian cultivars.
49 S. spontaneum accessions ranged from 0.42 (IND
00-997 with IND 00-1048, IND 00-998 with IND 00- Clustering Analysis
1048, IND 00-998 with IND 00-1051) to 0.78 (clones Clusters was constructed using UPGMA, based on
IND 00-1024 and IND 00-1025) with an average of the Jaccard’s similarity coefficients to estimate the
0.58. The overall diversity among the accessions was genetic relationships among the 49 S. Spontaneum
found to be high, which is much higher than the accessions (Fig. 2). STMS markers were found to be

Fig. 2 — Dendrogram of 49 accessions of S. spontaneum L. collected from Arunachal Pradesh.

 APPUNU et al.: VARIABILITY AND MOLECULAR DIVERSITY OF WILD SUGARCANE 165

adequate for assessing genetic diversity. All these plains. Geographical distribution is in agreement with
accessions distinctly clustered into two groups, which earlier studies (Mary et al. 2006, Fan et al. 2013). The
shared a common node at a similarity coefficient of genetic relationship between the accessions of S.
0.70. Cluster I and II consisted of 45 and 4 accessions, spontaneum was related to geographical distribution
respectively. The cluster I was further divided into and ecological conditions. All of these studies
two sub-clusters. One sub-cluster consisted of four indicated that geographic factors play an essential role
sub-clusters (A, B, C, D) and another consisted of two in the pattern of the genetic structure within S.
sub-clusters (E and F). Of S. spontaneum accessions, spontaneum.
15 were in sub-cluster A, six in B, one in C, nine in D,
seven in E and F each, which shared a common node Population Structure
at similarity coefficient of 0.75. The result shows that The two optimum number of clusters were
most of the accessions collected from the same decided using the value of ∆K distribution (Fig. 3,
area/habitats or adjacent area gathered in the same Supplementary Table 3). From the total clones
cluster. Geographical factors play an important role in investigated 46 clones had more than 60%
the pattern of accessions genetic structure47. The membership in the given cluster. Three clones IND
accessions IND 00-1036 - IND 00-1044 from high 00-1001, IND 00-1012 and IND 00-997 share
altitude regions (> 1000 m, supplementary Table 1) similar membership coefficients in both the clusters
were formed a sub-cluster A. The IND 00-1012 - IND and considered as admixtures. The alpha value 0.112
00-1022 from neighboring regions classified into sub- indicating most of the individuals are pure and only
cluster B and a clone IND 00-1024 classified into sub- few admixtures. The mean FST values with 0.58
cluster C. The clones IND 00-1025, IND 00-1026, (cluster 1) and 0.09 (cluster 2) confirmed the
IND 00-1027, IND 00-1030, IND 00-1032, IND 00- existence of differences among clusters. It is
1034, IND 00-1035, IND 00-1045 and IND 00-1046 generally accepted that FST values under 0.05
from Paya-Tiding and Tiding regions classified into indicate negligible genetic differentiation while
sub-cluster D. those over 0.25 indicate a great deal of genetic
The accessions IND 00-1056, IND 00-1057, IND differentiation51. The average distances (expected
00-1058, IND 00-1059, IND 00-1060, IND 00-1061 heterozygosity) among individuals in same cluster
and 00-1062 from Kherum, Medo, Kamlang and were 0.13 (cluster 1) and 0.27 (cluster 2). High
Wakro regions were classified into sub-cluster E, and heterozygosity means lots of genetic variability, we
IND 00-1063, IND 00-1064, IND 00-1067, IND 00- found moderate to low variability in the accessions
1068, IND 00-1069, IND 00-1070 and IND 00-1071 studied. The present study of 49 S. spontaneum from
from Pokri, Mahadevpur and Derakgade Namsi were Lohit and Changlang regions of Arunachal Pradesh,
classified into sub-cluster F. One accession each from North Eastern India based on morphological traits
Khamba (IND 00-1012), Hawa-Camp (IND 00-1045) (plant height, stalk diameter, single cane weight,
Namsai (IND 00-1046) grouped in different cluster internode number and internode length) and genetic
than clones from same region suggesting their distinct analysis has revealed that there is obvious high
genetic identity. The clones collected from high variation. These highly diverse S. spontaneum
altitude ranging from 1000-1640 m under severely accessions could be a potential source for
cold and windy conditions. Evaluation of these clones exploitation in sugarcane improvement programme
under low-temperature conditions revealed tolerant to for high biomass and cold tolerance. The present
cold stress conditions48 and thus these could be an study revealed variations existing among the
important source of genes for improving cold accssions collected from Lohit and Changlang
tolerance49-50. The cluster II further subdivided into regions of Arunachal Pradesh, which would
two subgroups, that is two accessions were in sub- definitely helpful in planning and utilizing them in
cluster A and B each. The accessions from Namsai sugarcane improvement programme.
(IND 00-1047) and Piyong (IND 00-1048) grouped
into sub-cluster A, and accessions IND 00-1051 Acknowledgments
(Innoa) and IND 00-1054 (Changlang) were classified This work was supported by Indian Council of
into sub-cluster B. These areas closely located, and Agricultural Research (ICAR), Government of India.
accessions belong to cluster II were from fertile The authors wish to thank Dr. G. Hemaprabha, Head,
166 INDIAN J BIOTECHNOL, JULY 2020

Fig. 3 — (a) Estimates of the rate of the slope of the log probability curve (ΔK) plotted against K. (b) Population structure of accessions
based on Bayesian assignment probabilities.

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