Parental materials
MCU 5, CO 14, CO 17, TCH 1828, KC 2, KC 3, GISV 323, GTHV 15-34 and RHC 1409 were used for the present study. They were obtained from the Tamilnadu Agricultural University, Department of Cotton, Coimbatore. They were raised during in August 2021 with two rows each with the spacing of 90 cm X 45 cm. Agronomical and cultural practices were followed as recommended and as per the schedule.
Molecular analysis
By generally following the process (Zhang and Stewart, 2000), genomic DNA was extracted from the cotton leaves of the parents, and the quantity and quality of DNA was verified for Polymerase Chain Reaction (PCR) using a NanodropTM 1000 spectrophotometer. After verification the amplified fragments from PCR (using the primers for the fibre quality and trichome hairiness traits) were separated through agarose gel electrophoresis and scoring of banding patterns and parental polymorphism identification.
PCR amplification
About 27 primers with high PIC values were obtained from the cotton marker database (CMD) (http://www.cottonmarker.org/) developed by Mainlab at Washington State University. These primers were commercially synthesized and procured from Eurofins Genomics Pvt. Ltd. 2X SMART PRIME PCR Master mixes were used for amplification. The following primers were used for the study (Table 1).
Table 1. Particulars of primer pairs used for molecular study
S.
No.
|
Marker Name
|
Primer sequence (5’-3’)
|
Annealing Temperature
(˚C)
|
1
|
BNL1153
|
F
|
CTTTATCCGGAGACGGAACA
|
55˚C
|
R
|
CTAACTTTTGCTCACCCCCA
|
2
|
CIR316
|
F
|
TTACAGGCACTACCACC
|
60˚C
|
R
|
CCTTTCTGGCGACTT
|
3
|
BNL1122
|
F
|
TCGATAACGGCTATAGTAATCTCTC
|
55˚C
|
R
|
CAACAAATAAGCAGCCAAGAAA
|
4
|
BNL3599
|
F
|
TTTAGCCCCAGTAACATGCC
|
55˚C
|
R
|
ACTGCAAGCTCTGCCCTAAA
|
5
|
BNL3147
|
F
|
ATGGCTCTCTCTGAGCGTGT
|
55˚C
|
R
|
CGGTTCAGAGGCTTTGTTGT
|
6
|
BNL1513
|
F
|
TTTCACAAGCACAACCATAGG
|
55˚C
|
R
|
AATACAGGTTCAAAGTTGATAGGG
|
7
|
BNL1440
|
F
|
CCGAAATATACTTGTCATCTAAACG
|
55˚C
|
R
|
CCCCCGGACTAATTTTTCAA
|
8
|
BNL1964
|
F
|
AGGGAGGGGGAGGTTCTC
|
55˚C
|
R
|
CGGTAGTCCTCCACCATGTT
|
9
|
BNL1417
|
F
|
TTATTCTAACCACCGCCTCC
|
55˚C
|
R
|
TGAGTGGATATGCTTGGCCT
|
10
|
CIR014
|
F
|
AGCTTGCCTCTTTCTG
|
60˚C
|
R
|
ACATTAGAACTCCCTGCT
|
11
|
BNL1878
|
F
|
TGCTTCAACTGCTCTTGCAT
|
55˚C
|
R
|
TCGATATCTGGAACACCCAC
|
12
|
BNL2568
|
F
|
GGGAAGAGAGGGAGACTAACG
|
55˚C
|
R
|
ATTTTGATAGGTTGGTTTTGTCC
|
13
|
BNL1059
|
F
|
CCTTCTCTGACACTCTGCCC
|
55˚C
|
R
|
TGTATTCTCTTCTTTTCCTTATACTTTT
|
14
|
BNL1693
|
F
|
CCCTTGGGAATAGC
|
55˚C
|
R
|
CATGTGTCTCCGTGTGTGTGTG
|
15
|
BNL2884
|
F
|
TCAACTCATACCAAATCAATTCC
|
55˚C
|
R
|
CCCTGTTTTGTTCAATGGGT
|
16
|
NAU1215
|
F
|
GAGTGAGACTGGAGCTGGTT
|
55˚C
|
R
|
CAAATCATTGTTTGCAGCAG
|
17
|
CIR199
|
F
|
CAGAATTTGACCGTTTC
|
50˚C
|
R
|
GCCATGATATTTCGGT
|
18
|
BNL3580
|
F
|
CTTGTTTACATTCCCTTCTTTATACC
|
55˚C
|
R
|
CAAAGGCGAACTCTTCCAAA
|
19
|
MUSB1035
|
F
|
TCATGATAGCAACGAGTGGTGC
|
58˚C
|
R
|
GTCGAGTTGTATTAGTGTGCCCG
|
20
|
CIR0407
|
F
|
GCACAGAACATCCATACA
|
50˚C
|
R
|
TCTCTCTCTCTTTCACACAC
|
21
|
BNL 1059
|
F
|
CCTTCTCTGACACTCTGCCC
|
55˚C
|
R
|
TGTATTCTCTTCTTTTCCTTATACTTTT
|
22
|
BNL 3255
|
F
|
GACAGTCAAACAGAACAGATATGC
|
60˚C
|
R
|
TGTATTCTCTTCTTTTCCTTATACTTTT
|
23
|
CIR 246
|
F
|
TTAGGGTTTAGTTGAATGG
|
55˚C
|
R
|
ATGAACACACGCACG
|
24
|
CIR 307
|
F
|
GACTTGAAAAGATTACACAC
|
55˚C
|
R
|
GAATTTGCTGGCTCT
|
25
|
JESPR 153
|
F
|
GATTACCTTCATAGGCCACTG
|
55˚C
|
R
|
GAAAACATGAGCATCCTGTG
|
26
|
JESPR 127
|
F
|
GATTTGGGTAACATTGGCTC
|
55˚C
|
R
|
CTGCAGTGTTGTGTTGGGTAGA
|
27
|
JESPR 154
|
F
|
GTTCCCTCAGTTGCTCAGAAG
|
55˚C
|
R
|
GGAGGAGTTGGCAGAAAATAGC
|
Separation of amplified fragments using Agarose gel electrophoresis
A 3% (w/v) agarose gel was casted in a chamber filled with 0.5 X TBE buffer, and the amplified products, together with a ladder of 100 base pairs, were loaded into the wells to determine the size of the amplified fragment of the product. For 2.30 hours, the PCR products were operated at 90 volts. After electrophoresis, the gel was taken from the tank, inspected under UV light, and photographed using a gel documentation equipment (Bio-rad Gel Doc XR imaging system).
Analysis and scoring of banding patterns
A total of 27 SSR primer pairs were used to study polymorphism among 9 parents. All 27 primers produced were clear, scorable and unambiguous bands and they were chosen for parental polymorphism analysis. DNA bands were scored which was present in the agarose gel for their presence and absence across the genotypes in the form of binary codes (presence – 1; absence – 0).
Number of Jassids per plant and Injury Grade
Hopper burn injury was assessed as per the Indian Central Cotton Committee (ICCC, 1960) methods and based on resultant symptoms of infestation. A visual rating of hopper injury on each entry was recorded on 15th, 30th, 45th, 60th and 75th days after sowing and means injury index (grade index) was calculated. The hopper burn grades consist of following grades.
Grades
|
Symptoms
|
1
|
Leaves free from crinkling or with no yellowing, bronzing and drying
|
2
|
Few leaves on lower portions of the plant curling, crinkling and slight yellowing
|
3
|
Crinkling and curling all over, yellowing, bronzing and browning in the middle and lower portion, plant growth hampered; and
|
4
|
Extreme curling, yellowing, bronzing and browning, drying of leaves and defoliation, stunted growth
|
A leafhopper resistance index was calculated as proposed by Nageswara Rao (1973), which is
G1 x P1 + G2 X P2 + G3 X P3 + G4 X P4
--------------------------------------
P1 + P2 + P3 + P4
Where G represented the number of the grade of ICCC and P the number of plants under the each entry. Grouping of injury index to categories of resistance was as follows.
Grade index
|
Category
|
- – 1.0
- – 2.0
2.1 – 3.0
3.1 – 4.0
|
Resistant
Moderately resistant
Susceptible
Highly susceptible
|
Trichome length and density analysis
The pubescence traits, involved in jassid resistance, were observed on the plants based on qualitative grading (Pubescence rating) and quantitative measurement (Trichome density and trichome length) as detailed by Bourland et al. (2003) and Hornbeck and Bourland (2007).
The number of trichomes/unit area were recorded in each genotype for Abaxial Leaf Pubescent Count (ALPC), Leaf Midrib pubescent Count (LMC), Leaf Vein pubescent Count (LVC). The trichome density was recorded on leaves based on the two criteria devised by Bourland et al. (2003). Qualitative grading system for trichomes: Upper, middle and lower portion of the selected plant leaves were assessed for the trichome density rating in random.
Trichome density scale
|
Category
|
1
3
5
7
9
|
Smooth
Lightly hairy
Hairy
Very hairy
Pilose
|
Quantitative measure of leaf trichomes: The same leaves taken for the study of qualitative grading were used for the quantitative measure of trichomes. In index card with an area of 0.1 cm2 was used for the observation. Data was recorded over the abaxial side, leaf veins and leaf midrib of each leaf from three different positions and averaged. Trichomes in the 0.1 cm2 area were counted with the aid of high magnifying power microscope (Stereo Zoom microscope). The mean for trichomes cm-2 was determined separately for each trichome count taken on three different positions of the plant.
Trichome length and density analysis
Aptina MT9M001 image sensor used with compound microscopes, producing clear images with very good contrast and color. With the ScopePhoto software the images were displayed on the slides for the leaf samples that were freshly collected at 50 DAS and cut to the size of one centimeter square and then directly viewed on the computer screen. Then still photographs were captured and then the pictures were saved for the trichome studies.