INTRODUCTION

Sorghum bicolor, commonly called sorghum and also known as durra or jowari, is a grass species cultivated for its edible grain. S. bicolor is typically an annual, but some cultivars are perennial. It grows in clumps that may reach over 4 meters high. The grain is small, rang-

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*Corresponding Author: sgangurde40@gmail.com Received 18th March, 2016

Accepted after revision 14th June, 2016 BBRC Print ISSN: 0974-6455 Online ISSN: 2321-4007

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ing from 3 to 4 mm in diameter. Sorghum was domes- ticated in North East Africa near the equator (De wet, 1978). It belongs to family Poaceae, subfamily Panicoi- dae, tribe Andropogonae and the subtribe Sorghastrae (Price et al., 2005). It is predominantly a self pollinated crop. Sorghum [Sorghum bicolor (L.) Moench] is the fifth most important cereal crop globally after wheat, maize,

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rice and barley (FAO, 2006). Sorghum grain is one of the main staples for the world’s poorest and most food- insecure people. Sorghum is now grown in more than 80 countries, mostly in tropical and sub-tropical regions.

Shoot fly (Atherigona soccata) is a major grain yield limiting factor that causes damage when sowings are delayed in rainy season (Ashok Kumar, 2008).The early sown crop escapes from shoot fly damage but the late sown crop in most cases is affected. Shoot fly infesta- tion is high when sorghum sowings are staggered due to erratic rainfall distribution which is common in the semi arid tropics ( Kumar et al., 2008). In India, the losses due to shoot fly damage have been estimated to reach as high as 90% of grain, and 45% of fodder yield (Sukhani and Jotwani,1980; Jotwani, 1982). Shootflies of the genus Atherigona are known to cause ‘deadhearts’ in a number of tropical grass species (Deeming 1971).

To understand the nature of gene action for inherit- ance of shoot fly resistance, 10 parents was evaluated, 45 F1’s and their reciprocals in replicated trials during the rainy and postrainy seasons. The genotypes ICSV 700, Phule Anuradha, ICSV 25019, PS 35805, IS 2123, IS 2146, and IS 18551 exhibited resistance to shoot fly damage across seasons. Crosses between susceptible parents were preferred for egg laying by the shoot fly females, resulting in a susceptible reaction. ICSV 700, ICSV 25019, PS 35805, IS 2123, IS 2146, and IS 18551 exhibited significant and negative general combining ability ( gca) effects for oviposition, deadheart inci- dence, and overall resistance score. (Mohammad et al. 2016).

Before the development of molecular markers, mor- phological markers were found to be useful in varietal identification and assessing genetic diversity, but they had certain limitations. Later markers based on protein variants were used with limited success. Isoelectric vari- ants of proteins, referred to as isozymes or allozymes, were found to be useful and there were many studies made to assess genetic diversity of different crops using such protein markers (Morden et al., 1989). Marker- assisted selection (MAS) is a new paradigm in plant breeding. It involves the selection of genotypes carry- ing a desirable gene, or gene combination, via linked marker(s). Breeders practice marker-assisted selection when an agronomically important trait that is difficult to assess. Through MAS, the transfer of traits from exotic donor parents to more elite locally adapted crop culti- vars is possible. Backcrossing has been a widely used technique in plant breeding for almost a century.

The shootfly dead heart percentage has been esti- mated to be 43.5% during the Rabi 2012-13 in Maha- rashtra (Anonymous 2013). Given the economic impact of shoot fly, the improvement of genetic resistance to this pest is one of the major goals in sorghum breeding

programs in India. In this context, a better understand- ing of the inheritance of resistance and identification of genomic regions/QTL that influence resistance can help the breeders to develop more efficient and effective breeding and selection schemes through marker-assisted selection (MAS).

MATERIAL AND METHODS

The experimental sorghum seed material comprised of backcross populations developed from the cross AKSV- 13R X IS-18551. Experimental seed material was devel- oped by crossing BC2 with recurrent parent (recipient parent) to obtain BC3 seed in kharif 2013 and BC4 seed was obtained by crossing BC3 with recurrent parent (recipient parent) in rabi 2013-14. The experiment was carried out at Sorghum Research Unit, Dr. PDKV, Akola. Plant protection measures are avoided for recording morphological observations. Developed population was grown at a spacing of 45 cm X 15 cm with simple lat- tice design.

Following observations were recorded on each of the parent and the individual plants in each population. Pink pigment on plumule and leaf sheath was assessed at 5 DAE at 1 to 5 rating scale as per Sharma et al. (1997) as (1-Plumule and leaf sheath with dark pigment, 2- Plu- mule and leaf sheath with fair pink pigment, 3- Plumule and leaf sheath with light pink pigment, 4-Plumule and leaf sheath with very light pink pigment, 5-Plumule and leaf sheath with green colour).

Leaf glossiness was evaluated on a 1 to 5 rating as given below at 10 DAE in the early morning hours. When there was maximum reflection of light from the leaf sur- faces as 1- Highly glossy (Light green, shinning, narrow and erect leaves), 2 - Glossy (Light green, less shinning and erect leaves), 3 - Moderate glossy (Fair green, light shinning, medium leaf width and less dropping leaves), 4 - Moderate non glossy (Green, broad, dropping leaves), 5 - Non glossy (Dark green, dull, head and dropping leaves).

The observations of dead heart count were taken on

14th and 21st DAE for each entry. Dead Heart percentage was calculated as the number of dead hearts with respect to total number of plants in the entry multiplied by 100. The observations were taken on 14th and 21st DAE for each entry. Seedling with eggs laid by shoot fly larvae was expressed in terms of percentage of the plants. The seedlings with eggs percentage was calculated by num- ber of seedlings with eggs with respect to total number of plants in the entry multiplied by 100.

Observations Trichome density (mm2) were taken on

14th DAE, on fifth leaf from apical point as per the proce- dure outlined by Sharma et. al. (1997). leaf samples were

kept in 20 ml of acetic acid: alcohol (2:1) solution for overnight then transferred in 90 percent lactic acid in small vials and stored for further observations. The leaf samples were mounted on a slide in a drop of water and observed under a binocular microscope at magnification of 10x. The number of trichomes per mm2 was counted on abaxial (lower side) leaf surface.

For Genotypic observations, genomic DNA was iso- lated from each of the genotype using a modified cetyl tri methyl ammonium bromide (CTAB) method (Sharma et al., 2002). For DNA extraction two gram leaf samples

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of three to four weeks old seedlings of each BC3 and BC4 population were used. DNA quantified on 0.8% agarose gel followed by Ribonuclease A treatment.

PCR AMPLIFICATION

SSR primer pairs were used for the present investigation. The list of forward and reverse primers used for ampli- fication of the genomic DNA with their sequences, is given in table 1. The synthesis of following primers was done from Genaxy Scientific Pvt. Ltd., India.

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The PCR was carried out in small reaction tubes, con- taining a reaction volume typically of 16 μl that was inserted into a thermal cycler (Applied Biosystems Gene Amp PCR System 2700) that heated and cooled the reac- tion tubes within it to the precise temperature required for each step of the reaction. The working concentration of master mix for 16 μl (final volume) was prepared as 0.9mM 10 X Taq polymerase assay buffer with MgCl2, 2.4mM dNTPs and 0.5 U Taq polymerase. 2 μl of DNA sample was used with 1.5 μl of each primer. The ampli- fied products were resolved on 10% PAGE on a Genei’s Vertical Gel Electrophoresis System (glass plate size 24 cm x 19 cm).To resolve the SSR products the silver stain- ing was carried out following Tagelstrom (1992).

DATA ANALYSIS

The banding patterns obtained from PCR amplifica- tion of the various SSR primer in each of the BC3 and BC4 population was visualized with silver staining were scored as ‘A’ [Homozygote for the allele for parental strain P1 (IS18551)], ‘B’ [Homozygote for the allele for recurrent parental strain P2 (AKSV 13 R)], ‘H’ (Heterozy- gote carrying the alleles from both P1 and P2 parental strains), ‘-’ (Missing data for the individual).

Phenotypic correlation

Phenotypic correlation coefficients were estimated using the formula of Singh and Choudhary (1996).

The observed values of correlation coefficients were compared with the tabulated values for (n-2) degrees of freedom to test their significance.

RESULTS AND DISCUSSION

The BC3 population was derived from crossing BC2 with recipient parent and BC4 population derived from BC3 population crossed with recipient parent. To identify the reaction linked to the shoot fly tolerance components traits SSR markers were used and morphological obser- vations were recorded. The experiment was conducted in late kharif and late rabi 2013 at Sorghum Research Unit, Dr. Panjabrao Deshmukh Krish Vidyapeeth Akola. The results obtained from morphological and molecular

analysis using specific SSR primers for shoot fly resist- ance are discussed in detail in this chapter.

Phenotypic performance of parents AKSV- 13R and IS 18551 revealed that both the parents differ for the shootfly resistance contributing traits like leaf glossiness (1-5 scale), pigmentation (1-5 scale), percent seedling with eggs at 14th and 21st days after emergence, dead heart percentage at 14th and 21st days after emergence and trichome density measured on abaxial surface per mm2 of leaf. The mean performance of parents for these traits are summarized below:

Table 2: Mean phenotypic performance for shoot fly resistance component traits in the parents.

The resistant donor parent IS18551 recorded rat- ing of one on scale five leaf glossiness as compared to rating four leaf glossiness in parent AKSV-13R on 1-5 scale. Similarly differences for mean performances for pigmentation was recorded in two parents as IS18551 (1.5) and AKSV-13R (4) on 1-5 scale. The mean percent seedlings with eggs recorded after 14th and 21st days after emergence were 10 and 12 percent respectively in IS 18551. Which were numerically lower than AKSV- 13R as 45 and 50 percent after 14th and 21st days after emer- gence respectively. Similarly dead heart percentage at 14th and 21st days after emergence varied in two parents, AKSV- 13R (47% and 52% on 14th and 21st DAE) and IS18551 (9% and 11% on 14th and 21st DAE). Trichome density was higher in IS 18551 (28 per mm2) as com- pared to AKSV -13R (3 per mm2).

Total 136 plants of BC3 and 30 plants of BC4 popu- lation were studied. BC3 population was studied for phenotypic traits related to shoot fly resistance such as glossiness, pigmentation, trichome density, egg laying percent, dead heart percent and yield per plant. Mean phenotypic performance for shoot fly resistance com- ponent traits in the BC3 of cross AKSV-13R X IS18551.

In BC3 population of AKSV -13 R x IS18551 cross, pigmentation ranged from 1.5 to 4.5. Highest pigmenta- tion was recorded in plant no. BC3-7 which was 1.5 and lowest pigmentation observed in plant no BC3-111 which was 4.5. Whereas pigmentation in remaining plants

ranged from 2.5 to 4. In BC3 population of AKSV-13 R x IS18551 cross, glossiness was ranged from 1 to 4. Out of the 136 plants, plant no. BC3- 111, BC3-127, BC3-128, BC3-129, BC3-131, BC3-132, BC3-133, BC3-136 showed lowest glossiness rating 4 and plant no. BC3-7 and BC3- 113 showed highest glossiness rating 1. Whereas glossi- ness in remaining plants ranged from 1 to 4.

In BC3 population of AKSV -13 R x IS18551 cross, trichome density ranged from 1 to 56.33 trichomes/mm2. Highest trichome density of 56.33 trichomes/mm2 was found in plant no. BC3-7 and lowest trichome density of 1 trichome/mm2 was found in plant no. BC3-111. Seed- lings with eggs on abaxial surface were recorded on 14th and 21st day after emergence (DAE). The presence of eggs on 14th and 21st DAE was found in plants BC3- 38, BC3- 84, BC3-111, BC3-127, BC3-132, BC3- 136. Whereas the presence of eggs on 21st DAE was found in BC3-4, BC3-5, BC3-18, BC3-19, BC3-31, BC3-32, BC3-128.

Total dead heart damage observed at 14th DAE and

21st DAE. The presence of dead heart damage on 14th and 21st DAE was found in plants BC3- 38, BC3-84, BC3- 111, BC3-127, BC3-132, BC3- 136. Whereas the presence of dead heart damage on 21st DAE was found in BC3-4, BC3-5, BC3-18, BC3-19, BC3-31, BC3-32, BC3-128. Yield per plant in BC3 population ranged from 22 gm to 86 gm. Plant no. BC3-7 and BC3-113 showed highest yield 86 gm whereas plant no. BC3-136 showed lowest yield 22 gm. Total 69 plants showed more yield than mean yield that is 53.47.

Mean phenotypic performances for shoot fly resist- ance component traits in the BC4 of cross AKSV-13R X IS18551. In BC4 population of AKSV -13 R x IS18551

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cross, pigmentation ranged from 1.5 to 4. Highest pig- mentation was recorded in plant no. BC4-14 and BC4-20 which was 1.5 however lowest pigmentation observed in plant no. BC4-18 which was 3.5. In BC4 population of AKSV -13 R x IS18551 cross, glossiness was ranged from 1 to 3.5. Out of the thirty plants, plant no. BC4- 18 showed lowest glossiness of 3.5 and plant no. BC4- 14 and BC4-20 showed highest glossiness of 1 where as glossiness in remaining plants ranged from 1 or 3.

In BC4 population of AKSV -13 R x IS18551 cross, trichome density ranged from 4 to 59.33 trichome/mm2. Highest trichome density was found in plant no. BC4- 14 as 59.33 trichomes/mm2 and lowest trichome den- sity was found in plant no. BC4-18 as 4 trichomes/mm2. Seedlings with eggs on abaxial surface were recorded on 14th and 21st day after emergence (DAE). The presence of eggs on 14th and 21st DAE was found in plant BC4-

18.Whereas presence of eggs on 21st DAE was found

in plants BC4-22 and BC4-27. Total dead heart damage observed at 14th DAE and 21st DAE. The presence of dead heart damage on 14th and 21st DAE was found in plant

BC4-18. Whereas presence of dead heart damage on 21st DAE was found in plants BC4-22 and BC4-27.

Yield per plant in BC4 population ranged from 12 gm to 83 gm. Plant no. BC4-14 showed highest yield 83 gm and plant no BC4-18 showed lowest yield 12 gm. Total

13plants BC4-1, BC4-2, BC4-4, BC4-11, BC4-12, BC4-14, BC4-15, BC4-16, BC4-17, BC4- 20, BC4-21, BC4-24, BC4-28 showed more yield than mean yield that is 48.83.

Simple linear correlation coefficient between shoot fly resistance components were estimated based on each plant of BC3 and BC4. This helps to understand the

varying degree of association and contribution of each character in building up total genetic architecture of resistance considering the varying degree of phenotypic values.

Marker-assisted selection appeared as a promising tool for breeding quantitative resistance. Two problems are inherent in backcross breeding, one being the large number of generations required to recover the genome of the inbred parent and the other being linkage drag, i.e. the presence of the inbred portions of the donor’s genome (which can be linked to non-favourable alleles) surrounding the introgressed allele. As previously described by Tanksley et al. (1996), the main advantage of using DNA markers as opposed to conventional selec- tion is to accelerate the fixation of recipient alleles in non target regions and to identify the genotypes con- taining crossovers close to target genes.

In present investigation a set of markers are used to screen the parents of the backcross populations AKSV- 13R and IS18551 (resistance) to identify polymorphic one for further genotyping the BC3 and BC4 popula- tion. Screening was done on 10% PAGE gel where sil- ver staining recipe was adopted. Marker showed poly- morphism between parent were selected for screening of the BC3 and BC4 population. Total 17 markers were used for parental screening out of these 8 showed poly- morphism. Recurrent parent, AKSV 13R, susceptible for shoot fly while being agronomically superior were back-

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crossed with donor parent IS 18551. All the backcross progenies obtained for AKSV 13R were screened with foreground markers to identify the progenies with target QTLs.

The target QTL from donor parents is insured in recur- rent parent background by selecting for heterozygous for the flanking markers. A total 136 plants of the BC3 population screened with six polymorphic foreground markers (Xtxp-37, Xtxp-75, Xtxp-141, Xtxp-94, Xtxp- 15, Xisp-10258). The band in the gels was scored as A, B, H and “_” based on the pattern compared with those of the parents. Only the plants with marker status ‘H’ for the all target marker were scored and selected. Out of the 136 plants, only plant no. BC3-7, BC3-16, BC3-113, were found to be heterozygous (H) for six primers for target QTL region and plant no. BC3-49 showed heterozygous banding pattern in five primers highlighted in table 5.

Foreground selection for QTL introgression in AKSV

13R X IS 18551 BC4 populations:

A total 30 plants of the BC4 populations screened with nine polymorphic foreground markers (Xtxp-37, Xtxp- 75, Xtxp-141, Xtxp-94, Xtxp-15, Xisp-10258, Xisp 10263, Xgap 1, Xtxp 65). Only the plants with marker status ‘H’ for the all target marker were scored and selected. Out of the 30 plants, plant no. BC4-14 showed heterozygous banding pattern in seven primers while plant no. BC4-24 showed heterozygous banding pattern in six primers highlighted in table 6.

QTL specific allele recovery from donor parent in BC3 and BC4 population of various crosses

All the BC3 and BC4 progenies were tested for the pres- ence of trichome density and glossiness specific donor allele from donor parent using polymorphic foreground markers. The data is scored for presence and absence for specific allele.

QTL specific allele recovery from donor parent IS18551 in BC3 population of AKSV-13R x IS 18551:

Allele specific for trichome density and glossiness were recovered using six polymorphic foreground markers Xtxp-37, Xtxp-75, Xtxp-141, Xtxp 15, Xtxp 94, Xisp 10258. Plant no. BC3-50, BC3-75 and BC3-133 showed presence of specific allele using all the primers with expected size.

QTL specific allele recovery from donor parent IS18551 in BC4 population of AKSV-13R x IS 18551:

Allele specific for trichome density and glossiness were recovered using nine polymorphic foreground markers Xtxp-37, Xtxp-75, Xtxp-141, Xtxp-94, Xtxp-15, Xisp- 10258, Xisp 10263, Xgap 1, Xtxp 65. Plant no. BC4-6, BC4-7 and BC4-17 showed maximum alleles using all the primers with expected size.

CONCLUSION

The results obtained from morphological observations revealed that the two parents differed considerably for all the observed shoot fly resistance component traits and the wide variation was observed among the BC3 and BC4 population for shoot fly resistance component traits. Phenotypic correlations between the component traits of shoot fly resistance were estimated based on individual progeny from BC3 and BC4 means. The results so obtained can be briefly summarized as yield showed highly signifi- cant and positive correlation with trichome density, leaf glossiness and pigmentation in BC3 and BC4 population of AKSV-13R X IS 18551. For molecular marker studies, genomic DNA was isolated from the parent and all BC3 and BC4 population using CTAB method. DNA quality was confirmed by running them on 0.8 per cent agarose gel stained with ethidium bromide. DNA samples were treated with RNase A (10 mg/ml) @100 μl / ml of sample. Quan- tification of DNA was done and accordingly, the samples were diluted to a final concentration of 15 ng/μl. 30 ng of DNA was used for each PCR reaction.

Foreground selection using specific markers for glossiness and trichome density QTLs of BC3 and BC4 plants were carried out. Out of 136 BC3 populations, Plant no. BC3-7, BC3-16, BC3-113 were found to be het- erozygous for six primers and plant no. BC3-49 showed

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heterozygous banding pattern in five primers. Whereas in BC4 population plant no. BC4-14 showed heterozy- gous banding pattern in seven primers and plant no. BC4-24 showed heterozygous banding pattern in six primers. QTL specific allele recovery from donor par- ent IS 18551 in BC3 and BC4 population was carried out. Out of 136 BC3 population plant no. BC3-50, BC3-75 and BC3-133 showed presence of specific alleles using six polymorphic foreground markers. Whereas plant no. BC4-6, BC4-7 and BC4-17 showed maximum alleles using all the primers with expected size in BC4 population.

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