Evaluation of F5 Individuals Obtained from B28×Kunduru-1149 Reciprocal Cross Population by Functional Markers

In the study, B28 and Kunduru-1149 durum wheat genotypes were crossed as reciprocal in 2012-2013 cropping season. 13 (B28/Kunduru1149 and Kunduru-1149/B28) reciprocal crosses were obtained and were used as materials at F5 stage. The cross combinations and the parents were screened with 10 DNA markers to determine alleles of gluten strength (Bx7OE), Yellow rust (Sun104, Xgwm18, Xwgp115 and Xgwm47), stem rust (Sun209 and Sun479), high protein ratio (UHW89), powdery mildew (Xgwm66) and leaf rust (Xgwm130). In the study, the average polymorphism information content (PIC) was calculated as 0.98 and the lowest PIC value was obtained from Xwgp115 marker with 0.96, while the rest of the markers had 0.99 PIC values. Stem rust resistance allele Sr49 was detected in B28/Kunduru-1149_F5_4 (Sun479) and B28/Kunduru-1149_F5_1 (Sun209) genotypes. One of the yellow rust resistance alleles Yr15 (Xgwm18) was detected in B28/Kunduru-1149_F5_2 and B28/Kunduru-1149_F5_3 genotypes, while Yr51 (Sun104) was identified in B28/Kunduru-1149_F5_3, B28/Kunduru-1149_F5_6, B28/Kunduru-1149_F5_7, Kunduru-1149/B28_F5_2 and Kunduru1149/B28_F5_6 genotypes. A dendrogram was created to determine kinship of the individuals with the parents. The highest genetic similarity was observed between B28 / Kunduru-1149_F5_6 and Kunduru-1149 / B28_F5_2 genotypes with 0.714, while the most diverse ones were Kunduru-1149 and B28/Kunduru_F5_7 with 0.10. Research Article


INTRODUCTION
Wheat (Triticum spp.) is an annual cereal crop consumed as major food source for centuries (Sevinç, 2010). Turkey is one of the centers of origin of the durum wheat which is originated in Karacadağ location of South-East Anatolia Region (Anonymous, 2016).
Wheat is grown in 219.3 mil. ha and 757.7 mil. ton produced world-wide, while 7.2 mil. ha grown and 20 mil. ton produced in Turkey (TUIK, 2018). World durum wheat production is approximately 40.2 mil. ton, while 3.6 mil. ton in Turkey (TUIK, 2018). Turkey is one of the durum wheat producers in the world especially in South Anatolia Region.
Plant breeders have put effort on developing high quality and high yielding cultivars for many years and different breeding techniques are used to improve crop-plants. Crossing is one of the breeding techniques used to combine traits from both parents.
Landraces have been sources to expand genetic diversity for disease resistance, drought tolerance, quality traits and many more traits. Besides favorable characteristics, they have some negative traits such as lodging, low grain yield etc. which must be eliminated by improving the traits.
Molecular marker technology is used widely in breeding programs with recent developments in biotechnology. Markers assisted selection (MAS) has been used to detect alleles related to the traits such as disease, quality parameters and agronomic traits. It provides earlier and precise selection especially for quantitative traits. Functional markers have been developed after many efforts on quantitative trait locus (QTL) studies and available for many genes and traits. Marker assisted selection have now been used widely due to its accurate, rapid, reproducible and cost effective solutions for the breeding programs.
Kunduru-1149 is a cultivar developed from selection of landraces. It has many advantages besides disadvantages such as lodging and lower quality parameters. On the other hand B28 is a landrace obtained from USDA National Small Grains Collection, Aberdeen, USA gene bank and has higher quality traits and disease resistance. Those genotypes crossed as reciprocal and 13 genotypes at F5 stages selected from both combinations (B28 × Kunduru-1149 and Kunduru-1149 × B28). In the study it was aimed to identify some disease and quality traits by allele specific markers to determine genotypes related with those gluten strength, high protein ratio, yellow rust, stem rust, powdery mildew and leaf rust diseases genes. For this purpose, the genotypes were screened with 10 allele specific DNA markers.

MATERIALS and METHOD
In the study, 13 genotypes obtained from reciprocal crosses of B28 and Kunduru-1149 and the parents were used as plant material. Seeds of the genotypes were planted and two leaves seedling were harvested to extract DNAs (Dumlupinar, 2016). The DNA content and purity were determined by spectrophotometer (Thermo-Scientific Nanodrop 2000 spectrophotometer).
Allele specific markers of gluten strength (Bx7 OE ), Yellow rust (Sun104, Xgwm18, Xwgp115 and Xgwm47), stem rust (Sun209 and Sun479), high protein ratio (UHW89), powdery mildew (Xgwm66) and leaf rust (Xgwm130) were screened on Qiagen Qiaxcel Fragment Analyzer (Table 1). The data obtained from fragment analyzer were scored and the genetic similarity of the genotypes were determined by Dice index (Dice, 1945) using NTSYSpc 2.21q software (Rohlf, 2005). Polymorphism information content (PIC) was determined by using the formula described by Weir (1996), where PIC=1-∑Pi 2 , where Pi is the frequency of the i th allele in the 15 durum wheat genotypes studied.

RESULTS and DISCUSSION
Marker assisted selection (MAS) studies in wheat have been accomplished in many plant breeding programs worldwide and succeeded on many agronomical traits. Based on genotypic data obtained from durum wheat segregation populations was investigated. The allele numbers of the primers, figures of the primers Xgwm18 and Xgwm66 were shown in Figures 1, 2 and 3 respectively. The dendrogram was generated from the marker data of durum wheat genotypes is shown in Fig. 4. The allele specific markers interrogated on the durum wheat genotypes were indicated in Table 2. KSU J. Agric Nat 23 (4)   In the study the functional markers for gluten strength (Bx7OE), Yellow rust (Sun104, Xgwm18, Xwgp115 and Xgwm47), stem rust (Sun209 and Sun479), high protein ratio (UHW89), powdery mildew (Xgwm66) and leaf rust (Xgwm130) were used. According to the results a total number of 50 alleles were produced by 10 DNA primers and, average allele number per primer was 5. The average polymorphism information content of the study was determined as 98%, and the lowest PIC value was obtained from Xwgp115 marker with 96%, while the rest of the primers had a 99% PIC values (Table 2). In a previous study Maccaferri et al. (2003) conducted a research on genetic diversity of a durum wheat set derived from Mediterranean basin using microsatellites and reported a mean diversity index (DI) of 56%. Moragues et al. (2007) (Randhawa et al., 2014). Gungor (2019) also detected 225 bp alleles using the Sun104 marker for yellow rust on a durum wheat panel. Yan et al. (2003) reported a marker-trait relation for Sun104 marker and yellow rust resistance gene Yr51. In addition, B28/Kunduru-1149_F5_2 and B28/Kunduru-1149_F5_3 genotypes had alleles for Xgwm18 markers related with yellow rust resistance gene Yr15 and Yr26. However, the other markers related with yellow rust resistance such as Xwgp115 and Xgwm47 had no alleles among the genotypes, though Cowger et al., 2012) reported a marker-trait relation for Xgwm47 for Yr64 and Yr66 yellow rust resistance and Gungor (2019) indicated an allele for Xwgp115 marker in durum wheat cultivars (Table 2). Of the Sun209 and Sun479 markers linked to stem rust resistance gene Sr49, Sun209 had allele on B28/Kunduru-1149_F5_1 genotype at 148bp, while Sun479 amplified allele on B28/Kunduru-1149_F5_4 genotype at 200 bp. Bansal et al. (2015) and Gungor (2019) reported relation for the Sun209 and Sun479 markers with Sr49 stem rust gene at 148 bp and 200 bp respectively, which is consistent with our findings.
On the other hand, some of the markers such as Xgwm130 marker linked to the leaf rust resistance gene, Bx7 OE marker for gluten strength, UHW89 marker which is carrying high protein content gene Gpc-B1 and Xgwm66 marker which is involved with powdery mildew resistance gene used in the study had no alleles on the genotypes. However, Butow et al. A dendrogram was created using the whole alleles produced by DNA markers. According to the dendrogram two main groups obtained. The first one was consisted of B28/Kunduru-1149_F5_1, B28/Kunduru-1149_F5_2, B28/Kunduru-1149_F5_3 and Kunduru-1149/B28_F5_3 genotypes, while the parents were took place on the other group. The second group divided into two groups including parents in each group. The most similar genotypes were found as Kunduru-1149/B28_F5_2 and B28/Kunduru-1149_F5_6 genotypes with the 71% genetic similarity