Genetic Affinity, ß ODAP, Homoarginine and Asparagine Contents of Turkish Grass Pea Landraces

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1 Genetic Affinity, ß ODAP, Homoarginine and Asparagine Contents of Turkish Grass Pea Landraces Z. Acar 1, N. Onar 1, H. Uysal 2, M. Can 1, F. Kumbasar 1, E. Tuzen 1 1 Ondokuz Mayis University, Samsun- Turkey 2 Adnan Menderes University, Aydin-Turkey

Lathyrus natural distribution is primarily

65 species, 23 of them endemic (Gunes and Ozhatay

2 Lathyrus natural distribution is primarily centered in the Eastern Mediterranean region (Cocks et al., 2000). Turkey is an important center for Lathyrus with 65 species, 23 of them endemic (Gunes and Ozhatay 2000). In Anatolia grasspea has been cultivated for thousands years (Turk et al., 2007).

4 Advantages of grass pea; Drought and water logging tolerance, Low input requirements, Insect and pest resistance, Adaptation to a variety of climates and soils, Nutritional value of seeds, up to 35% protein content (Rosa et al., 2000).

5 L. sativus seeds has been consumed as a food legume in human and animal diet, but ıt contains some free amino acids. Such as; ß- N-oxalyl-L-α,ß-diaminopropionic acid (ß ODAP) is suggested to be the causative agent for a paralysis of the lower limbs, known as "neurolathyrism".

6 Homoarginine, which also play an important role in physiological and biochemical processes. Homoarginine has been proposed to modulate the toxicity of ß ODAP (Shamin et al., 2002). Asparagine is essential for healthy brain development in children and ıt also affect some body functions.

7 The aim of this paper was to provide information on the ß ODAP, homoarginine and asparagine contents and genetic affinity of grass pea populations cultivated in Turkey.

8 Methods From various regions of Turkey, 51 grass pea and one Lathyrus clymenum local landraces were collected and cultivated in the experimental fields of Ondokuz Mayis University. One released cultivar was also included in the research. The seeds harvested in the year of 2011 were analysed for ß ODAP, homoarginine and asparagine by a new, validated and simple capillary electrophoretic method.

9 Figure 1. The regions grasspea seeds collected from

10 Figure 2. Peak time and area of a) homoarginine, b) asparagine, c)α-odap and d, β -ODAP.

11 DNA analysis was performed in 5 plant stools by using appropriate primers and the method of ISSR to examine the genetic proximity.

12 Results and Discussion The highest values of both β-odap, homoarginine and asparagine belonged to the same genotype (LS 1501). The most abundant of the three measured amino acids was homoarginine.

13 Figure 3. Proportions of ß -ODAP, homoarginine and asparagine in seed extracts of 52 grass pea populations Concentrations; ß ODAP 1.04 to 8.68 mg/g Homoarginine 2.14 to 1.27 mg/g Asparagine 0.06 to 4.74 mg/g

14 This amino acid was followed by ß ODAP and asparagine. Polignano et al. (2005) reported ß -ODAP grass pea seed concentrations as % in 47 progenies. Fikre et al. (2008) indicated that homoarginine was up to 0.8%, ß -ODAP was between 0.02% and 0.54% while asparagine content was between 0.01% and O.06% in 9 genotypes.

15 There was a medium level positive correlation between β- ODAP and homoarginine concentrations (r 2 =0.649). Seeds containing high levels of β-odap, were found to have enhanced homoarginine content. This is very important because homoarginine can modulate the toxicity of ß ODAP (Shamin et al., 2002).

16 Figure 4. Correlation betweenβ-odap and homoarginin

17 This finding is in agreement with literature information; Piergiovanni & Damascelli (2011) also found a significant positive correlation between homoarginine and β- ODAP contents of seven Italian grass pea ecotypes cultivated in two locations of southern Italy.

18 Also a correlation between asparagine and β-odap levels was observed in some accessions. Comparison of these results with the data of this work reveals that homoarginine, ß -ODAP and asparagine concentrations of 52 Lathyrus local landraces seeds spread from low to high levels.

19 The ß -ODAP content of grass pea is known to vary widely, both among genotypes and environments (Campbell, 1997). We compared the data of ß -ODAP with the data published by Basaran et al. (2011) that belongs to the same Lathyrus local landraces (used the same analysis method). A statistical difference between ß -ODAP contents of 2011 harvest and collected samples in 2007 was found at 95% confidence level. The ß -ODAP data of this work reflect the differences due to the environment, because grass pea seeds of this study were obtained by cultivation of samples collected by Basaran et al. (2011).

20 The concentrations of the isolated DNA samples ranged between 17.3 ng/ml and ng/ml. In the used primers, the band sizes in each genotype were obtained ranging between 0.23 kilo base (kb) and 1.28 kb. The DNA samples from grass pea genotypes were analysed through the program of NTSYSpc 2.0 after screening and scoring via ISSR primers.

21 Table 1. According to AMOVA (Analysis of Molecular Variance) proximity values of Lathyrus sativus genotypes ELZ01 ELZ02 ELZ03 MLT01 MLT02 MLT03 MLT04 ADI01 ADI02 NEV01 NEV02 NEV03 NEV04 NEV05 NEV06 BRD01 BRD02 BRD03 MUG01 DEN01 DEN02 DEN03 DEN04 DEN05 DEN06 DEN07 DEN08 USK01 USK02 USK03 USK04 USK05 USK06 USK07 USK08 USK09 USK10 USK11 USK12 USK13 USK14 USK15 USK16 KUT01 BUR01 BUR02 BUR03 BUR04 CNR01 CNR02 CNR03 SAM01 GURBUZ ELZ01 1,000 ELZ02 0,722 1,000 ELZ03 0,662 0,661 1,000 MLT01 0,661 0,660 0,716 1,000 MLT02 0,613 0,630 0,605 0,684 1,000 MLT03 0,672 0,709 0,678 0,650 0,639 1,000 MLT04 0,646 0,706 0,620 0,682 0,500 0,605 1,000 ADI01 0,652 0,588 0,624 0,600 0,542 0,614 0,713 1,000 ADI02 0,675 0,689 0,614 0,650 0,569 0,696 0,783 0,726 1,000 NEV01 0,637 0,607 0,635 0,648 0,481 0,686 0,739 0,595 0,756 1,000 NEV02 0,593 0,631 0,667 0,667 0,449 0,640 0,825 0,766 0,774 0,718 1,000 NEV03 0,579 0,568 0,561 0,555 0,466 0,609 0,615 0,557 0,574 0,538 0,614 1,000 NEV04 0,541 0,595 0,438 0,420 0,522 0,623 0,373 0,464 0,448 0,395 0,378 0,429 1,000 NEV05 0,610 0,610 0,552 0,622 0,532 0,616 0,645 0,581 0,680 0,532 0,631 0,634 0,503 1,000 NEV06 0,599 0,585 0,500 0,585 0,479 0,568 0,579 0,460 0,585 0,633 0,565 0,621 0,459 0,643 1,000 BRD01 0,515 0,591 0,436 0,542 0,589 0,552 0,561 0,467 0,573 0,496 0,573 0,595 0,456 0,657 0,626 1,000 BRD02 0,573 0,576 0,544 0,597 0,512 0,564 0,621 0,633 0,703 0,606 0,649 0,563 0,471 0,644 0,603 0,714 1,000 BRD03 0,578 0,587 0,481 0,495 0,449 0,551 0,543 0,493 0,567 0,477 0,553 0,520 0,504 0,591 0,500 0,722 0,755 1,000 MUG01 0,427 0,478 0,443 0,424 0,446 0,482 0,510 0,463 0,466 0,353 0,461 0,409 0,382 0,471 0,443 0,520 0,462 0,486 1,000 DEN01 0,588 0,579 0,548 0,681 0,504 0,611 0,598 0,618 0,632 0,604 0,642 0,595 0,460 0,624 0,545 0,637 0,728 0,705 0,486 1,000 DEN02 0,648 0,671 0,527 0,677 0,542 0,627 0,727 0,621 0,674 0,593 0,671 0,671 0,455 0,673 0,640 0,685 0,684 0,693 0,556 0,720 1,000 DEN03 0,568 0,589 0,536 0,651 0,463 0,634 0,732 0,638 0,715 0,636 0,731 0,652 0,412 0,631 0,562 0,644 0,699 0,627 0,488 0,807 0,743 1,000 DEN04 0,609 0,676 0,547 0,688 0,500 0,603 0,768 0,621 0,706 0,662 0,732 0,604 0,394 0,626 0,622 0,667 0,685 0,630 0,637 0,733 0,786 0,798 1,000 DEN05 0,542 0,685 0,467 0,588 0,519 0,580 0,551 0,533 0,475 0,478 0,600 0,556 0,458 0,481 0,543 0,620 0,529 0,623 0,504 0,647 0,709 0,639 0,681 1,000 DEN06 0,541 0,594 0,563 0,656 0,528 0,545 0,667 0,545 0,612 0,535 0,719 0,693 0,314 0,560 0,509 0,626 0,598 0,587 0,392 0,612 0,631 0,703 0,641 0,557 1,000 DEN07 0,504 0,625 0,386 0,462 0,466 0,471 0,590 0,436 0,574 0,562 0,577 0,681 0,442 0,649 0,716 0,678 0,595 0,559 0,452 0,488 0,652 0,544 0,692 0,651 0,522 1,000 DEN08 0,598 0,690 0,543 0,642 0,474 0,612 0,633 0,540 0,644 0,486 0,596 0,622 0,429 0,589 0,533 0,604 0,602 0,559 0,392 0,613 0,619 0,718 0,692 0,526 0,627 0,436 1,000 USK01 0,661 0,688 0,549 0,575 0,487 0,531 0,659 0,632 0,594 0,482 0,586 0,660 0,473 0,579 0,563 0,619 0,585 0,596 0,444 0,595 0,652 0,581 0,631 0,630 0,620 0,593 0,629 1,000 USK02 0,708 0,680 0,535 0,528 0,457 0,667 0,717 0,698 0,660 0,623 0,646 0,633 0,505 0,614 0,576 0,635 0,629 0,660 0,496 0,741 0,682 0,673 0,688 0,582 0,522 0,593 0,755 0,667 1,000 USK03 0,635 0,706 0,462 0,644 0,563 0,611 0,732 0,613 0,678 0,635 0,656 0,660 0,514 0,634 0,653 0,648 0,642 0,588 0,540 0,662 0,738 0,667 0,718 0,591 0,615 0,660 0,571 0,653 0,810 1,000 USK04 0,595 0,630 0,551 0,521 0,500 0,596 0,571 0,583 0,571 0,536 0,559 0,510 0,500 0,516 0,496 0,520 0,582 0,549 0,448 0,612 0,638 0,639 0,632 0,709 0,481 0,577 0,526 0,589 0,615 0,645 1,000 USK05 0,644 0,596 0,624 0,607 0,579 0,691 0,667 0,607 0,660 0,639 0,626 0,530 0,400 0,567 0,571 0,539 0,650 0,624 0,462 0,646 0,712 0,648 0,620 0,591 0,449 0,541 0,535 0,597 0,723 0,613 0,651 1,000 USK06 0,651 0,600 0,566 0,547 0,500 0,583 0,637 0,612 0,641 0,558 0,629 0,626 0,460 0,576 0,544 0,613 0,681 0,649 0,400 0,718 0,680 0,718 0,625 0,577 0,714 0,516 0,649 0,674 0,725 0,678 0,660 0,629 1,000 USK07 0,620 0,607 0,549 0,533 0,395 0,596 0,676 0,644 0,644 0,574 0,574 0,563 0,458 0,577 0,560 0,602 0,648 0,679 0,500 0,710 0,653 0,683 0,647 0,643 0,553 0,515 0,621 0,634 0,750 0,667 0,629 0,727 0,660 1,000 USK08 0,636 0,598 0,578 0,482 0,494 0,653 0,590 0,585 0,614 0,563 0,589 0,519 0,500 0,482 0,559 0,561 0,630 0,558 0,417 0,667 0,574 0,679 0,569 0,493 0,582 0,375 0,612 0,507 0,712 0,600 0,638 0,711 0,682 0,692 1,000 USK09 0,763 0,690 0,707 0,649 0,630 0,697 0,574 0,598 0,609 0,604 0,551 0,504 0,554 0,543 0,517 0,522 0,583 0,603 0,449 0,616 0,636 0,593 0,531 0,527 0,500 0,386 0,568 0,584 0,766 0,673 0,614 0,755 0,612 0,685 0,753 1,000 USK10 0,697 0,643 0,621 0,706 0,568 0,642 0,743 0,633 0,655 0,667 0,673 0,581 0,435 0,710 0,708 0,677 0,704 0,649 0,574 0,692 0,767 0,740 0,786 0,696 0,471 0,667 0,567 0,578 0,714 0,752 0,636 0,718 0,611 0,682 0,629 0,696 1,000 USK11 0,549 0,598 0,544 0,566 0,449 0,600 0,634 0,603 0,677 0,597 0,648 0,619 0,503 0,658 0,617 0,657 0,745 0,671 0,547 0,673 0,731 0,690 0,740 0,589 0,561 0,620 0,600 0,634 0,681 0,654 0,575 0,695 0,638 0,671 0,578 0,560 0,734 1,000 USK12 0,588 0,568 0,497 0,612 0,532 0,586 0,605 0,557 0,586 0,527 0,606 0,639 0,446 0,591 0,533 0,667 0,634 0,619 0,503 0,687 0,700 0,720 0,691 0,652 0,564 0,571 0,636 0,632 0,673 0,597 0,529 0,639 0,636 0,652 0,532 0,571 0,754 0,704 1,000 USK13 0,542 0,611 0,517 0,525 0,452 0,588 0,647 0,589 0,631 0,601 0,646 0,659 0,483 0,698 0,647 0,700 0,658 0,587 0,514 0,689 0,709 0,676 0,704 0,592 0,571 0,662 0,626 0,593 0,671 0,698 0,551 0,611 0,601 0,663 0,575 0,532 0,732 0,765 0,735 1,000 USK14 0,609 0,594 0,520 0,610 0,514 0,571 0,596 0,541 0,588 0,672 0,600 0,520 0,469 0,637 0,601 0,594 0,724 0,647 0,482 0,719 0,754 0,699 0,706 0,585 0,545 0,623 0,511 0,561 0,708 0,703 0,667 0,709 0,597 0,686 0,642 0,691 0,786 0,728 0,629 0,700 1,000 USK15 0,534 0,483 0,515 0,600 0,463 0,516 0,617 0,513 0,583 0,548 0,627 0,621 0,417 0,587 0,561 0,658 0,617 0,557 0,418 0,659 0,607 0,716 0,634 0,561 0,659 0,480 0,688 0,577 0,556 0,550 0,556 0,571 0,603 0,642 0,639 0,523 0,650 0,648 0,706 0,690 0,568 1,000 USK16 0,623 0,574 0,495 0,520 0,478 0,590 0,487 0,543 0,548 0,521 0,553 0,660 0,505 0,552 0,538 0,549 0,597 0,661 0,431 0,679 0,629 0,629 0,603 0,495 0,587 0,447 0,667 0,619 0,694 0,629 0,520 0,648 0,595 0,661 0,660 0,602 0,569 0,667 0,658 0,688 0,671 0,608 1,000 KUT01 0,489 0,587 0,495 0,529 0,333 0,510 0,620 0,563 0,563 0,549 0,583 0,577 0,388 0,545 0,505 0,583 0,653 0,591 0,371 0,655 0,661 0,693 0,667 0,658 0,638 0,556 0,590 0,537 0,750 0,630 0,579 0,627 0,575 0,727 0,576 0,596 0,640 0,639 0,636 0,637 0,667 0,571 0,548 1,000 BUR01 0,552 0,523 0,492 0,561 0,437 0,516 0,532 0,471 0,554 0,526 0,588 0,567 0,370 0,632 0,582 0,676 0,667 0,655 0,443 0,696 0,679 0,685 0,622 0,505 0,593 0,481 0,571 0,491 0,651 0,632 0,513 0,604 0,614 0,691 0,628 0,594 0,752 0,655 0,711 0,677 0,721 0,699 0,619 0,655 1,000 BUR02 0,567 0,512 0,523 0,536 0,366 0,597 0,526 0,523 0,537 0,505 0,534 0,583 0,444 0,560 0,544 0,618 0,642 0,678 0,364 0,702 0,615 0,687 0,623 0,538 0,523 0,505 0,658 0,545 0,654 0,544 0,577 0,577 0,655 0,612 0,652 0,610 0,585 0,647 0,667 0,696 0,629 0,617 0,699 0,674 0,678 1,000 BUR03 0,585 0,622 0,607 0,578 0,426 0,568 0,652 0,565 0,619 0,592 0,667 0,659 0,459 0,673 0,519 0,652 0,698 0,667 0,517 0,757 0,681 0,757 0,714 0,604 0,706 0,429 0,667 0,676 0,780 0,656 0,597 0,642 0,720 0,800 0,657 0,595 0,717 0,750 0,814 0,796 0,694 0,808 0,754 0,632 0,800 0,667 1,000 BUR04 0,633 0,582 0,638 0,533 0,412 0,645 0,667 0,634 0,629 0,610 0,640 0,613 0,434 0,523 0,497 0,536 0,595 0,603 0,440 0,671 0,632 0,706 0,591 0,545 0,655 0,438 0,620 0,586 0,705 0,615 0,703 0,760 0,655 0,717 0,753 0,694 0,650 0,616 0,579 0,588 0,634 0,667 0,689 0,667 0,603 0,643 0,747 1,000 CNR01 0,581 0,590 0,576 0,576 0,600 0,675 0,407 0,500 0,500 0,594 0,430 0,394 0,521 0,348 0,417 0,385 0,462 0,430 0,327 0,514 0,517 0,510 0,455 0,500 0,444 0,366 0,419 0,493 0,604 0,556 0,644 0,623 0,591 0,523 0,632 0,789 0,484 0,475 0,521 0,475 0,587 0,429 0,537 0,478 0,400 0,557 0,390 0,618 1,000 CNR02 0,557 0,500 0,466 0,522 0,400 0,466 0,646 0,567 0,590 0,651 0,667 0,532 0,313 0,504 0,547 0,598 0,605 0,598 0,523 0,617 0,655 0,707 0,737 0,611 0,623 0,591 0,483 0,613 0,597 0,605 0,554 0,593 0,657 0,650 0,509 0,526 0,719 0,661 0,617 0,580 0,641 0,622 0,526 0,627 0,598 0,521 0,667 0,619 0,491 1,000 CNR03 0,470 0,458 0,480 0,488 0,395 0,590 0,481 0,389 0,496 0,571 0,495 0,400 0,444 0,384 0,425 0,433 0,460 0,400 0,420 0,515 0,500 0,553 0,527 0,469 0,491 0,429 0,479 0,429 0,554 0,584 0,585 0,518 0,583 0,516 0,613 0,560 0,515 0,483 0,456 0,434 0,574 0,482 0,490 0,382 0,451 0,515 0,441 0,569 0,623 0,656 1,000 SAM01 0,528 0,617 0,467 0,500 0,421 0,608 0,525 0,533 0,596 0,571 0,591 0,632 0,425 0,515 0,464 0,529 0,547 0,526 0,389 0,597 0,574 0,667 0,632 0,578 0,642 0,557 0,603 0,554 0,657 0,593 0,743 0,535 0,568 0,642 0,592 0,593 0,579 0,583 0,615 0,611 0,594 0,559 0,540 0,667 0,518 0,637 0,613 0,652 0,613 0,590 0,545 1,000 GURBUZ 0,466 0,476 0,543 0,405 0,244 0,486 0,429 0,419 0,396 0,378 0,454 0,366 0,431 0,319 0,358 0,381 0,382 0,348 0,375 0,426 0,354 0,440 0,444 0,364 0,348 0,339 0,408 0,536 0,542 0,508 0,557 0,379 0,515 0,533 0,549 0,559 0,480 0,408 0,370 0,347 0,455 0,424 0,379 0,388 0,386 0,476 0,484 0,441 0,462 0,462 0,577 0,522 1,000

22 Figure 5. Genetically similarity dendrogram created with ISSR markers for grass pea genotypes

23 The lowest genetic relationship was determined between the genotype numbered 4402 and cultivar Gurbuz with the value of The maximum genetic relationship was determined between the genotype numbered 4404 and the genotype numbered 5002 with the value of

24 The average values of genetic distances among all genotypes identified as Ten main groups were formed in dendrogram. The genotypes collected from similar ecological conditions have been found to combine as the basis of the subgroup of genotypes. Probably there has been seed exchange among the local farmers.

25 Conclusions The Lathyrus local landraces were collected from traditional farmers in various regions of Turkey. The data of this work reflects that ß -ODAP, homoarginine, asparagine contents and genetic affinity of grass pea local landraces vary in a wide range from very low to high levels. Although all of these accessions should be preserved as a genetic material, the grass pea genotypes with higher yield and low ß - ODAP content are relevant for future genetic and breeding studies.

26 A positive correlation between homoarginine and ß -ODAP quantities in seeds of 52 Lathyrus local landraces was proven in this work. This is very important because homoarginine can modulate the toxicity of ß ODAP (Shamin et al., 2002). The results obtained from this work and the other study conducted with the same landraces showed that environment affected ß -ODAP content of Lathyrus seeds.

VARIABILITY IN FORAGE QUALITY OF TURKISH GRASS PEA (Lathyrus sativus L.) LANDRACES

Turkish Journal of Field Crops, 2011, 16(1): 9-14 VARIABILITY IN FORAGE QUALITY OF TURKISH GRASS PEA (Lathyrus sativus L.) LANDRACES Uğur BAŞARAN 1 Hanife MUT 1 Özlem ÖNAL-AŞCI 2 Zeki ACAR 3 İlknur AYAN