ISSN 1313 - 8820Volume 7, Number 1

March 2015

2015

Scope and policy of the journalAgricultural Science and Technology /AST/ – an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues.

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Editors and Sections

Genetics and Breeding

Atanas Atanasov (Bulgaria)Nikolay Tsenov (Bulgaria)Max Rothschild (USA)Ihsan Soysal (Turkey)Horia Grosu (Romania)Bojin Bojinov (Bulgaria)Stoicho Metodiev (Bulgaria)

Nutrition and Physiology

Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Bogdan Szostak (Poland)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)Martin Banov (Bulgaria)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2015

ISSN 1313 - 8820 Volume 7, Number 1March 2015

Interspecific hybridization in cotton and its use in breeding

A. Stoilova*, I. Saldzhiev

Field Crops Institute, 6200 Chirpan, Bulgaria

Abstract. Interspecific hybridization in cotton is of great importance with a view to exploit the diversity of species in the genus Gossypium L. The aim of this study was to use the genetic potential of wild species of the genus Gossypium L. to improve productivity, fiber quality and resistance to some stressors of the modern Bulgarian cotton varieties. Interspecific hybridization of Gossypium hirsutum L. species (2n=52) with the wild diploid species G. sturtii F. Muell., G. thurberi Tod., G. davidsonii Kell. and G. raimondii Ulbr.(2n=26) was realized. To overcome sterility, caused by incompatibility of the genomes, the growing tips of the F plants in phase cotyledons were treated with 0.1% solution of colchicine for 12 hours. Amphidiploids of the G. hirsutum × G. sturtii, G. hirsutum × G. 1

thurberi and G. hirsutum × G. davidsonii, and trispecific hybrids G. hirsutum - G. arboreum - G. raimondii, G. hirsutum - G. arboreum - G. thurberi and G. hirsutum - G. thurberi - G. raimondii were obtained. To overcome the undesirable qualities that hybrids inherited together with desirable ones from the wild species two- or three-times backcrossing in C and F (at the triple hybrids) and in the later generations was applied. Studies carried out with this hybridization 1 1

revealed a number of opportunities such as to increase productivity, to improve fiber quality, resistance to low positive temperatures, drought tolerance, to obtain new valuable traits. After backcrossing to the cultivated species valuable introgressed forms having high productivity (from the hybridization with G. thurberi), high strength of the fiber and resistance to aphids and thrips (from the hybridization with G. sturtii), drought-tolerance or resistance to low positive temperatures, etc., were obtained.

Keywords: G. hirsutum L., interspecific hybridization, amphidiploids, trispecific hybrids, backcrosses

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 7, No 1, pp , 201549 - 60

Tod., G. trilobum Skovsted – resistant to low temperatures (-7 to -Introduction10°C). G. davidsonii, G. thurberi and G. trilobum are referred to the group of Mexican cotton, G. harknessii is widespread in Baja Wild species of the genus Gossypium L. possess a valuable California, the islands of Santa Maria and Carmen, and G. sturtii genetic potential that many researchers have used in the genetic belongs to the Australian group (Ter-Avanesyan, 1973).and breeding researches (Mehetre et al., 2003; Gawander et al.,

The aim of this study was to reveal the possibilities of using the 2003). They are carriers of genes for such valuable characters which interspecific hybridization of the species G. hirsutum L. with some are not or rarely found in the cultivated species. Some authors wild diploid species in the cotton breeding in our country to improve (McFadden et al., 2004; Agudelo et al., 2005) reported that in their productivity, fiber quality and resistance to some stress factors of the genome genes for resistance to diseases and pests are localized, modern varieties.others (Mehetre et al., 2003; McCarty, Jenkins, 2006; Linghe et al.,

2011) emphasized their importance as donors of fiber quality – strength, brilliance, natural color. According to BaoLiang et al. (2003)

Material and methodsG. anomalum species has the greatest potential. Chaoyou et al. (2006) after screening of 155 introgressed lines, using SSR molecular markers, concluded that different wild species have In hybridization with the cultivated G. hirsutum L. species different capacity as regards fiber quality, disease resistance, (2n=52) the wild diploid species G. sturtii F. Muell., G. thurberi Tod., tolerance to abiotic and biotic stress. G. davidsonii Kell. and G. raimondii Ulbr. (2n=26) were included. In

In China, by remote hybridization (Gossypium hirsutum × G. crosses with these species the G. hirsutum L. varieties Chirpan-433, anomalum) F × (G. hirsutum × G. thurberi) F variety Jinmian 21 Tashkent-1 (Uzbek), Beli izvor (432), Garant (996), Balkan (442) and 3 4

lines Nos. 9736 and 241 were used. Later, the varieties Avangard-resistant to bollworm (Helicoverpa zea), cotton aphid (Aphis 264, Perla-267, Vega (412), line 413, etc., with introduced gossypii), fusarium (Fusarium wilt) and verticillium (Verticillium wilt) germplasm of the species G. barbadense L., were also involved. was created (SuiLan et al., 2003). ZhengLan et al. (1999) after Crosses were made under field conditions as the G. hirsutum L. backcrossing of F plants Gossypium hirsutum × G. sturtii with the G. 1

species was used as a maternal form and for backcrosses. Varieties hirsutum species registered variety QinYuan 4 possessing high of different genotypes were used to improve the crossability of G. yield, good quality of fiber and multiple resistances to biotic factors. hirsutum L. species with G. thurberi Tod. Hybrids F of G. hirsutum L. In Belgium for more than 40 years one of the world's largest 1

× G. barbadense L., later varieties with introduced germplasme of G. collections of interspecific hybrids including species of A, B, C, D, E, barbadense L. were used to overcome its incrossability with G. F and G genomes was created. The latest program is about the use davidsonii Kell. To overcome sterility, caused by incompatibility of of Australian wild species (Mergeai, 2006). In this field of research the genomes, the growing tips of the F plants in phase cotyledons the works of Sarr et al. (2011) were focused on. 1

For the cotton breeding in our country of interest are the wild were treated with 0.1% solution of colchicine for 12 hours or 0.05% diploid species (2n=26): G. harknessii Brandg, G. davidsonii Kell., for 24 hours. To overcome the undesirable qualities that are inherited G. sturtii F. Muell for their disease and drought resistance; G. thurberi from the wild species along with the desired and to combine the

49

* e-mail: saldzhieva@abv.bg

50

valuable ones two- or three-times backcrossing in C and F (at the 1 1

triple hybrids) and in the subsequent generations with promising G. hirsutum varieties was applied.

The wild species, amphidiploids and F BC -BC hybrids were 1 1 2

planted in pots, in greenhouse. Thereafter the experimental work was continued in the field, where selected genotypes in F - or 2 2 3

in subsequent generations were sown in separate rows, 2.4 m long, and the selected progenies were sown in two rows, 8.2 m long with a sowing design 60×10×1.

Inheritance of productivity/plant, fiber quality parameters (length and strength), of different morphological characteristics, the resistance to aphids and thrips, low positive temperatures (in cool and wet years with insufficient temperature sum for cotton maturing), drought tolerance based on the depression of seedlings under osmotic stress (cultivation of seedlings in 1M solution of sucrose) were accounted.

Results and discussion

ybridi ation of G. hirsutum L. and G. sturtii . uellThe wild species G. sturtii . uell was hybridized easily with all

G. hirsutum L. varieties as the crossability usually was about 50%. The hybrids F G. hirsutum L. × G. sturtii having genomic formula 1

(AD) C , (2n 39) were sterile due to incompatibility of the genomes 1 1

of both species. Complete restoration of fertility was achieved by treating with colchicine. The amphidiploids (allohexaploids) 2[(AD) C ], (2n 78) obtained had restored fertility and combined 1 1

characters of both species (Figure 1). A detailed description of the plant morphology in F and C as well as data on economic qualities 1 1

of amphidiploids w re presented in our previous works (Stoilova and Savova, 1985 Stoilova, 2012). They had a very good tolerance to quickly its own genome exhibiting selectivity of own gametes and low temperatures and drought, and resistance to thrips and aphids elimination of the wild species traits. In the progeny of pentaploids, inherited from the wild species. The amphidiploids had shorter but as a result of different deviations in the meiosis, tetraploid, stronger and finer fiber than the parental forms of G. hirsutum heteroploid and aneuploid plants have occurred described in detail species and these properties were also of particular interest for the by Egamberiev and Filatova (1981). Euploid plants are productive, breeding purposes. Amphidiploids with Avangard-264, Perla-267 cultural type, with traits of the wild species. Backcrossing restores and Colorit (412) varieties were distinguished by higher values of the chromosome number and genotype of the species G. hirsutum L. fiber quality. but does not exclude introgression of genes from wild species.

The amphidiploid G. hirsutum G. sturtii is a constant, not segregating form and it could be defined as a new synthetic ybridi ation of G. hirsutum L. and G. thurberi Todhexaploid species. After backcrosses with the cultivated species G. Hibridization between G. hirsutum and this species was difficult. hirsutum, despite the high percentage of fertilization, the unripe Successful crosses were realized only with line 9736 and cv. Balkan capsules died on the 14 21 day. Many authors explained this (Stoilova and Savova, 1986), later with breeding lines with phenomenon by incompatibility of the embryo and endosperm. germplasm of the species G. barbadense L. The hybrids F G. 1

According to Daminova (1989) one of the reasons for normal hirsutum × G. thurberi having genomic formula (AD) D , (2n 39) 1 1

fertilization of the egg was the absence of synchronous functioning were also sterile. After the colchicine treatment their fertility was of the two genotypes during the mitosis in the early embryogenesis. restored. The amphidiploid 2[(AD) D ], (2n 78) had low fertility. Its 1 1To overcome this partial incompatibility embryo rescue method was fiber was fine, with brilliance, differing from that of G. hirsutum, by 2 successfully applied by Hadzhiivanova and Bozhanova (2009) on to 7 mm shorter than that of the parental forms of this species. A low the obtained amphidiploids. Twenty plants were regenerated from temperature resistance and natural leaf-fall like those in the wild 88 cultivated embryos. Two BC plants were adopted successfully 1 species were observed (Stoilova, 1988). The C amphidiploid 1and grown to maturity. (allohexaploid) was genetically unstable and segregated.

Successful backcrosses were realized as the G. hirsutum Cytological studies of Arutyunova (1960) cited from Volkova (1974) varieties and lines were used as maternal forms, amphidiploids as have shown that hexaploid chromosome number is stored only in the pollinators. Plants obtained by this approach were similar to the C and C in some cases. Fertility in C was low, photoperiodicity was 1 2 2cultural form (maternal type) the genome (AD) was in its own 1 strongly expressed, and the bolls were formed in late autumn. cytoplasm. Egamberdiev and Filatova (1981) reported that the Typical for C and C plants was that at the beginning of vegetation 1 2phenotypic expression of individual traits varied depending on what

they had slow growth. In the process of segregation forms (own or foreign) cytoplasm was the genome of the cultivated variety.

possessing valuable characters were observed. In C C from the 6 8G. hirsutum L. species used as a maternal form strives to restore

ure 1. phi iploi G. hirsutum (Chirpan-433) G. sturtii F. Muell ( ) , ( n= ) ith restore fertilit

51

crossing 9736 × G. thurberi plants with high productivity, large bolls associated with the accumulation of a large number of bolls, were and medium long fiber were observed. In some plants the fiber was observed.finer and longer than that of the maternal form. Others had big bolls After backcrosses of the allotetraploid 9736 × G. thurberi to the with very large seeds. Many of these characters were preserved in maternal line 9736, stabilization of the BC hybrids was observed in 3

the next progenies. The fiber lint percentage even in the later hybrid F BC , while after backcrosses with different varieties segregation 4 3

generations was low 30 – 35% and vegetation period was more than was more prolonged, but the process of formation of new forms was 120 days. more strongly expressed and occurred at a higher degree.

After backcrosses of the amphidiploid 9736 × G. thurberi with maternal form, in F BC highly productive progenies were obtained, 4 3 Hybridization of G. hirsutum L. and G. davidsonii Kell later among them high-yielding lines were selected (№ 440). The This species was crossed to cvs. Garant, Balkan and line 241 realization of introgressed lines is given in Figure 2. (G. hirsutum L.). However, unviable seeds which did not germinate

Line 9736 had lower productivity, short fiber and low lint were obtained. Their non-viability is determined by the lethal genes percentage than the modern varieties and in later generations for contained in both species. To overcome this incompatibility F 1

backcrosses new promising varieties and lines were used. Figure 3 presents F BC plants possessing comparatively high productivity 4 1

obtained after a single backcross of the amphidiploid C 9736 × G. 6

thurberi with line 364.Due to the limited number of plants in F BC backcrosses with 1 1

the species G. hirsutum L. were made in F BC and in later 2 1

backcross generations which also produced positive results (Table 1). On the one hand, this approach increases the resulting genetic material and the other more cultural genotypes are included in backcrosses and thus the selection process is accelerated. The backcrosses with different parents determined different combinations of the productivity, length and lint percentage of the fiber.

Economic characters were the highest in BC , which is 3

explained by an increase in the number of plants with characters of cultivated species. In the second and third generations the best earliness was found for the hybrids resulting after three times backcrossing. In these generations genotypes with high productivity,

Table 1. Economic characters of F1BC3 hybrids obtained after backcrosses of C5-C7 9736 × G. thurberi Tod. (Planted in field)

Hybrids F BC1 3

Number ofplants

Productivity/plant, g

Boll weight, g Fiber length, mm

lint percentage,%

Parents264 (Avangard-264)

361 (Natalia)

364 (Darmi)

L. 3 (Dorina)

MillenniumF BC1 3

Backcrosses in F BC 1 12[(C 9736×G.thurberi)×361]× 3616

2[(C 9736×G.thurberi)×361]× 3617

2[(C 9736×G.thurberi)×364]× 3646

2[(C 9736×G.thurberi)×364]× 3647

2[(C 9736×G.thurberi)×Mill]×Mill6

Backcrosses in F BC 2 12[F BC (C 9736×G.thurberi)×364]×3642 1 6

2[F BC (C 9736×G.thurberi)× Millennium] × Millennium2 1 6

Backcrosses in F BC 3 1 2[F BC (C 9736×G.thurberi) × Millennium] × Millennium3 1 6

2[F BC (C 9736×G.thurberi) × L.3]×L.33 1 5

Backcrosses in F BC 5 12[F BC (C 9736×G.thurberi) ×264]×2645 1 5

39

39

39

39

39

14

23

53

42

59

34

31

26

11

25

14.2

18.0

18.0

16.8

14.8

23.3

26.6

23.5

24.1

16.7

24.4

17.3

20.8

26.4

27.3

5.9

5.6

6.0

5.5

5.5

6.3

5.8

6.3

6.4

5.8

5.4

5.2

6.0

5.9

5.9

29.3

31.0

30.5

29.6

30.8

28.1

28.5

28.7

28.2

28.3

29.8

29.4

29.1

28.8

28.6

36.0

35.6

36.0

38.5

41.1

36.2

35.6

33.3

35.9

40.1

34.1

39.4

39.3

39.5

35.5

Figure 2. Creation of introgressed lines by interspecific hybridization of G. hirsutum L. 2(AD) , (2n=52) × 1

G. thurberi Tod. 2D , (2n=26)1

Fertile allohexaploid

2[(AD) D ]1 1

× G. hirsutum L. 2(AD)1 Self-pollination

2(AD) + 0 – n D1 12(AD) + 0 – n D 1 1

Pentaploid 2(AD) D1 1

Introgressed lines

G. hirsutum L.

2(AD)1

52

hybrids G. hirsutum × G. barbadense were involved in hybridization In F of the trispecific hybrids plants with comparatively high 3

with G. davidsonii ell. productivity were observed (Figure 6). After twice backcrossing of The hybrids F (G. hirsutum × G. barbadense) × G. davidsonii, the triple hybrids with Avangard-264 and Darmi varieties greater 1

whose genome was generally presented ADD , (2n 39) were length of the fiber was formed than with Chirpan-539, which is 3-d

explained by the genotypes of the varieties. The fiber lint percentage sterile. After colchicine treatment and chromosome doubling after twice backcrossing remained low even when parents with high amphidiploids (allohexaploids 2n 78) with high heterozygosity were values of this property were used for backcrossing 34.5 37.2% for obtained. In C a great diversity of morphological and other traits was 1

the backcrosses with Chirpan-539 and Chirpan-603 and 32.6 observed (Stoilova, 1995). Genome AD is complex and explains the 36.4% with Avangard-264 and Darmi, possessing longer fiber and observed variation in C , which is genetically unstable. Later, 1

lower lint percentage. Improvement in productivity was observed introgressed varieties of G. hirsutum × G. barbadense were involved after twice backcrossing in F -F and in later hybrid generations. 3 4in hybridization with G. davidsonii and new original amphidiploids When different parents were used for crossing and backcrossing, were obtained.the productivity, length and lint percentage of the fiber had higher Improving the earliness in this hybridization was observed with average values than when the parents were the same, which is every subsequent backcross but even in F -F BC generations the 2 3 3

explained by the strengthening of heterozygosity (Figure 7 and 8). number of plants with characters of the cultivated species was restricted and thus the opportunity for selection of valuable forms

ossibilities to exploit the interspecific hybridi ation of the G. was also limited (Stoilova, 2012). At this hybridization a greater hirsutum L. speciesnumber of subsequent backcrosses are required.

Three introgression methods are used for transferring genetic material from the wild species into the genome of the cultivated G. ynthetic allotetraploidshirsutum species depending on the affinities between the implicated Very valuable for the cotton breeding are allotetraploids: G. genomes: paraphyletic method which is similar to the natural arboreum L. × G. raimondii Ulbr. 2(A D ), (2n 52) G. thurberi Tod. × 2 5

processes for obtaining synthetic allotetraploids on the basis of two G. arboreum L. 2(A D ), (2n 52) and G. thurberi Tod. × G. raimondii 2 1diploid species, one of which has A-genome, and the other has D-Ulbr. 2(D D ), (2n 52). All of these species are diploids, each has two 1 5 genome pseudophyletic method based on direct crossing of the

chromosome sets - 2x, where x 13 or 2n 26. G. thurberi and G. tetraploid species with diploid ones having A- and D-genomes and

raimondii belong to the D genomic group, G. thurberi has genome transferring the allohexaploids - 2(A D D) or 2(A D A) on tetraploid 1 1 1 1D , and G. raimondii has genome D , (2n 26). The cultivated species 1 5 level by crossing them with diploid species having A- or D-genome,

G. arboreum refers to the A genomic group, genome A , (2n 26). 2 respectively aphyletic method crossing of the tetraploid species After crossing of the alotetraploids with the G. hirsutum L. species with distant diploid species unrelated to the tetraploids (genomes B, trispecific hybrids were obtained (Figure 4 and 5).

ure 3. lants F C (C 3 G. thurberi) 3 4 ith hi h pro ucti it4

ure 4. llotetraploi F G. thurberi G. raimondii an F plant of the trispecific h ri (G. thurberi G. raimondii)

Chirpan- 3

53

ure 5. riple h ri G. thurberi - G. arboreum - G. hirsutum

54

ure . F plants of the trispecific h ri (F G. thurberi G. raimondii) -3 - G. hirsutum L. ith hi h pro ucti it 3

ure . F C plants of triple h ri s here ifferent arieties ere use for ac crosses

55

E, F, C and G) and subsequent colchicine treatment to double the (D ) exclusively drought resistant.7

number of chromosome sets (Ndungo et al., 1988). The paraphyletic The trispecific hybrid G. hirsutum G. arboreum G. thurberi and pseudophyletic methods based on the synthesis of natural A D A D (2n 4x 52) has two subgenomes A and D of G. hirsutum, 1 1 2 1 1 1

allotetraploids with genomic formula 2(AD) lead to the production of one D -genome of G. thurberi, and one A -genome of G. arboreum. 1 2

trispecific hybrids which are highly heterozygous forms and The genomes A (A and A ) have high affinity and the D-genomes 1 2

therefore in the backcross generations are observed as early as F 1 have relatively high. Chromosomes Ah or Dh of G. hirsutum could processes of segregation. The aphyletic method allows the form bivalents, respectively with A of G. arboreum and D of G. 2 1

exploitation of all the genomes of the diploid species. thurberi. The presence of homology between Ah and A , Dh and D 2 1The affinity of subgenome A of the amphidiploid species with the contributes to the emergence of recombinants. Of all D-genomes of

genome A of the diploid species is very strong. Concerning the the diploid species the genomes D (G. thurberi) and D (G. trilobum) 1 8affinity of D-subgenome of the amphidiploid species with D-genome have the best compatibility with the subgenome D of G. hirsutum. 1of the diploid species there are three groups: strong (with G. thurberi The wild species G. trilobum (D ) possesses cold resistance and it is 8 2D ) not very strong (with G. raimondii 2D ) causing lethality 1 5also of interest for the cotton breeding in our country.(with G. davidsonii 2D ) (Louant, 1973 Valicek, 1978).3-d The trispecific hybrid G. hirsutum G. thurberi G. raimondii

The trispecific hybrid G. hirsutum G. arboreum G. raimondii A D D D (2n 4x 52) has two subgenomes A and D from the G. 1 1 1 5 1 1A D A D (2n 4x 52) has two subgenomes A and D of G. hirsutum, 1 1 2 5 1 1 hirsutum species, one genome of G. thurberi D and one genome 1one A -genome of G. arboreum and one D -genome of G. raimondii. 2 5 of G. raimondii D , i.e. one A-genome and three D-genomes. The 5The subgenome A of G. hirsutum is compatible with the genome A 1 2 subgenome A and D and D genomes have insulating mechanisms, 1 1 5of G. arboreum, which means that the chromosomes of both species but it is possible to have high con ugation of chromosomes from the could form bivalents. There is a high degree of heterogeneity (non-four different genomes. Mehetre et al. (1993) at G. hirsutum 2(AD) × 1homology) between the subgenome D of G. hirsutum and the 1G. raimondii (2D ) amphihaploid observed 4 to 6 bivalents as a result 5genome D of G. raimondii. The productivity of the three-species 5of pairing between Ah and D chromosomes, respectively from G. 5hybrid G. hirsutum G. arboreum G. raimondii largely depends on hirsutum and G. raimondii species, as well as of Ah with Dh and Dh the genotype of G. hirsutum varieties. Under this scheme with D -chromosomes. The formation of pairs between non-5(paraphyletic method) in hybridization with the G. hirsutum species homologous chromosomes showed that the chromosomes of the other very valuable for the cotton breeding in our country wild genome D G. raimondii have close homology with the AhDh 5 species of the D-genome group may be included, such as: G. chromosomes of G. hirsutum. The results obtained from studies with har nesii (D ), G. armourianum (D ), G. aridum (D ), G. lobatum 2-2 2-1 4

ure . F C plant o taine after ac crossin of the 3

triple h ri (F G. thurberi G. raimondii) ealan ith

Chirpan- 3 ariet

ure . ro en associate stron fi er, o taine fro the interspecific h ri i ation of the G. hirsutum × G. sturtii and ac cross, si ilar to G. hirsutum species

56

molecular markers (Reinsch et al., 1994 Lacape et al., 2003) and allotetraploid (AD) C C with the species G. hirsutum 2(AD) it is 1 1 1 1

QTL analyses of the same characters of chromosome possible the gene introgression from C -genome to (AD) -genome to 1 1

supplemented lines (Endrizzi et al., 1984) supported the existence of strengthen and the frequency of chromosomal translocations a homologous connection between the Ah and Dh-subgenomes. between the genomes of wild species and this of G. hirsutum

At the trispecific hybrid G. hirsutum G. thurberi G. raimondii species to increase. At the hybridization of G. hirsutum × G. sturtii D-genomes of the two diploid species have high-affinity, and each of the number of chromosome associations ACA and CAC was them has a comparatively high affinity with the subgenome Dh. Ah significant, while those of CDC and DCD types were less frequent. subgenome has a partial compatibility with both diploid D-genomes These types of associations showed translocations between the and Dh-subgenome, i.e. Ah subgenomic chromosomes may form chromosome C and A, and C and D (Ndungo et al., 1988).chromosome associations with the three D-genomes. The strategy Effectiveness of the selection process greatly depends on the to produce trispecific hybrids possessing three D and one A-genome genotype of the parental varieties included in crosses with the wild is of great interest from the viewpoint of genetics and breeding, as species and the genotype of varieties used for backcrosses. The well as to clarify the affinity of Ah-genome to the three D-genomes. wild species G. davidsonii, which is not crossed with G. hirsutum L. This method could also be implemented to obtain triple hybrids by and rarely crossed with G. barbadense, has very good crossability crossing G. hirsutum species with allotetraploids of diploid species with introgressed varieties and lines obtained from the crosses of the from the B-genomic group G. anomalum, possessing very high two cultivated tetraploid species. The triple hybrids produced from quality of the fiber. B-genomes have good affinity to Ah-genome and the crossing of the synthetic allotetraploids with the species G. can replace it. hirsutum form highly heterogeneous populations with low

The presence in the G. hirsutum genome of a D-subgenome, productivity in the early generations. The low productivity is due to which has very little affinity to the other genomes of the genus genotypic imbalance specific for the early generations of Gossypium L. (A, B, C, E, F and G) restricts the efficiency of the interspecific hybrids.paraphyletic and pseudophyletic methods to apply the synthetic Detailed results of the application of different backcross allotetraploids and to use all wild diploid species. The application of approaches are presented in a previous study (Stoilova, 2012). the allohexaploidity based on the aphyletic method (Ndungo et al., Backcrossing with Chirpan-539 and varieties of this type (Chirpan-1988) allows the exploitation of all the genomes of wild diploid 603, Beli Iskar) led to increase in the productivity and lint percentage species. of the fiber. To obtain forms with longer fiber the backcrossing with

Of interest is the translation of the allohexasaploid G. hirsutum introgressed varieties and lines with germplasm of the species G. × G. sturtii 2[(AD) C ], (2n 26) on tetraploid level by its crossing with 1 1 barbadense Avangard-264, Pearla-267, line 364 (Darmi variety), the wild species G. sturtii 2C , (2n 26). After backcrossing of the line 361 (Natalia variety) was of greater importance. The hybrid 1

ure 1 . F C pro enies o taine after t ice ac crossin of the a phi iploi C 3 G. thurberi o . ith Millenniu4

ree ariet

57

populations with Chirpan-539 variety were earlier than those with Avangard-264 variety. Simultaneously, improvement of length and lint percentage of the fiber was found for the hybrids obtained after backcrossing with line 3 (Dorina variety) with longer fiber and higher lint percentage. Using different genotypes for backcrosses heterozygosity increases and the processes of segregation become complicated, but this makes possible economically valuable characters and qualities to combine in different ways on the one hand, and on the other, the emergence of transgressive variation. Backcrossing in the later and in the backcross generations allows for more efficient use of the resulting genetic material, which at the interspecific hybridization is very limited.

Breeding results from the interspecific hybridi ation of G. hirsutum

After backcrossing of the amphidiploid G. hirsutum × G. sturtii, forms similar to the species G. hirsutum, having high strength of the fiber, were obtained (Figure 9). Plants in some traits and qualities differed from the cultural form. Internodes of their sympodial branches were more elongated, fertility was lower, the fiber was shorter, but as already noted very strong, leaves three-five lobed, in the wild type they are small, entire, and in the cultural form medium large, five lobed.

In F BC of the combination line 65 × (Balkan × G. sturtii) 4 1

progenies (2, 249, 250 and 251) with fine, long and very strong fiber were selected. Long fiber was inherited from line 65, and its strength from the wild species. Two lines possessing natural early leaf-fall,

originating from line 64 × G. sturtii 250 Gy and line 267 × G. sturtii 250 Gy were obtained. Their fiber is long with low lint percentage, as in the mother forms. Lines 64 and 267 are of interspecific origin G. hirsutum L. × G. barbadense L. Sympodial branches are long with highly elongated internodes. These lines are used in breeding programs as donors of natural early leaf-fall.

After two- and three-times backcrossing of the obtained amphidiploids G. hirsutum × G. thurberi and G. hirsutum × G. davidsonii, and trispecific hybrids, in F BC -BC valuable genotypes 2 2 3

were selected and in F -F BC -BC progenies and lines possessing 4 6 2 3 hirsutum × G. thurberi hybridization form and retain a large number valuable characters of the wild species that are at different stages of of bolls and have a faster rate of their ripening inherited from the wild the selection process were also selected (Figure 10, 11, 12, 13 and species. Plants develop big bush and form a large number of 14). monopodial branches. Progenies selected from the hybridization of

Progenies possessing high productivity obtained after G. G. hirsutum × G. davidsonii, having high productive potential, are

ure 11. F C pro enies o taine after t ice 4

ac crossin of the a phi iploi C 3 G. thurberi o .

ith ar i ariet

ure 1 . F C pro enies o taine interspecific 4

h ri i ation of the G. hirsutum L. ith the il species G. davidsonii Kell. an t ice ac crossin ith atalia ariet

58

late in maturity, especially in years with insufficient temperature sum. period the wild species, except G. thurberi, amphidiploids and the All lines have shown resistance to cotton aphids, inherited from the studied introgressed lines were resistant (Rashev et al., 2002). wild species. Critical phase in cotton for attack by aphids is from Under osmotic stress, the introgressed lines were characterized by germination to 3 4 leaf stage. The results showed that during this relatively low rates of depression in the growth of the aerial parts of

young cotton seedlings (from 37 to 56.9%) and can be characterized as more drought tolerant than the standard variety Chirpan-539 (Bozhanova and Stoilova, 2002).

In very cold and wet years when cotton strong late in ripening, some introgressed lines exhibited very high cold tolerance and were highly superior to the standard in seed cotton yield, by 40.0 to 77% for the lines from the G. hirsutum × G. sturtii hybridization, and 34.4 to 86.7% for the lines from the hybridization of G. hirsutum × G. thurberi (Stoilova, 2012).

Of lines obtained by remote hybridization and included in competition variety trial in 2012 2013, two were distinguished by higher productivity than the standard Chirpan-539 and other six showed greater length of the fiber (Table 2). Numbers 421 and 424 were obtained from the hybridization of G. hirsutum L. and G. thurberi Tod. after twice backcrossing with the hirsutum L. Number 429 was obtained by hybridization of G. hirsutum L. and G.

ure 13. F C pro enies o taine interspecific h ri i ation of the G. hirsutum L. ith the il species G. davidsonii 4

Kell. an t ice ac crossin ith atalia ariet an Chirpan- 3 ariet

ure 14. F C pro enies o taine fro the crosses of G. hirsutum L. species - Chirpan- 3 ariet ith the allotetraploi 4

G. thurberi Tod. G. raimondii Ulbr. an t ice ac crossin

59

davidsonii Kell., and twice and three times backcrossing with the Gossypium longicalyx and interspecific cotton hybrids. Journal of hirsutum L.; Numbers 449 and 457 were realized from [(G. turberi × Nematology, 37, 4, 444-447.G. raimondii) × Darmi] × Darmi. BaoLiang Z, Song C, XinLian S, XiangGui Z and ZhenLin Z,

2003. Construction of gene pools with superior fiber properties in upland cotton through interspecific hybridization between Gossypium hirsutum and Gossypium wild species. Acta Agronomica

Conclusion Sinica, 29, 514-519.Bozhanova V and Stoilova A, 2002. Study on drought tolerance of

Тhe studies and results obtained show that the use of alien cotton genotypes by the depression in the growth of seedlings under germplasm in the cotton breeding is very reliable. Through the osmotic stress. Biology and the Future I, 2-4, 117-119(Bg).alloploidity possibilities for transferring valuable characters and Chaoyou P, Xiongming D and Zhiying M, 2006. Evaluation of the qualities from the wild species into the genome of cultivated cotton introgressed lines and screening for elite germplasm in Gossypium. G. hirsutum L. have been revealed. With the inclusion of G. hirsutum Chinese Science Bulletin, 51, 304-312.L. varieties possessing germplasm of the G. barbadense L. species, Daminova DM, 1989. Physiological concept of incrossability in in hybridization with the wild diploid species G. sturtii F. Muell., G. interspecific cotton hybrids. Proceedings Conference of Young thurberi Tod. and G. davidsonii Kell., new original amphidiploids Scientists on topical issues of cotton. Tashkent, July 12-13.(allohexaploids) have been obtained. The resulting amphidiploids Egamberdiev EA and Filatova PC, 1981. Cotton hybrids resistant are of great importance for the cotton genetics and breeding. to vilt. Hlopkovodstvo, 9, 24.

The allotetraploids G. arboreum L. × G. raimondii Ulbr., G. Endrizzi JE, Turcotte EL and Kohel RJ, 1984. Qualitative genetics, thurberi Tod. × G. raimondii Ulbr. and G. thurberi Tod. × G. arboreum cytology and cytogenetics. Published in Cotton. American Society of L. are very valuable for the cotton breeding as donors of fiber quality. Agronomy, Madison, Wis, USA, 81-129.The crossing of allotetraploids with the G. hirsutum L. species Gawander VL, Aher AR and Shinde GC, 2003. Cytomorphology of resulted in the obtaining of trispecific hybrids. The last ones are interspecific hybrids between Gossypium hirsutum L., its haploid highly heterozygous forms and in backcross generations are and Gossypium raimondii. Indian Journal of Genetics and Plant observed as early as F processes of segregation.1 Breeding, 63, 319-324.

The diversity of biotypes arose in backcross generations and Hadzhiivaniva B and Bozhanova V, 2009. Overcoming of the emergence of plants similar to G. hirsutum L. associated incompatibility between amphidiploid cotton (Gossypium characters of the wild species allows us to recommend the use of hirsutum×Gossypium sturtianum) and some cultivated species amphidiploids obtained in the breeding programs with cotton as through embryo rescue method. Genetics and Breeding, 38, 71-donors to improve productivity (with G. thurberi), fiber quality, its 74(Bg).strength (with G. sturtii) and length (with G. thurberi and G. raimindii) Lacape JM, Nguyen TB, Thibivilliers S, Bojinov B, Courtois B, of the modern varieties, their resistance to thrips and aphids, Cantrell RG, Burr B and Hau B, 2003. A combined RFLP-SSR-tolerance to drought and low positive temperatures. AFLP map of tetraploid cotton based on a Gossypium hirsutum ×

By interspecific hybridization and backcrosses valuable source Gossypium barbadense backcross population. Genome, 46, 612-material for the cotton breeding has been created. Valuable 626.introgressed lines having high productivity and high quality, which Linghe Z, Meredith Jr, William R and Boykin DL, 2011. are at various stages of testing have been realized. Germplasm potential for continuing improvement of fiber quality in

upland cotton: Combining Ability for Lint Yield and Fiber Quality. Crop Science, 51, 60-68.

References Louant BP, 1973. Recherchers sur les possibilites d'ameliorer le cotonnier par l'introgression directe de caracteres provenant de

Agudelo P, Robbins RT, Stewart JM, Bell A and Robinson AF, Gossypium raimondii Ulbr. Publ. Inst. Natl Agron. Congo Belge, 2005. Histological observations of Rotylenchulu reniformis on Serie Science, 116-177.

Table 2. Economic characters of lines obtained after interspecific hybridization of the G. hirsutum L. species, 2012–2013(average for two years)

Lines No. Lint percentage,%

Boll weight,g

In percent to seed

cotton yield

First pickingkg/ha

In percent to Chirpan-539

Seed cotton yieldkg/ha

Fiber length,mm

Chirpan-539

421

424

429

449

457

GD 5.0 %

GD 1.0 %

GD 0.1 %

1665

1634

1632

2040

1520

1936

196

260

336

100.0

98.1

98.0

122.5+++

91.3

116.3++

11.8

15.6

20.2

1433

1349

1403

1799+++

1247

1650+

205

272

351

86.1

82.6

86.0

88.2

82.0

85.2

-

-

-

5.3

5.4

5.3

5.1

5.1

5.2

0.3

0.4

0.5

26.2

27.2+++

27.5+++

26.9++

28.5+++

27.1+++

0.5

0.7

0.9

39.70039.000038.400038.800038.200037.8

0.5

0.7

0.9

60

McCarty JС, Wu J and Jenkins J, 2006. Genetic diversity for agronomic and fiber traits in day-neutral accessions derived from primitive cotton germplasm. Euphytica,148, 283-293.McFadden H, Beasley D and Brubaker C, 2004. Assessment of Gossypium sturtianum and G. australe as potential sources of Stoilova A, 1988. Hybridization of Gossypium hirsutum L. (2n=52) Fusarium wilt resistance to cotton. Euphytica, 138, 61-72. and Gossypium thurberi Tod. (2n=26). Development of the hybrids in Mehetre SS, Aher AR, Patil VR, Gawande VL, Mokate AS, F and the amphidiploid. In: Interspecific hybridization in plants. 1

Gomes M and Eapen S, 2003. Cytomorphological and molecular Interspecific hybridization Symposium with international bases of interspecific hybrids between Gossypium davidsonii and G. participation. Sofia, September 29-30, 1988.anomalum. SABRAO Journal of Breeding and Genetics, 35, 43-56. Stoilova A, 2012. Breeding of new cotton varieties and germplasm Mergeai G, 2006. Cotton improvement through interspecific based on improved fiber quality. Thesis for DSc (Bg). hybridization. / Introgressions interspécifiques chez le cotonnier. Stoilova A and Savova N, 1985. Hybridization of Gossypium Cahiers Agricultures. Montrouge: John Libbey Eurotext, 15, 135- hirsutum × G. sturtii. Development of the F hybrids and 1

143. amphidiploid. In: Ndungo V, Demol J and Marechal R, 1988. L'amelioration du cotonier Gossypium hirsutum L. par hybridation interspecifique. I. La SuiLan H, BaoDe G, LiXia J and YongZhang L, 2003. Selection of phylogenie et la speciation du genra Gossypium. Bull. Rech. Agron. the very early high quality new cotton variety Jinmian 21. China Gembloux-Belgique, 23, 27-49. Cotton, 30, 30.

Toussaint A, Palm R, Ahoton L, Baudoin JP and Mergeai G, 2011. Isolation of five new monosomic alien addition lines of Gossypium australe F. Muell in G. hirsutum L. by SSR and GISH analyses. Plant Breeding (Berlin) 130, 60-66.

stReinisch A, Dong JM, Brubaker CL, Stelly DM, Wendel JF and Valiceк P, 1978. Wild and cultivated cotton] (1 part). Cotton Fibers Paterson AH, 1994. A detailed RFLP map of cotton Gossypium Tropicale, 33, 363-387. hirsutum × Gosypium barbadense: chromosome organization and evolution in a disomic polyploid genome. Genetics, 138, 829-847.Sarr D, Lacape JM, Rodier-Goud M, Jacquemin JM, Benbouza H, Toussaint APalm R, Ahoton L, Baudoin JP and Mergeai G, ZhengLan L, RuQin J, WenNan Zh, LiMin L, ZengXin W, Yuan W, 2011. Isolation of five new monosomic alien addition lines of YunShen Hu and YinXia D, 1999. Study on the interspecific Gossypium australe F. Muell in G. hirsutum L. by SSR and GISH hybridization of Gossypium hirsutum × G. sturtianum and selection analyses. Plant Breeding (Berlin) (Eng) 130, 60-66. of a new variety Qinyuan 4. Scientia Agricultura Sinica, 32, 14-19.

and Gossypium davidsonii Kell. (2n=26). Jubilee conference with international participation "70 years Institute of Cotton and Durum Wheat - Chirpan", 27-28.09.1995 Proceedings "Problems of fiber and grain bread cultures", Stara Zagora, 135-139.

Youth Conference of Genetics' 84, Sofia, Bulgarian Academy of Science, 189-192.

Rashev S, Stoilova A and Saldzhiev I, 2002. Test of new Ter-Avanesyan DC, 1973. Cotton (Hlopchatnik), L. "Kolos"germplasm in cotton to cotton aphid (Aphis Gossypii Glov.) resistance. Jubilee Conference titled "Academic Pavel Popov and achievements of the Crop Science in Bulgaria" November 22 Scientific works, XLVII, AU-Plovdiv, 141-144.

Wolkova LA, 1974. Cytological study of allohexaploids/ amphidiploids in cotton and F progenies from back crosses. Thesis 1

for PhD, Tashkent (Ru).

Stoilova A, 1995. Hybridization of Gossypium hirsutum L. (2n=52)

Review

Genetics and Breeding

Nutrition and Physiology

Production Systems

Molecular mechanisms and new strategies to fight stresses in egg-producing birds E. Shatskikh, E. Latypova, V. Fisinin, S. Denev, P. Surai

Gene action in the inheritance of date to ear emergence and time to physiological maturity in bread wheat crosses (Triticum aestivum L.)N. Tsenov, T. Gubatov, E. Tsenova

Productivity and stability of the yield from common winter wheat cultivars developed at IPGR Sadovo under the conditions of Dobrudzha region P. Chamurliyski, E. Penchev, N. Tsenov

Effectof black (stem) rust (Puccinia Graminis F.SP. Tritici) attack to the spike characteristics in Polishwheat (Triticum Polonicum L.)H. Stoyanov

Analysis of DNA polymorphism of CAST gene in Local Karnobat and Stara Zagora sheep breedsD. Hristova, S. Georgieva, S. Tanchev

Correlation between grain yield and yield components in winter barley varieties N. Markova Ruzdik, D.Valcheva, D.Vulchev, Lj. Mihajlov, I. Karov, V. Ilieva

Genetic diversity in different accessions of oat (Avena sativa L.)T. Savova, B. Dyulgerova, G. Panayotova

Interspecific hybridization in cotton and its use in breedingA. Stoilova, I. Saldzhiev

Influence of the direction of crossing on heterosis and transgression events in relation to the length of the vegetative period of Burley tobaccos variety group Y. Dyulgerski, T. Radoukova, L. Dospatliev

The performance of female dairy calves fed texturized starters with different protein sources E. Yavuz, N. Todorov, G. Ganchev, K. Nedelkov

Feeding value estimation of spring forage pea (Pisum sativum L.) in organic cultivationI. Nikolova, N. Georgieva, Y. Naydenova

Treatment of post harvest residues with cellulose decomposing preparationsI. Effect on grain yield from wheatG. Milev, I. Iliev, A. Ivanova

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Seasonal dynamics of important for Coriandrum sativum virus pathogensB. Dikova, H. Lambev

Crop relationship "yield-evapotranspiration" for green beanR. Kalaydzhieva, D. Davidov, A. Matev, V. Kuneva

Species composition and density of weeds in a wheat crop depending on the soil tillage system in crop rotationP. Yankov, M. Nankova, M. Drumeva, D. Plamenov, B. Klochkov

Assessment of Bulgarian Black Sea coastal water using the biological quality element phytoplanktonD. Petrova, D. Gerdzhikov

Evaluation on reaction of late maturing maize hybrids and lines to Fusarium ear rotM. Haddadi, M. Zamani

Contemporary state of macrophytobenthos along the Bulgarian coast of the Black SeaD. Petrova, V. Vachkova, D. Gerdzhikov

Bioconversion of nitrogen in eco-technical system for eggs productionA. Gencheva

Mixed viral infections in tomato as a precondition for economic lossN. Petrov

Storage and its effect on the antioxidant capacity of dried Bulgarian Chrysanthemum balsamita L.A. Popova, D. Mihaylova, I. Alexieva

Correcting the breadmaking quality of flour damaged by Sunn pest (Eurygaster integriceps) by using apple pectinI. Stoeva

Investigation the influence of dietary fiber on the rheological properties of alginate beadsZ. Manev, N. Petkova, P. Denev, D. Ludneva, S. Zhelyazkov

Occurrence of Pseudomonas syringae pv. tomato in BulgariaM. Stoyanova, K. Aleksandrova, D. Ganeva, N. Bogatzevska

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thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, September 11–14, Berlin, Germany.Thesis:Hristova D, 2013. Investigation on genetic diversity in local sheep breeds using DNA markers. Thesis for PhD, Trakia University, Stara Zagora, Bulgaria, (Bg).

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