# Apple Cultivation and Breeding in Afghanistan:S-RNase Genotypes and Search System for Suitable Cultivar Combination

Authors: Matiullah Akbari; Mao Yamaguchi; Tsutomu Maejima; Shungo Otagaki; Katsuhiro Shiratake; Shogo Matsumoto
Journal: International Journal of Agronomy
 (2016)

---

## Abstract

We investigatedS-RNase genotypes of nine useful Afghan apple cultivars including six original cultivars and one rootstock. We also determinedS-RNase genotypes of 11 apple cultivars and lineages and seven rootstocks in Japan. We speculated regarding the unidentified parents of cultivars and lineages from theS-RNase genotypes and their fruit and branch characteristics and also identified mistaken parents. We compiled a database of the appleS-RNase genotypes of 622 apple cultivars investigated, which included a survey system of cultivar combinations showing those that were fully incompatible, semicompatible, and fully compatible, written in the Pashto language.


---

## Body

## 1. Introduction

The origin of apple is in Central Asia, and many areas of Afghanistan have native apples. Apple is one of the most cultivated horticultural crops in this country since the climate is appropriate for it. The major areas of apple production, that is, areas producing more than 1,000 metric tons per year, are Ghazni, Kabul, Kandahar, Kapisa, Logar, Paktika, Paktya, Panjshir, Parwan, Uruzgan, and Wardak [1]. According to the forecast of the Ministry of Agriculture, Irrigation, and Livestock (MAIL), apple production in Afghanistan increased five to 10% from 2012 to 2013 [1]. It was 77,000 metric tons in 2013/14 (April/March) [1].Self-incompatibility is a genetic mechanism that prevents inbreeding and promotes outcrossing in many flowering plants [2]. The gametophytic self-incompatibility (GSI) system in apple is controlled by the multiallelicS-locus [2, 3], so that when the pollenS-haplotype matches one of the pistilS-haplotypes, the pollen tube growth is arrested in the style. In apple GSI, F-box gene called SFBB (S-locus F-box brothers) andS-RNase gene act as pollen and pistil factors, respectively [4, 5]. As fertilization of apple needs at least two cultivars having no or one commonS-haplotype, accurate knowledge of theS-genotype that is identified by theS-RNase alleles is important for stable apple production.Afghan apple growers know about the self-incompatibility of apple cultivars, but unfortunately they know nothing about theirS-RNaseallele genotypes. Currently in Afghanistan, the apple growers design their apple orchard based on the fruit skin colors of different apple cultivars. However, sometimes the combination of two apple cultivars having different skin color results in full-incompatibility due to their identicalS-genotype, likeS
2
S
3 of “Jester” (red skin) and “Golden Delicious” (yellow skin). Current apple production in Afghanistan is mainly from exotic cultivars imported around 20 years ago [1]. Among various apple cultivars including “Discovery” (S
10
S
24) [6], “Fiesta” (S
3
S
5) [7], “Fuji” (S
1
S
9) [8, 9], “Gala” (S
2
S
5) [7], “Golden Delicious” (S
2
S
3) [7], “Granny Smith” (S
3
S
23) [6], “Jester” (S
2
S
3) [10], “July Red” (S
24
S
25) [http://www.agr.nagoya-u.ac.jp/~hort/apple/], “Mutsu” (S
2
S
3
S
20) [9, 11], “Double Red Delicious” (S
9
S
28) [12], “Red Devil,” “Saturn,” “Spartan” (S
9
S
10) [13], and “Summerred” (S
2
S
9 in Belgium,S
10
S
24 in Japan) [6, 14], three cultivars “Double Red Delicious,” “Golden Delicious,” and “Granny Smith” (known as Green Delicious in Afghanistan) are mainly grown commercially [15]. Though the cost of apple production in Afghanistan is lower than in other countries, their apple quality is higher than the ones imported from Pakistan or Iran. Developing original new and high-quality cultivars using Afghan original cultivars seems to be important for Afghan apple growers who wish to compete in local and international markets.In this study, we investigated theS-genotypes of apple cultivars and lineages including Afghan original cultivars for apple breeding and cultivation. In addition, we translated the appleS-RNase allele genotype database to the Pashto language and made a homepage where Afghan apple growers can search for suitable cultivar combination. Indeed, Pashto is Afghanistan’s official language and is used by more than 60% of the country’s populations.


## 2. Materials and Methods

### 2.1. Plant Material

Young leaves of six Afghan original cultivars and a rootstock, that is, “Jawrasi Golden,” “Kado Seb,” “Mahali” (rootstock), “Nazak Badan,” “Parachinari,” “Swati,” “Tur Kulu,” “Red Devil,” and “Saturn” [15, 16], were collected from either Badam Bagh, Kabul Province, or Wardak Province in Afghanistan. Remaining apple cultivars, lineages, and rootstocks used in this study were from collections at the Iwate Agricultural Research Center, Japan, the Nagano Fruit Tree Experiment Station, Japan, or Koukaen, Esashi-ku, Oshu City, Japan. Young leaves were stored at –80°C until use.


### 2.2.S-RNase Allele Amplification and Allele-Specific PCR-RFLP Analyses

Total DNA was isolated from the leaves of individual plants [17]. The primers and conditions used forS-allele-specific PCR-RFLP analyses were those described by Broothaerts et al. (for S
2 and S
3) [18], Janssens et al. (for S
7 and S
9) [7], Kitahara et al. (for S
24) [19], Kitahara and Matsumoto (for S
10) [9], Matsumoto et al. (for S
5 and S
7) [20], Matsumoto et al. (for S
1 and S
20) [21], and Matsumoto and Kitahara (for S
28) [22].


## 2.1. Plant Material

Young leaves of six Afghan original cultivars and a rootstock, that is, “Jawrasi Golden,” “Kado Seb,” “Mahali” (rootstock), “Nazak Badan,” “Parachinari,” “Swati,” “Tur Kulu,” “Red Devil,” and “Saturn” [15, 16], were collected from either Badam Bagh, Kabul Province, or Wardak Province in Afghanistan. Remaining apple cultivars, lineages, and rootstocks used in this study were from collections at the Iwate Agricultural Research Center, Japan, the Nagano Fruit Tree Experiment Station, Japan, or Koukaen, Esashi-ku, Oshu City, Japan. Young leaves were stored at –80°C until use.


## 2.2.S-RNase Allele Amplification and Allele-Specific PCR-RFLP Analyses

Total DNA was isolated from the leaves of individual plants [17]. The primers and conditions used forS-allele-specific PCR-RFLP analyses were those described by Broothaerts et al. (for S
2 and S
3) [18], Janssens et al. (for S
7 and S
9) [7], Kitahara et al. (for S
24) [19], Kitahara and Matsumoto (for S
10) [9], Matsumoto et al. (for S
5 and S
7) [20], Matsumoto et al. (for S
1 and S
20) [21], and Matsumoto and Kitahara (for S
28) [22].


## 3. Results and Discussion

### 3.1.S-RNase Genotypes of Apple Cultivars Including Afghanistan Original Cultivars

We analyzed theS-RNase genotypes of apple cultivars and lineages for using them in a breeding programme.S-RNasegenotypes of “Iwate 4 Gou,” “Natsuakari,” K-20, K-21, K-22, KAD-14, KAD-17, KAD-19, KAD-20, and “Saturn” (Table 1, numbers 1, 2, 6–8, 12–15, and 25) matched one of theS-RNasegenotypes deduced from their reputed parentage. In contrast,S-RNase genotypes of “Oirase,” K-23, K-24, K-25, and “Red Devil” (Table 1, numbers 4, 9–11, and 24) were inconsistent with the one from its reputed parents. “Natsuotome” (Table 1, number 3) was identified asS
7
S
9, and it was suggested that “Fuji” (S
1
S
9) or “Alps Otome” (S
1
S
9) seemed to be used as a paternal parent, judging from its fruit and branch characteristics. As the average fruit weight of “Natsuotome” (195 g) is lighter than that of its maternal parent “Sansa” (280 g), its characteristics might be from “Alps Otome” (50 g), not from “Fuji” (300 g). On the other hand, the characteristics of concentrically cracking at peduncle end in “Natsuotome” are similar to “Shinano Hoppe” (Akane × Fuji), a pedigree of “Fuji” (Figure 1). “Oirase” (S
9
S
28) and K-24 (S
1
S
3) (Table 1, numbers 4 and 10) might be a sport of “Starking Delicious” (S
9
S
28) and “Shinano Gold” (S
1
S
3), respectively, judging from theirS-genotypes. The reputed paternal parent “Tsugaru” for “Oirase” and an unknown pollen parent for K-24 seemed to be unrelated to their production. The paternal parents of “Suwakko” (Table 1, number 5) are unknown. “American Summer Pearmain” (S
1
S
20) or “Indo” (S
7
S
20) or “Kitanosachi” (S
7
S
20) might be used as a paternal parent of “Suwakko” (S
3
S
20), given itsS-RNase genotype and resembling its branch characteristics to “Indo” (Figure 1). In the case of K-23 (Table 1, number 9), either its maternal or its paternal parents were wrong. Since theS-RNase genotype of K-25 (Table 1, number 11) was identified asS
9
S
x, its maternal parent “Shinano Gold” (S
1
S
3) might have been wrongly identified. One unknownS-RNase allele was present in three (JM1, JM7, and Mark [Table 1, numbers 16–18]) out of seven rootstocks in Japan.Table 1
S-RNase genotypes of 20 apple cultivars and 7 rootstocks including their lineage.
Number
Cultivar
Reputed parentage
S-RNase genotype
1
“Iwate 4 Gou”
“Fuji” (S
1
S
9)  ×  “Delicious” (S
9
S
28)
S
1
S
28
2
“Natsuakari”
“Sansa” (S
5
S
7)  ×  “Yoko” (S
3
S
9)
S
3
S
7
3
“Natsuotome”
Chance seedling of “Sansa” (S
5
S
7)
S
7
S
9
4
“Oirase”
“Starking Delicious” (S
9
S
28)  ×  “Tsugaru”+ (S
3
S
7)
S
9
S
28
5
“Suwakko”
“Sekaiichi” (S
3
S
9)  ×  Unknown≠
S
3
S
20
6
K-20
“Shinano Gold” (S
1
S
3)  ×  “Beniroman” (S
3
S
5)
S
1
S
5
7
K-21
“Shinano Gold” (S
1
S
3)  ×  “Beniroman” (S
3
S
5)
S
1
S
5
8
K-22
“Orin” (S
2
S
7)  ×  “Beniroman” (S
3
S
5)
S
3
S
7
9
K-23
“Gunma Meigetsu”+ (S
1
S
3)  ×  “Shinano Gold”+ (S
1
S
3)
S
1
S
x
10
K-24
“Shinano Gold” (S
1
S
3)  ×  U
n
k
n
o
w
n
+
≠
S
1
S
3
11
K-25
“Shinano Gold”+ (S
1
S
3)  ×  “Fuji” (S
1
S
9)
S
9
S
x
§
12
KAD-14
JM1 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
13
KAD-17
JM1 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
14
KAD-19
JM1 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
15
KAD-20
JM7 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
16
JM1
Malus prunifolia  ×  M.9 (S
3
S
9)
S
9
S
x
§
17
JM7
Malus prunifolia  ×  M.9 (S
3
S
9)
S
9
S
x
§
18
Mark
M.9 (S
3
S
9)  ×  Unknown≠
S
3
S
x
§
19
“Jawrasi Golden”
Unknown¶  ×  Unknown≠
S
28
S
x
§
20
“Kado Seb”
Unknown¶  ×  Unknown≠
S
28
S
x
§
21
“Mahali”
Unknown¶  ×  Unknown≠
S
5
S
28
22
“Nazak Badan”
Unknown¶  ×  Unknown≠
S
2
S
9
23
“Parachinari”
Unknown¶  ×  Unknown≠
S
x
S
y
§
24
“Red Devil”
“Discovery” (S
10
S
24)  ×  “Kent”+ (S
3
S
9)
S
2
S
24
25
“Saturn”
“Jonathan” (S
7
S
9)  ×  “Golden Delicious” (S
2
S
3)
S
2
S
7
26
“Swati”
Unknown¶  ×  Unknown≠
S
3
S
28
27
“Tur Kulu”
Unknown¶  ×  Unknown≠
S
2
S
28
+Incorrect parentage indicated by this work is in  bold.
≠Unknown: pollen parent is unknown.
§
S
x, S
y ≠ S
1
,
S
2
,
S
3
,
S
5
,
S
7
,
S
9
,
S
10
,
S
20
,
S
24
,
S
28.
¶Unknown: ovule parent is unknown.Figure 1
A mature fruit and a shape of tree of “Natsuotome” ((a), (b)) and “Swakko” ((c), (d)) produced at Nagano Fruit Tree Experiment Station.
(a)
(b)
(c)
(d)Within the six original Afghan cultivars, “Nazak Badan,” “Swati,” and “Tur Kulu” (Table1, numbers 22, 26, and 27) were determined asS
2
S
9,S
3
S
28, andS
2
S
28, respectively. We also identifiedS-genotypes of rootstock “Mahali” (Table 1, number 21) asS
5
S
28, but one or two unknownS-RNase alleles were present in “Jawrasi Golden” (S
28
S
x), “Kado Seb” (S
28
S
x), and “Parachinari” (S
x
S
y) (Table 1, numbers 19, 20, and 23). This is the first case to analyzeS-genotypes of Afghan cultivars.


### 3.2. Database ofS-RNase Allele Genotypes and the Search System for Suitable Apple Cultivar Combination Using the Pashto Language

We previously established a search system for apple cultivar combination, written in English on the homepage (http://www.agr.nagoya-u.ac.jp/~hort/apple/) [23]. In this study, we have developed a Pashto language version of the search system homepage for apple growers in Afghanistan. At first all names of cultivars and their parents in the database were translated from English to the Pashto language. Then, we changed the search system homepage to a Pashto language version. As the writing of the Pashto language reads from right to left, the new homepage looks like an opposite of the English version (Figure 2). All apple growers in Afghanistan can access the homepage at the URL (http://www.agr.nagoya-u.ac.jp/~hort/apple/ps/). In the homepage of this database, there is a box called the search box. When you write the first letter of a cultivar name in the search box, a list of similar cultivar names will appear from which you can select the target cultivar easily (Figures 2(a) and 2(b)). Afterward, before clicking the search button, you must decide if the cultivar is used as an ovule parent or pollen parent. Then click the search button. Actually, the search system ofS-genotype database is developed for suitable combinations of apple cultivars. The search system ofS-genotype database indicates three different types of combinations between apple cultivars: full-incompatibility, semicompatibility, and full-compatibility. Therefore, you will find three different open windows reflecting the 3 compatibilities mentioned above (Figures 2(c)–2(f)).Figure 2
Pashto language version of representative screenshots of the search system. “Fuji” was selected for searching cultivars on the English version (a) and Pashto version (b). Search results: cultivars incompatible with “Fuji” on the English version (c) and Pashto version (d), cultivars semicompatible with “Fuji” on the Pashto version (e), and cultivars fully compatible with “Fuji” on the Pashto version (f).
(a)
(b)
(c)
(d)
(e)
(f)


## 3.1.S-RNase Genotypes of Apple Cultivars Including Afghanistan Original Cultivars

We analyzed theS-RNase genotypes of apple cultivars and lineages for using them in a breeding programme.S-RNasegenotypes of “Iwate 4 Gou,” “Natsuakari,” K-20, K-21, K-22, KAD-14, KAD-17, KAD-19, KAD-20, and “Saturn” (Table 1, numbers 1, 2, 6–8, 12–15, and 25) matched one of theS-RNasegenotypes deduced from their reputed parentage. In contrast,S-RNase genotypes of “Oirase,” K-23, K-24, K-25, and “Red Devil” (Table 1, numbers 4, 9–11, and 24) were inconsistent with the one from its reputed parents. “Natsuotome” (Table 1, number 3) was identified asS
7
S
9, and it was suggested that “Fuji” (S
1
S
9) or “Alps Otome” (S
1
S
9) seemed to be used as a paternal parent, judging from its fruit and branch characteristics. As the average fruit weight of “Natsuotome” (195 g) is lighter than that of its maternal parent “Sansa” (280 g), its characteristics might be from “Alps Otome” (50 g), not from “Fuji” (300 g). On the other hand, the characteristics of concentrically cracking at peduncle end in “Natsuotome” are similar to “Shinano Hoppe” (Akane × Fuji), a pedigree of “Fuji” (Figure 1). “Oirase” (S
9
S
28) and K-24 (S
1
S
3) (Table 1, numbers 4 and 10) might be a sport of “Starking Delicious” (S
9
S
28) and “Shinano Gold” (S
1
S
3), respectively, judging from theirS-genotypes. The reputed paternal parent “Tsugaru” for “Oirase” and an unknown pollen parent for K-24 seemed to be unrelated to their production. The paternal parents of “Suwakko” (Table 1, number 5) are unknown. “American Summer Pearmain” (S
1
S
20) or “Indo” (S
7
S
20) or “Kitanosachi” (S
7
S
20) might be used as a paternal parent of “Suwakko” (S
3
S
20), given itsS-RNase genotype and resembling its branch characteristics to “Indo” (Figure 1). In the case of K-23 (Table 1, number 9), either its maternal or its paternal parents were wrong. Since theS-RNase genotype of K-25 (Table 1, number 11) was identified asS
9
S
x, its maternal parent “Shinano Gold” (S
1
S
3) might have been wrongly identified. One unknownS-RNase allele was present in three (JM1, JM7, and Mark [Table 1, numbers 16–18]) out of seven rootstocks in Japan.Table 1
S-RNase genotypes of 20 apple cultivars and 7 rootstocks including their lineage.
Number
Cultivar
Reputed parentage
S-RNase genotype
1
“Iwate 4 Gou”
“Fuji” (S
1
S
9)  ×  “Delicious” (S
9
S
28)
S
1
S
28
2
“Natsuakari”
“Sansa” (S
5
S
7)  ×  “Yoko” (S
3
S
9)
S
3
S
7
3
“Natsuotome”
Chance seedling of “Sansa” (S
5
S
7)
S
7
S
9
4
“Oirase”
“Starking Delicious” (S
9
S
28)  ×  “Tsugaru”+ (S
3
S
7)
S
9
S
28
5
“Suwakko”
“Sekaiichi” (S
3
S
9)  ×  Unknown≠
S
3
S
20
6
K-20
“Shinano Gold” (S
1
S
3)  ×  “Beniroman” (S
3
S
5)
S
1
S
5
7
K-21
“Shinano Gold” (S
1
S
3)  ×  “Beniroman” (S
3
S
5)
S
1
S
5
8
K-22
“Orin” (S
2
S
7)  ×  “Beniroman” (S
3
S
5)
S
3
S
7
9
K-23
“Gunma Meigetsu”+ (S
1
S
3)  ×  “Shinano Gold”+ (S
1
S
3)
S
1
S
x
10
K-24
“Shinano Gold” (S
1
S
3)  ×  U
n
k
n
o
w
n
+
≠
S
1
S
3
11
K-25
“Shinano Gold”+ (S
1
S
3)  ×  “Fuji” (S
1
S
9)
S
9
S
x
§
12
KAD-14
JM1 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
13
KAD-17
JM1 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
14
KAD-19
JM1 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
15
KAD-20
JM7 (S
9
S
x)  ×  Mark (S
3
S
x)
S
3
S
9
16
JM1
Malus prunifolia  ×  M.9 (S
3
S
9)
S
9
S
x
§
17
JM7
Malus prunifolia  ×  M.9 (S
3
S
9)
S
9
S
x
§
18
Mark
M.9 (S
3
S
9)  ×  Unknown≠
S
3
S
x
§
19
“Jawrasi Golden”
Unknown¶  ×  Unknown≠
S
28
S
x
§
20
“Kado Seb”
Unknown¶  ×  Unknown≠
S
28
S
x
§
21
“Mahali”
Unknown¶  ×  Unknown≠
S
5
S
28
22
“Nazak Badan”
Unknown¶  ×  Unknown≠
S
2
S
9
23
“Parachinari”
Unknown¶  ×  Unknown≠
S
x
S
y
§
24
“Red Devil”
“Discovery” (S
10
S
24)  ×  “Kent”+ (S
3
S
9)
S
2
S
24
25
“Saturn”
“Jonathan” (S
7
S
9)  ×  “Golden Delicious” (S
2
S
3)
S
2
S
7
26
“Swati”
Unknown¶  ×  Unknown≠
S
3
S
28
27
“Tur Kulu”
Unknown¶  ×  Unknown≠
S
2
S
28
+Incorrect parentage indicated by this work is in  bold.
≠Unknown: pollen parent is unknown.
§
S
x, S
y ≠ S
1
,
S
2
,
S
3
,
S
5
,
S
7
,
S
9
,
S
10
,
S
20
,
S
24
,
S
28.
¶Unknown: ovule parent is unknown.Figure 1
A mature fruit and a shape of tree of “Natsuotome” ((a), (b)) and “Swakko” ((c), (d)) produced at Nagano Fruit Tree Experiment Station.
(a)
(b)
(c)
(d)Within the six original Afghan cultivars, “Nazak Badan,” “Swati,” and “Tur Kulu” (Table1, numbers 22, 26, and 27) were determined asS
2
S
9,S
3
S
28, andS
2
S
28, respectively. We also identifiedS-genotypes of rootstock “Mahali” (Table 1, number 21) asS
5
S
28, but one or two unknownS-RNase alleles were present in “Jawrasi Golden” (S
28
S
x), “Kado Seb” (S
28
S
x), and “Parachinari” (S
x
S
y) (Table 1, numbers 19, 20, and 23). This is the first case to analyzeS-genotypes of Afghan cultivars.


## 3.2. Database ofS-RNase Allele Genotypes and the Search System for Suitable Apple Cultivar Combination Using the Pashto Language

We previously established a search system for apple cultivar combination, written in English on the homepage (http://www.agr.nagoya-u.ac.jp/~hort/apple/) [23]. In this study, we have developed a Pashto language version of the search system homepage for apple growers in Afghanistan. At first all names of cultivars and their parents in the database were translated from English to the Pashto language. Then, we changed the search system homepage to a Pashto language version. As the writing of the Pashto language reads from right to left, the new homepage looks like an opposite of the English version (Figure 2). All apple growers in Afghanistan can access the homepage at the URL (http://www.agr.nagoya-u.ac.jp/~hort/apple/ps/). In the homepage of this database, there is a box called the search box. When you write the first letter of a cultivar name in the search box, a list of similar cultivar names will appear from which you can select the target cultivar easily (Figures 2(a) and 2(b)). Afterward, before clicking the search button, you must decide if the cultivar is used as an ovule parent or pollen parent. Then click the search button. Actually, the search system ofS-genotype database is developed for suitable combinations of apple cultivars. The search system ofS-genotype database indicates three different types of combinations between apple cultivars: full-incompatibility, semicompatibility, and full-compatibility. Therefore, you will find three different open windows reflecting the 3 compatibilities mentioned above (Figures 2(c)–2(f)).Figure 2
Pashto language version of representative screenshots of the search system. “Fuji” was selected for searching cultivars on the English version (a) and Pashto version (b). Search results: cultivars incompatible with “Fuji” on the English version (c) and Pashto version (d), cultivars semicompatible with “Fuji” on the Pashto version (e), and cultivars fully compatible with “Fuji” on the Pashto version (f).
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(b)
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## 4. Conclusions

We have investigated 27S-RNase genotypes of apple cultivars and lineages including the nine most useful Afghanistan apple cultivars. We developed a Pashto language version of theS-genotypes database of apple cultivars, along with a convenient search system for cultivar combination showing three different types of compatibilities, that is, full-compatibility, semicompatibility, and full-incompatibility. The translated Pashto version of the search system will enable apple growers in Afghanistan to select suitable and compatible apple cultivars for successful combination in an orchard to ensure stable fruit production and breeding.



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*Source: 3101864-2016-01-20.xml*