**4. Morphological characters of triploid loquats**

As compared to diploidy loquat, the triploids have stronger growth vigor, characterized by the thick trunk and branches as well as larger leaves. Moreover, phenotypic differences are also found in flowers, triploid plants usually have larger flowers and flower buds than diploid ones. The transverse and length diameters of flowers, flower buds, anthers and ovaries of triploidy loquat were significantly larger than those of diploids. And fruits of triploidy loquat are significantly larger and seedless, with an edible rate of more than 80% (Table2-4, Figure 1). However, the morphological characters of different seedlings have a wide range of variation. For example, the triploid seedlings of Changbai No.1, Q21 has a ovoid-shaped fruits, whereas plant Q27 and Q11 have long ovoid fruits and the fruit weight of Q27 was significantly heavier than that of Q11 and Q21(Table 4).

#### **5. GISH (Genomic** *in situ* **hybridization) analysis of triploid loquats**

GISH is an efficient and accurate technique for the determination of levels and incorporation positions of alien chromatin. This technique has been widely applied to numerous interspecific and intergenomic plant hybrids (Snowdon et al., 1997). The GISH analysis of


Table 2. Comparison of plant morphology of diploid loquats and their related triploid seedlings. z Mean separation of 2x and related 3x means at 5% level (Liang et al., 2011).

frequency of natural triploid individuals exists in loquat germplasm. Since then, massive screening have been conducted in Chinese loquat germplasm, which has led to the selection of 352 natural triploid individuals out of 99,542 seeds in 36 varieties, the frequency of

Crop No. 2n × 2n seedlings Distribution of seedlings, percentage and number

Loquat 99,542 99.57 0.35 (352) 0.07 (74) 0.01 (10) Table 1. Comparison between apple and loquat for the frequency of ploidy levels in natural

As compared to diploidy loquat, the triploids have stronger growth vigor, characterized by the thick trunk and branches as well as larger leaves. Moreover, phenotypic differences are also found in flowers, triploid plants usually have larger flowers and flower buds than diploid ones. The transverse and length diameters of flowers, flower buds, anthers and ovaries of triploidy loquat were significantly larger than those of diploids. And fruits of triploidy loquat are significantly larger and seedless, with an edible rate of more than 80% (Table2-4, Figure 1). However, the morphological characters of different seedlings have a wide range of variation. For example, the triploid seedlings of Changbai No.1, Q21 has a ovoid-shaped fruits, whereas plant Q27 and Q11 have long ovoid fruits and the fruit weight

GISH is an efficient and accurate technique for the determination of levels and incorporation positions of alien chromatin. This technique has been widely applied to numerous interspecific and intergenomic plant hybrids (Snowdon et al., 1997). The GISH analysis of

> No. branches

Dawuxing (2x) 25.5bz 7.2a 5.3b 27.5b 7.3b 3.8a 3x seedling 50.0a 3.6b 7.1a 45.8a 15.6a 2.9b Longquan No.1 (2x) 29.7b 6.0a 6.5b 22.2b 7.0b 3.2b 3x seedling 50.0a 2.4b 8.5a 36.3a 13.4a 2.7a Jinfeng (2x) 24.9b 7.0a 6.4 25.6b 7.2b 3.5a 3x seedling 45.0a 3.0b 7.5 43.2a 15.6a 2.8b Zaohong No.3 (2x) 36.0b 7.0a 5.8b 25.7b 7.7b 3.4a 3x seedling 49.0a 4.0b 7.4a 38.7a 14.1a 2.8b

Table 2. Comparison of plant morphology of diploid loquats and their related triploid seedlings. z Mean separation of 2x and related 3x means at 5% level (Liang et al., 2011).

Annual branch diam (cm) Leaf

Width (cm)

Leaf index

Length (cm)

Apple 6,825 99.63 0.28 (19) 0.09 (6)

**4. Morphological characters of triploid loquats** 

of Q27 was significantly heavier than that of Q11 and Q21(Table 4).

(cm)

**5. GISH (Genomic** *in situ* **hybridization) analysis of triploid loquats** 

Diploid Triploid Tetraploid Pentaploid

occurrence is about 0.35% (Table 1).

Cultivar Trunk circum.

crosses.


Table 3. The comparison of flowers of natural triploid and diploid loquats. The lower cases in the table means of the significant level ( P<5%) of multiple range test.


Table 4. Characteristics of fruit in diploid loquats and their related triploids. 1LO= long ovoid, O= ovoid, R=roundish; 2OY= orange yellow, W=white;

3Maximum fruit weight (Liang et al., 2011).

Fig. 1. Compariation of morphological characteristics in leaf, flower and fruit between triploid seedling and diploid (left: 3x, right: 2x)

the natural triploid loquat revealed three types of hybrid (Table 5). In the first type, hybrid signals were detected throughout all 51 chromosomes. In the second type, only 34 chromosomes were detected with hybrid signals. In the third one, only areas around the centromere of all the 51 chromosomes showed hybrid signals. These results showed that these triploids were either homogenous or heterogenous triploids. The different types of hybrid signals also indicated the genetic diversity of natural triploid loquats. Wang (2008) used GISH analysis on artificial triploid loquats, the results showed that the source of hybrid somatic chromosomes can be accurately distinguished using genome DNA of one parent as probe, revealing 17 chromosomes from male parent, and 34 from maternal parent. No significant variation in chromosome structure, such as interchange and inversion was found.


Table 5. The results of GISH and composing of genomes of different varieties of natural triploid loquats. "+, -" means of different genomes. (Liang, 2006)

#### **6. Molecular marker analysis of natural triploid loquats**

Molecular marker is an effective technique to assess genetic diversity both dominant and codominant molecular markers such as ISSR (inter-simple sequence repeat), AFLP (amplified fragment length polymorphism), MSAP (methylation sensitive amplified polymorphism) and SSR (simple sequence repeat) have been used in genetic diversity analysis of loquat germplasm, including natural triploid loquats.

Fig. 1. Compariation of morphological characteristics in leaf, flower and fruit between

variation in chromosome structure, such as interchange and inversion was found.

Dawuxing (A322, A376), Longquan No.1 (B4-331, B316, B349, B352, B378), Jinfeng (D425), Zaohong No.3 (E39), Ganlu No.1 (I315), Huangrou (G320),

Dawuxing (A368, A379), Longquan No.1 (B347,

Dawuxing (A332), Longquan No.1 (B38, B329, B333, B338, B339, B345, B350, B351, B356, B375), Zaohong No.3 (E310), Ruantiaobaisha (H324), Longquan No.5 (K381, K459), Longquan No.6 (J367), Donghuzao, Jianyangtezao, Xiangzhong No.11, Zaozhong No.6

Table 5. The results of GISH and composing of genomes of different varieties of natural

Molecular marker is an effective technique to assess genetic diversity both dominant and codominant molecular markers such as ISSR (inter-simple sequence repeat), AFLP (amplified fragment length polymorphism), MSAP (methylation sensitive amplified polymorphism) and SSR (simple sequence repeat) have been used in genetic diversity analysis of loquat

the natural triploid loquat revealed three types of hybrid (Table 5). In the first type, hybrid signals were detected throughout all 51 chromosomes. In the second type, only 34 chromosomes were detected with hybrid signals. In the third one, only areas around the centromere of all the 51 chromosomes showed hybrid signals. These results showed that these triploids were either homogenous or heterogenous triploids. The different types of hybrid signals also indicated the genetic diversity of natural triploid loquats. Wang (2008) used GISH analysis on artificial triploid loquats, the results showed that the source of hybrid somatic chromosomes can be accurately distinguished using genome DNA of one parent as probe, revealing 17 chromosomes from male parent, and 34 from maternal parent. No significant

Individuals Genome

composing

+++

++-

+++

triploid seedling and diploid (left: 3x, right: 2x)

Luzhou No.6 (C321)

triploid loquats. "+, -" means of different genomes. (Liang, 2006)

**6. Molecular marker analysis of natural triploid loquats** 

B372, B374, B441)

germplasm, including natural triploid loquats.

Type of GISH

Ⅰ

Ⅱ

Ⅲ

#### **6.1 ISSR (Inter-simple sequence repeat) analysis of natural triploid loquats**

Liang (2006) used ISSR markers to analyze genetic diversity of loquat. Result based on twelve ISSR primers showed that similarity of 'Dawuxing', 'Longquan No.1', 'Longquan No.5', 'Longquan No.6' ranged from 0.65 to 0.98, 0.64 to 0.95, 0.76 to 0.96 and 0.83 to 0.93 respectively. As showed by the amplification pattern of primer 835, unique bands were detected in some seedlings (Figure 2, Table 6), indicating significant genetic differentiation among the triploidy accessions.

Fig. 2. The amplification pattern of ISSR primer 835. (M: λDNA/*Hind* III + *EcoR* I marker)


Table 6. The characteristic bands of primer 835.

#### **6.2 AFLP (amplified fragment length polymorphism) analysis of natural triploid loquats**

Wang (2008) assessed the effectiveness of AFLP markers in loquat using 6 natural triploid loquats and their maternal parents. With results indicated that, 12 pairs of AFLP primers amplified 2454 bands, as contrasted with maternal parent, there were 112 added bands and 96 lost bands of triploids, the number of amplified bands also differed among the clones of triploid loquats (Table 7). A369 has the greatest difference from its parents (25 additional bands and 19 missing sites), followed by A348 (20 new bands and 22 missing sites), A35 (22 new bands and 14 missing sites), A368 and A322 have the same number of different sites


(A368 has more missing bands and A322 has more new bands), and A313 has 12 new bands and 10 missing bands (Wang et al., 2011).

Table 7. Statistics of polymorphic bands of natural triploids by AFLP analysis. a the ratios of one type of polymorphic bands and total polymorphic bands; b the ratios of polymorphic bands and total bands.

#### **6.3 MSAP (methylation sensitive amplification polymorphism) analysis of natural triploid loquats**

A total of 3879 bands were amplified with 12 pairs of primers within the group of six natural triploid loquat clones and their maternal plant, in which, 363 bands were fullly methylated and 241 bands were hemimethylated. The methylation ratios of six triploid lines were between 12.9% and 18.3%, 15.8% on average, full methylation ratios were between 7.6% and 11.7%, 9.7% on average, hemimethylation ratios were between 4.7% and 6.1%, 5.6% on average. All these belong to four patterns of methylation, monomorphism, demethylation, hyper-methylation and hypo-methylation, the number of sites and frequency were 251 and 29.2%, 171 and 19.9%, 334 and 38.9%, 103 and 12.0% respectively, and all of them exisited in all triploid lines (Table 8). All these indicted that, during the process of genome recombination and triploidization, a great number of hyper-methylation, demethylation, hypo-methylation and maintained methylation were proceeded (Wang, 2008).


Table 8. Genomic DNA methylation of natural triploid loquats and their female parent. a including full methylated and hemimethylated sites; b Full methylation denotes 5′-C<sup>m</sup> CGG-3′in double strands; c Hemimethylation denotes 5′ <sup>m</sup>C CGG-3′in single strand.

(A368 has more missing bands and A322 has more new bands), and A313 has 12 new bands

Missing 2454

Table 7. Statistics of polymorphic bands of natural triploids by AFLP analysis. a the ratios of one type of polymorphic bands and total polymorphic bands; b the ratios of polymorphic

**6.3 MSAP (methylation sensitive amplification polymorphism) analysis of natural** 

A total of 3879 bands were amplified with 12 pairs of primers within the group of six natural triploid loquat clones and their maternal plant, in which, 363 bands were fullly methylated and 241 bands were hemimethylated. The methylation ratios of six triploid lines were between 12.9% and 18.3%, 15.8% on average, full methylation ratios were between 7.6% and 11.7%, 9.7% on average, hemimethylation ratios were between 4.7% and 6.1%, 5.6% on average. All these belong to four patterns of methylation, monomorphism, demethylation, hyper-methylation and hypo-methylation, the number of sites and frequency were 251 and 29.2%, 171 and 19.9%, 334 and 38.9%, 103 and 12.0% respectively, and all of them exisited in all triploid lines (Table 8). All these indicted that, during the process of genome recombination and triploidization, a great number of hyper-methylation, demethylation,

2x A348 A368 A313 A35 A322 A369

C CGG-3′in single strand.

bands 20 14 12 22 19 25 112 53.9%

bands 22 18 10 14 13 19 96 46.1%

Total 42 32 22 36 32 44 208 8.5%b

hypo-methylation and maintained methylation were proceeded (Wang, 2008).

Total methylated bandsa 573 551 567 583 539 531 535 Full methylated bandsb 44 69 43 59 63 51 53 Hemimethylated bandsc 37 32 30 34 33 25 31 Total methylated bands 81 101 73 93 96 76 84 Full methylation ratios(%) 7.7% 12.5% 7.6% 10.1% 11.7% 9.6% 9.9% Hemimethylation ratios(%) 6.5% 5.8% 5.3% 5.8% 6.1% 4.7% 5.8% Total methylation ratios(%) 14.1% 18.3% 12.9% 16.0% 17.8% 14.3% 15.7%

Table 8. Genomic DNA methylation of natural triploid loquats and their female parent. a including full methylated and hemimethylated sites; b Full methylation denotes 5′-C<sup>m</sup>

m

3′in double strands; c Hemimethylation denotes 5′-

Numbers of polymorphic bands Numbers of

A348 A368 A313 A35 A322 A369 bands

polymorphic loci and their ratiosa

Total

CGG-

and 10 missing bands (Wang et al., 2011).

Types

Added

bands and total bands.

**triploid loquats** 

#### **6.4 SSR (simple sequence repeat) analysis of natural triploid loquats**

Fifty five pairs of polymorphism primers were screened, and a total of 135 alleles were detected with ten clones of 'Dawuxing'. The allele with 222 base pairs of CH01h02 was only found in the triploids. And there's three alleles of 238 bp, 236 bp and 230 bp with primer Hi15h12 of A322 (Figure 3). Similar results were obtained by Watanabe et al. (2008). New alleles emerged as compared diploid and each one of triploid individuals, indicating foreign genes maybe introgressed along with the formation progress of triploid individuals. All ten clones were completely distinguished from each other, the highest SM similarity coefficient was between A2x and A313, with 0.926, and the contrast one was between A313 and A332, with the similarity coefficient of 0.496. Principal component analysis divided 10 strains into three groups (Figure 4), Group I, including the A484, A376, A379 and A368 of four lines, A35, A322 and A332 are three lines constitute the group II, group III consists of A2x, A313 and A484 (He, 2010).

Fig. 3. The amplification pattern of SSR primer Hi15h12.

Fig. 4. Two dimensional plot of the principal components analyzed of 10 loquat individuals with 55 primer pairs, using the similarity matrix obtained with SM coefficient.
