B chromosomes in Conifers (Study Case of the Genus Picea A. Dietr.)

Data on B chromosomes in the genus Picea (Spruce) species are presented. Species of this genus are good objects for studying many problems on the role and origin of supernumerary chromosomes in conifers. Classical methods for studying chromosomes with staining of preparations with acetohematoxylin and the method of fluorescent in situ hybridization (FISH) were used. We report the B chromosomes occurrence in five Picea species and confirmed the data of different authors for seven species. At present, supernumerary chromosomes are found in 23 Picea species including the interspecies hybrid P. × fennica . B chromosomes of Picea species are smaller than A chromosomes; their size (4-6 µm) is 25-30% of the size of A chromosomes. The B chromosomes of spruce species are either meta-or submetacentric, which have been designated as B 1 and B 2 types respectively. The number of B chromosomes per cell in spruce species varies from one to six. It is generally agreed that B chromosomes are derived from A chromosomes through chromosome arrangements They differ from A chromosomes in sequence composition. The results allow us to consider that B chromosomes are of importance for organisms and possibly may play a role in their adaptation .


Materials and Methods
In present review materials consist of the publications on chromosome numbers of conifers.The information has been compiled from own publications on this subject and different papers and reviews on conifer cytogenetics and karyology (Khoshoo, 1961(Khoshoo, , 1962;;Mehra, 1968;Hizume, 1988;Jones, 2017;Rastogi & Ohri, 2020;Ohri, 2021;and others).Furthermore, to obtain these data electronic databases were also used (B-chrom, n.d.; Chromosome Counts Database, n.d.; Index to Plant Chromosome Numbers, n.d.;The Plant rDNA Database, n.d.).The method is the process of reading literature sources and extracting the names of Picea species with B chromosomes from these papers.
B chromosomes are part of the evolution system of the eucaryotic genome.It is generally agreed that B chromosomes are derived from A chromosomes through chromosome arrangements: Fragmentation, duplications, amplifications, and gene silencing (Jones & Rees, 1982;Jones & Houben, 2003;Jones et al., 2008;Kunakh, 2010;Houben et al., 2014;Borisov & Myshliavkina, 2019).After the divergence of B chromosomes from A chromosomes their evolution was independent.B chromosomes differ from A chromosomes in the organization of genetic material.B chromosomes of some plants and animals include many highly repeated DNA sequences and often do not encode genes.They may contain ribosomal, mitochondrial, and virus-type sequencies and possess A chromosome derived pseudogenes.Studies on some species have provided information about the occurrence of different repetitive DNA families, such as AT-rich sequences, C-CMA3 -DAPI bands, Ty3/gypsy retroelements, Gypsy LTR-RTs, and others (Jones et al., 2008;Kunakh, 2010;Houben et al., 2014;Borisov & Myshliavkina, 2019).
B chromosomes of Picea do not bear the secondary constrictions.Silver-staining has revealed bright bands on B chromosomes in Siberian spruce (P.obovata) indicating the possibility of its nucleolar-organizing ability (Vladimirova, 2002).However, application of fluorescent in situ hybridization (FISH) with probes of 5S and 45S ribosomal RNA genes in P. obovata and P. pungens has revealed absence of these genes on B chromosomes (Figure 2).These data are in agreement with results of Japanese investigators: B chromosomes of P. hondoensis (P.jezoensis var.hondoensis), P. engelmannii, P. koyamae also did not have loci of 5S rDNA or 18S rDNA.Fluorescent banding for B chromosomes of P. glauca and P. sitchensis displayed CMA bands.The B chromosomes of P. glehnii, P. koyamae, P. likiangensis, P. obovata revealed DAPI-bands.In B chromosomes of P. brachytyla, P. jezoensis, P. hondoensis no CMA and DAPI bands were observed (Hizume & Kuzakawa, 1995;Shibata & Hizume, 2008;Hizume et al., 1988Hizume et al., , 1989;;Hizume, 2017).DAPI is fluorescent stain for AT-rich regions in DNA, CMA (chromomycin A3) is a stain for GC-rich regions.

B Chromosomes in Mitosis, Meiosis, Inheritance, Effects on the Organism
In the mitosis of P. obovata with B chromosomes irregularities as lagging chromosomes, three poles and disorderly distribution of the chromosomes in anaphase I, single and double bridges, fragments and some other anomalies were revealed.Regular division of B chromosomes was noted; in many cases they moved to the poles first, ahead of other chromosomes.The percentage of mitosis anomalies in trees with B chromosomes was higher and their spectrum was wider (Muratova et al., 2001;Vladimirova, 2002).
At meiosis they affect the distribution and frequency of chiasmata and don't recombine with A chromosomes.In Siberian spruce (P.obovata) and Sitka spruce (P.sitchensis) in metaphase I in pollen mother cells B chromosomes formed bivalents and did not pair with A chromosomes.Their behavior at metaphase I suggested that they are distributed at random at the first meiotic division (Kean et al., 1982;Kruklis, 1982).Studies on the B chromosomes transmission in P. obovata and P. sitchensis revealed that they are transmitted through both female and male tracks.However, in P. obovata B chromosomes mainly inherited from mother plants (Kruklis, 1982;Muratova & Vladimirova, 2001b).
Usually, the presence of B chromosomes does not affect the phenotype.In many cases a large number of supernumerary chromosomes have negative influence on fertility and viability (Teoh & Rees, 1977;Jones & Rees, 1982).Siberian spruce trees with 3-4 B chromosomes were characterized by reduced pollen fertility, a smaller number of seeds, and a slight reduction in growth, 1 or 2 supernumerary chromosomes did not affect these features (Kruklis, 1971(Kruklis, , 1982)).In P. glauca, the seeds of trees with B chromosomes in the karyotype germinated on average one day earlier (Teoh & Rees, 1977).Studies of P. sitchensis did not reveal the of B-chromosomes on the growth rate (Moir & Fox, 1976).
In western populations of Siberian spruce B chromosomes are rare.Populations from the Southern Urals and East Kazakhstan are the most western where B chromosomes have been found.In related species P. abies growing in southwestern Europe (Bulgaria) 1-4 B chromosomes were found.P. × fennica, a hybrid between P. obovata and P. abies, has one B chromosome.Accessory chromosomes often occur in trees from eastern parts of the range, in Central and Eastern Siberia especially, in extreme conditions.B chromosomes have a widespread distribution in North American species of spruce: P. sitchensis and P. glauca.In P. sitchensis 29 out of 40 populations contained them.The highest frequency of B chromosomes is noted in the south of Vancouver Island, where the flora was adapted to high summer temperatures and low rainfall.P. glauca B chromosomes were found in 48 out of 51 studied populations.The highest frequency of B chromosomes was observed in the northeastern part of Vancouver Island and nearby areas of the continental America, characterized by low rainfall and high summer insolation (Moir & Fox 1977;Teoh & Rees 1977;Fox, 1987).
In P. obovata and P. glauca a higher frequency of B chromosomes was found among decorative forms, in introduced plants and urban stands, in contrast with natural populations (Butorina & Bogdanova, 2001;Muratova et al., 2002;Vladimirova, 2002;Borisov & Muratova, 2010).Examination of Siberian spruce (P.obovata) in different regions of Krasnoyarsk showed the presence of B chromosomes in all studied areas.Possibly, the appearance of B chromosomes in large industrial centers is a result of A chromosome mutations caused by industry emissions.Many authors assumed that the presence of B chromosomes in conifers could be connected with unfavorable ecological conditions of the areas where they grow (Teoh & Rees, 1977;Fox, 1987;Sedel'nikova et al., 2011;and others).
The results obtained by many authors allow us to consider that and B chromosomes are of importance for organisms and possibly may play a role in their adaptations.Plants with B chromosomes are more polymorphic and adaptive to changing environment (Jones & Rees, 1982;Kunakh, 2010;Borisov, 2014;Borisov & Myshliavkina, 2019;and others).Origin and fixing of B chromosomes give the possibility to raise the adaptive possibilities of organisms.But this hypothesis needs to be confirmed on new investigations.

Possible Origin of B Chromosomes of the Genus Picea
The similarity of B chromosomes of different Picea species in morphology, their occurrence in Siberian, Far Eastern and North American species suggest common ancient origin of the B chromosome.Broka (1990) proposed that the B chromosome arose in East Asia in the center of conifer origin.After that, B chromosomes spread to North America via the Beringian land bridge and to the Asian part of the eastern hemisphere.Fossil evidences showed that spruce entered North America in the middle of the Tertiary period (Wright, 1955).Analysis of new data obtained by sequencing nuclear, mitochondrial and chloroplast genomes confirmed the monophyletic origin of the genus Picea (Lockwood et al., 2013).These studies indicated that the genus split from a common ancestor of Pinaceae about 180 million years ago.
It clearly distinguishes three clades: I-Asian and European species; II-North American species; III-Asian and North American species P. breweriana.The version of Wright (1955) about two independent migrations of spruce from Asia to North America, which probably occurred 25 and 20 million years ago, is also confirmed.

Conclusion
Supernumerary chromosomes are reported for 23 Picea species including interspecies hybrid P. × fennica.The number of Picea species with B chromosomes to grow.Studies on the role of B chromosomes in evolution, and their influence on organisms require further investigations, including their molecular organization of genomes and reproductive biology.

Figure 2 .
Figure 2. Fluorescent in situ hybridization (FISH) of P. koyamae with probes of 5S and 45S ribosomal RNA genes.B chromosome (arrow point) has no FISH signals.Photo by O. V. Goryachkina.